key: cord- -amvlm p authors: pauli, eva-k.; schmolke, mirco; wolff, thorsten; viemann, dorothee; roth, johannes; bode, johannes g.; ludwig, stephan title: influenza a virus inhibits type i ifn signaling via nf-κb-dependent induction of socs- expression date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: amvlm p the type i interferon (ifn) system is a first line of defense against viral infections. viruses have developed various mechanisms to counteract this response. so far, the interferon antagonistic activity of influenza a viruses was mainly observed on the level of ifnβ gene induction via action of the viral non-structural protein (ns ). here we present data indicating that influenza a viruses not only suppress ifnβ gene induction but also inhibit type i ifn signaling through a mechanism involving induction of the suppressor of cytokine signaling- (socs- ) protein. our study was based on the observation that in cells that were infected with influenza a virus and subsequently stimulated with ifnα/β, phosphorylation of the signal transducer and activator of transcription protein (stat ) was strongly reduced. this impaired stat activation was not due to the action of viral proteins but rather appeared to be induced by accumulation of viral ′ triphosphate rna in the cell. socs proteins are potent endogenous inhibitors of janus kinase (jak)/stat signaling. closer examination revealed that socs- but not socs- mrna levels increase in an rna- and nuclear factor kappa b (nf-κb)-dependent but type i ifn-independent manner early in the viral replication cycle. this direct viral induction of socs- mrna and protein expression appears to be relevant for suppression of the antiviral response since in socs- deficient cells a sustained phosphorylation of stat correlated with elevated expression of type i ifn-dependent genes. as a consequence, progeny virus titers were reduced in socs- deficient cells or in cells were socs- expression was knocked-down by sirna. these data provide the first evidence that influenza a viruses suppress type i ifn signaling on the level of jak/stat activation. the inhibitory effect is at least in part due to the induction of socs- gene expression, which results in an impaired antiviral response. influenza a viruses are negative-stranded rna viruses that belong to the family of orthomyxoviruses. the segmented genome of influenza a virus encodes for up to viral proteins. as many other viruses, influenza viruses have evolved strategies to counteract cellular antiviral responses, especially to circumvent the type i ifn system as a first line of defense against the pathogenic invader. among the influenza viral proteins, the ns has been identified as the main type i ifn antagonistic factor. so far two major mechanisms have been described by which ns suppresses the initial expression of ifnb. on the one hand ns inhibits vrnamediated induction of the transcription factors interferon regulatory factor- (irf- ), activating protein- (ap- ) and nf-kb that target the ifnb promoter. this most likely occurs via binding to the rna-sensor retinoic acid inducible gene (rig-i) and inhibition of rig-i-mediated signaling in response to viral rna [ , ] . on the other hand ns inhibits maturation [ , ] and nuclear export of host mrnas [ ] . other functions of the multifunctional protein include block of activation of the dsrnaactivated protein kinase pkr by direct interaction [ ] or activation of the phosphatidylinositol- kinase pi k/akt pathway to prevent premature apoptosis induction [ , ] . while the ns -mediated antagonistic activities of influenza viruses mainly affect the induction of genes such as ifnb, so far no viral suppression of ifn signaling has been described. ifn are among the first molecules synthesized in response to viral infections [ ] . the ifn family includes three classes. type i comprises the well known ifna and ifnb. the only member of type ii ifn is ifnc. type iii ifn comprises ifnl , -l , and -l . all classes of ifn bind to different receptors and are structurally not related [ , ] . type i ifn belong to the key cytokines produced by influenza a virus-infected epithelial cells [ , ] . the antiviral activity of type i ifn is mediated by a set of ifn-induced genes (isgs). binding of ifna/b to its receptor is the initial step in this signaling process, followed by activation of the jak family and subsequent activation of stat proteins [ ] . ligand binding leads to dimerisation of the type i ifn receptor subunits ifnar and ifnar and causes their conformational change. the jak kinase tyk , which is constitutively bound to ifnar , phosphorylates the receptor at tyrosine residues and creates a docking site for stat . subsequently, tyk phosphor-ylates stat at y . at the same time the receptor-bound jak phosphorylates stat at y [ , ] . the phosphorylated transcription factors dimerise and bind to irf- [ ] . the newly formed heterotrimer, called ifnstimulated gene factor (isgf ), translocates into the nucleus and binds to ifn-stimulated response elements (isre), to initiate gene transcription of isgs. treatment of cells with type i ifn upregulates expression of an array of genes including sp , irf- and many others [ ] . among these isgs the , oligoadenylate synthetase (oas ), the mx proteins and the dsrna-activated protein kinase (pkr) are described to directly interfere with viral replication [ ] . both, pkr and the oas / rnasel system are capable of inhibiting cellular and viral translation. ifn-induced jak/stat signaling can be inhibited at different levels by several viral and cellular factors through various mechanisms. the large t-antigen of murine polyomavirus (mpyv) binds to jak and inhibits downstream signaling [ ] , whereas the vp of ebola virus (ebov) binds to karyopherina- thereby blocking nuclear accumulation of stat [ ] . endogenous cellular key regulators, capable of negatively regulating jak/stat-mediated signal transduction, include suppressor of cytokine signaling (socs) proteins, protein tyrosine phosphatases (ptp) and protein inhibitor of activated stats (pias). the family of socs proteins comprises eight members (cytokine-inducible sh domain-containing protein (cis) and socs - ). all members contain a central sh domain, an nterminus of variable length and sequence and a c-terminal amino-acid module called socs box [ ] . the socs box is necessary for recruitment of the ubiquitin transferase system and for stabilization and/or degradation of socs proteins [ ] [ ] [ ] . the n-terminus contains a kinase inhibitory region (kir), which functions as pseudo substrate for the jak [ ] . socs- and socs- differ in their mode of action. for inhibition of the kinase activity of jaks, socs- binds directly to the activation loop of jaks [ ] [ ] [ ] . in contrast, socs- first binds to the receptor [ , ] . induction of socs- gene transcription by viruses was reported for hsv- , hcv [ ] [ ] [ ] and for respiratory viruses, such as sars and rsv [ , ] . the level of induction of socs- by hsv- seems to determine whether infection turns to acute or persistent progression [ ] . for hcv it has been suggested that upregulation of socs- may contribute to the non-responsiveness of hcv patients to ifn therapy [ , [ ] [ ] [ ] . elevated socs- mrna levels during rsv infection were linked to th cell-mediated immune disease as atopic dermatitis and asthma [ , ] . in the present study we show that influenza a virus can be added to the list of viruses that induce socs- expression. the protein functionally interferes with viral replication by providing a virus-supportive ifn-antagonistic activity on the level of type i ifn-signaling that has not been described so far. phosphorylation of stat and stat by members of the jak tyrosine kinase family is a prerequisite for activation of these transcription factors to drive type i ifn-induced gene expression. therefore, we analyzed whether stat phosphorylation patterns are altered in influenza a virus infected cells that were stimulated with ifn at different time points post infection (p.i.). the human alveolar epithelial cell line a was infected with the influenza a virus strain a/puerto-rico/ / (h n ) (pr ) ( figure a ). cells were subsequently stimulated with ifnb at given time-points p.i. and stat phosphorylation was assessed in western blots. both stat and stat were readily phosphorylated upon cytokine stimulation in uninfected cells or in infected cells up to h p.i. ( figure a ). furthermore, virus infection alone resulted in a significant induction of stat phosphorylation - h p.i., presumably caused by virus-induced ifn expression. however, at later time points ( - h p.i.), in a cells both virus-and ifn-induced stat and stat phosphorylation was markedly reduced ( figure a ). similar patterns were observed upon stimulation of cells with ifna or upon infection with other viruses, such as the human influenza virus a/victora/ / (h n ) (data not shown). in addition, this phenomenon could also be detected in other epithelial cells such as the human embryonic kidney cell line hek ( figure e ) or the human umbilical vein endothelial cells (huvec) ( figure s b ). inhibition was not caused by indirect disturbing effects on cellular metabolism or enzyme activities due to ongoing virus replication, since ifnc-induced stat phosphorylation was not affected at all ( figure c ). finally, involvement of any auto-or paracrine action of virus-induced type i ifn could be ruled out, as the inhibitory effect was also observed in vero cells lacking functional type i ifn genes ( figure e ). with regard to the molecular basis of impaired ifna/b-induced stat phosphorylation in infected cells it was striking that the inhibitory effect correlated with the accumulation of viral proteins, as monitored in pb western blots ( figures a and e) . thus, the question arose whether individual expression of viral proteins may result in the interference with stat phosphorylation. out of the viral proteins of pr we choose the nucleoprotein (np), the ns protein, the matrix protein (m ) (figure a ) and the subunits of the viral polymerase, pa, pb and pb ( figure c ), for a representative experiment. these proteins are known to bind to vrna/rnps or to interfere with the rna-mediated innate immune response. for efficient transfection of the expression the type i interferon (ifn) system is one of the most powerful innate defenses against viral pathogens. most rna viruses are sensitive to the action of type i ifn. therefore, these pathogens have evolved strategies to evade this response. for example, influenza viruses express a viral protein, the non-structural protein (ns ), that suppresses production of ifnb by lowering cellular sensitivity to viral nucleic acid as a pathogen pattern. here we present data indicating that influenza a viruses are not only capable of suppressing production of the ifnb gene but also inhibit action of this antiviral cytokine on cells. this occurs by viral induction of a cellular protein, the suppressor of cytokine signaling (socs)- , a potent endogenous inhibitor of ifn signaling. this is a novel mechanism by which influenza viruses inhibit the antiviral response of the host and paves the path to efficient virus replication. this may be especially relevant for influenza viruses that induce high cytokine responses (cytokine burst), such as highly pathogenic avian influenza viruses of the h n subtype. induction of socs- expression would allow efficient replication despite high ifn and cytokine levels. constructs we used the highly susceptible cell line hek that also exhibits impaired ifnb-induced stat phosphorylation at later stages of infection ( figure e ). h post transfection cells were stimulated with ifnb and stat phosphorylation was monitored in western blots (figures a and c) . expression of none of the viral proteins resulted in a significant decrease of ifnb-induced stat or stat phosphorylation (figures a and c ). similar results were obtained in the human bronchial epithelial cell line h when expressing m , ns or np alone or in different combinations (data not shown). thus, we concluded that viral proteins most likely do not play a prominent role as blockers of ifna/b-induced jak/stat signaling. decrease of stat phosphorylation might also be due to the action of virus-induced phosphatases. on the one hand these enzymes may cause direct dephosphorylation of stat proteins. on the other hand phosphatases could act via an indirect mechanism by dephosphorylation and inactivation of jaks resulting in an attenuated phosphorylation of stats. several protein tyrosine phosphatases (ptps) are known to mediate dephosphorylation of both, jaks and stats [ ] . in order to investigate whether influenza a virus activates phosphatases that subsequently target jaks or stats, we treated infected or uninfected a cells with the well-known tyrosine phosphatase inhibitor sodium vanadate [ , ] . uninfected cells or cells infected with pr for h were incubated with increasing amounts of this compound min prior to stimulation with ifnb. this time point of infection was chosen since we observed considerable inhibition of ifn-induced stat phosphorylation in the course of infection ( figures a and e ). increasing concentrations of vanadate lead to a gradual shift of the steady state balance of phosphorylation/dephosphorylation. accordingly, a gradual increase of stat phosphorylation was observed that was similar in both infected and uninfected cells, albeit starting from different basal levels of phospho-stat ( figures a and b ). this is illustrated by an almost identical slope of the regression line in the graphical analysis of the band intensities of the ifnb stimulated samples ( figure b ). if the blockade of ifnb-induced stat phosphorylation would be mediated by specific virus-activated phosphatases, a much steeper slope for vanadate-treated infected cells would be expected. however, the result in figure b indicates that the virus-induced suppression of phosphorylation is not compensated by phosphatase inhibition and consequently no virusactivated phosphatase appears to be involved. in support of these data, phosphatase assays revealed that the overall activity of tyrosine phosphatases in infected cells was not elevated compared to uninfected cells. this is indicated by constant levels of free phosphates released from two different phospho-peptides that represent common tyrosine phosphatase substrates ( figure c ). thus, involvement of phosphatases in influenza virus-induced alteration of stat phosphorylation can be greatly ruled out. phosphorylation of stats in the ifnb signaling cascade may not only be counter-regulated by phosphatases but also by other cellular factors, such as proteins of the suppressors of cytokine signaling (socs) family. action of these proteins is mainly controlled on the level of transcriptional activation. socs proteins are described to have high affinity for jak and stat proteins and to inhibit the transmission of ifna and ifnb induced signaling [ , ] . to examine whether expression of socs genes is induced in influenza virus infected cells, a cells were infected with pr for different time points. subsequently total rna was analyzed for the amount of socs- and socs- mrna by means of quantitative real time-pcr (qrt-pcr). the mrna table for accession numbers of viral genes) using l according to manufacturer's instructions. note that the pol ii constructs in use also give rise to expression of second reading frames in the ns, m and pb genes (ns , m , pb -f ). h post transfection cells were stimulated with human ifnb ( u/ml) for minutes. total protein lysates were subjected to western blot analysis using anti-phospho-stat , anti-phospho-stat , anti-stat antibodies. expression of influenza viral proteins was monitored with antibodies against np, m , ns , pa, pb or pb . (e) hek cells were infected with the human influenza virus pr (h n ) (moi = ) for the indicated time points and were subsequently stimulated for min with either human ifnb at a concentration of u/ml. cell lysates were subjected to western blots as described. (b, d, f) quantification of relative pstat and pstat band intensities in a, c and e using aida software and d densitometry (fuji). doi: . /journal.ppat. .g levels of socs- and socs- differed notably in the time course ( figure a ). while socs- mrna is strongly and transiently elevated in the early phases of infection, socs- gene transcription is only significantly induced h p.i.. elevated socs- mrna levels were also observed in other host cell types, such as huvec starting h p.i. ( figure s a ). although elevation of socs- mrna levels in infected cells was rather transient, there appears to be a robust induction on protein level ( figure c ). first detected at h p.i., socs- protein levels increased and stayed on a high level throughout the observation period. strikingly, expression kinetics of the socs- protein perfectly matched the kinetics of virus-induced inhibition of stat phosphorylation ( figure c ), indicating that both processes are functionally linked. virus mediated socs- gene induction at early stages of infection ( figures a, c and s a) appeared to occur concomitant with an immediate and strong induction of ifnb ( figures b and s d) . this prompted us to analyze whether socs- transcription might be induced due to an auto-or paracrine action of ifnb expressed during infection. a cells were stimulated with ifnb for different time points and socs- gene induction was measured by qrt-pcr ( figure d ). as a control we monitored expression of , oligoadenylate synthetase (oas ) and mxa, genes that are typically induced by ifnb. while oas and mxa mrnas were readily upregulated upon ifnb treatment socs- mrna was not significantly elevated ( figure d ). similar results were obtained from huvec stimulated with ifnb ( figure s e ). to further confirm these results we knocked down the ifnar in a -cells by an sirna approach. although the knock down was efficient and leads to more than % inhibition of ifnb induced stat phosphorylation ( figure e ), the induction of socs- expression was not impaired ( figure f ). socs- levels in the knock down cells were similar compared to wild type cells and even higher than in the vector control ( figure f ). these results are consistent with data gained from previous experiments in vero cells ( figure e ) and indicate that neither induction of socs- mrna nor inhibition of stat phosphorylation is dependent on virus-induced type i ifn expression. since accumulation of viral rna in infected cells is a potent inducer of antiviral gene expression we investigated its ability to induce socs- gene transcription. as a source for viral rna, a cells were infected with influenza a virus for h and total rna from these cells was isolated. rna from uninfected a cells served as a negative control. different amounts of these rnas were used for stimulation of a cells for h (figures a, b and c). transfection of rna from uninfected cells did not result in an increase of socs- or socs- gene transcription ( figure a ) or ifnb induction as a control ( figure b ). however, transfection of rna from virally infected cells led to strongly elevated socs- mrna amounts while socs- mrna is only induced weakly ( figure a ). this dose dependent induction of socs- by stimulation with increasing amounts of rna from infected cells corresponds with a gradual decrease in the ability of this rna to induce or potentiate stat / phosphorylation ( figure c ). in contrast to cellular rna, influenza viral rna carries a triphosphate group at its terminus that was previously shown to be a major pathogen pattern that triggers cellular signaling [ ] . to verify that indeed the viral triphosphate rna in the pool of rnas from infected cells is the major trigger for induction of socs- expression, rna from infected or uninfected cells was treated with phosphatase to remove the triphosphate termini prior to stimulation of a cells ( figure e and f). the dephosphorylated viral rna was only poorly capable to induce socs- ( figure e ) or ifnb ( figure f ) mrna expression. in addition, poly(i:c) was transfected to mimic action of double-stranded (ds) rna ( figure e and f, right bars). however, the dsrna analog showed surprisingly little effects on socs- and ifnb mrna induction. since viral rna is able to induce ifnb gene transcription ( figure b and f) we again wanted to rule out that induction of socs- by viral triphosphate rna is mediated by auto-or paracrine action of de novo synthesized ifnb. in order to do so, cells were stimulated with viral rna after treatment with the protein synthesis inhibitor anisomycin at two different concentrations ( figure g ). socs- mrna was still induced to the same extent in the presence of the protein synthesis inhibitor, providing the ultimate proof that de novo protein synthesis is not required for socs- induction. so far, our data suggest that influenza virus-induced transcriptional upregulation of the socs- gene is not mediated by the . equivalent mrna amounts were normalized to gapdh mrna levels and calculated as n-fold of the levels of untreated cells that were arbitrarily set as . to detect socs- protein expression (c) cells were infected for time points indicated or left uninfected. total cell lysate was subjected to western blot analysis using anti-socs- antibody. to allow better comparison of socs- protein expression and stat phosphorylation phospho-stat and stat western blots from figure a are shown again here. (e) to functionally test effective knock down of the ifnar, a wild type, a vector control cells or a cells stably expressing ifnar ii- specific shrna were stimulated with human ifnb ( u/ml) for min. subsequently cells were lysed and levels of phospho-stat were determined by western blotting using specific antibodies. in addition, the relative pstat band intensities were quantified. doi: . /journal.ppat. .g autoregulatory action of type i ifns ( figure d and f) but is directly induced through accumulation of viral rna during infection. this raises the question, which rna-induced signaling pathways are responsible for socs- expression. the mkk/p mitogen activated protein kinase (mapk) pathway [ ] [ ] [ ] as well as the ikb kinase (ikk)/nuclear factor of kb (nf-kb) cascade [ ] [ ] [ ] are both known to be activated by rna or influenza virus infection and to be involved in the control of socs- expression. to assess whether the mkk /p -or the ikk/nf-kb-module is required for socs- gene induction, we generated a cell lines expressing dominant negative forms of either mkk (mkk ala) or ikk (ikk kd) (figure a to d). these mutants have been previously shown to efficiently block p or nf-kb signaling, respectively [ ] [ ] [ ] . to monitor socs- gene induction, wild type, vector or mutant expressing cell lines were infected with pr ( figure a ) or stimulated with rna from virally infected or uninfected a cells ( figure c ). induction of ifnb mrna was monitored as a control ( figure b and d) . while mkk ala expression did not result in significant reduction of socs- in either infected ( figure a ) or rna-stimulated cells ( figure c ), transcription is markedly reduced in ikk kd expressing cell lines. to obtain independent evidence for nf-kb dependence of socs- gene transcription, a wild type cells were incubated with the nf-kb specific inhibitor bay - prior to stimulation with rna from virally infected or uninfected a cells ( figure e ). again, ifnb mrna levels were assessed for control purposes ( figure f ). both, socs- and ifnb mrna levels were strongly reduced in bay - treated cells. this indicates that virus-induced socs- expression strongly depends on ikk and nf-kb activation, while the mkk /p appears not to play a prominent role. to further verify that influenza virus induces socs- via an rna sensory pathway and in an nf-kb dependent manner we infected cells with the influenza a virus mutant deficient for ns (dns ) (figure g and h) . the ns protein is known to block rna dependent signaling and nfkb activation [ ] . accordingly, infection of cells with the mutant virus resulted in a more pronounced and sustained, albeit delayed induction of socs- ( figure g ) if compared to infection with the isogenic wild type, that is a very poor inducer of socs- but still reasonably well induces ifnb. noteworthy, this isogenic wild type strain differs from the pr wild type virus used in the other experiments shown here (see materials and methods for details). to analyze whether nf-kb activation is sufficient for socs- gene induction we stimulated cells with il- b ( figure s a ) or tnfa ( figure s b ) that are both strong activators of the transcription factor. while mrna levels of il- , a strictly nf-kb dependent cytokine, are strongly elevated, socs- gene transcription is not significantly induced. under the assumption that these cytokines do not additionally induce counteracting processes one can conclude that nf-kb is required, yet not sufficient for the induction of socs- . thus viral induction of socs- may require additional factors that are only active in virus-infected cells. furthermore, these results rule out a potential role of virus-induced il- b or tnfa in the induction of socs- . this is supported by the observation that neither expression of il- to further assess a functional role of socs- in virus-induced suppression of stat phosphorylation we analyzed mouse cells with a targeted deletion of the socs- gene [ ] . wild type and socs- deficient mouse embryonic fibroblasts (mef) were infected for different time points with pr . the time of infection was prolonged in comparison to the infection of a cells because the human pr replicates less efficiently in mouse than in human cells. following infection lysates of these cells were assessed for stat phosphorylation ( figure a ). both cell types showed no phosphorylation of stat in the uninfected state. in contrast, infection of socs- knock out cells resulted in strongly elevated phosphorylation of stat in a sustained fashion. to rule out that this stat phosphorylation is due to altered secretion of ifnb or figure . enhanced stat phosphorylation in infected socs- deficient mef correlates with elevated induction of ifnb-stimulated genes. wild type mef and socs- knock out mef were infected with pr (moi = ) for the indicated times. subsequently, cell lysates were analyzed for stat phosphorylation (a). for control of productive virus replication, cell lysates were analyzed for viral protein pb expression. in (e, f, g) wild type and knock out cells were lysed at indicated time-points of infection. subsequently rna was subjected to reverse transcription. cdna was analyzed in quantitative real time pcr to assess mrna amounts of three prototype type i ifn-stimulated genes, sp (e), interferon regulatory factor- (irf- ) (f) and oas (g). equivalent mrna amounts were normalized to gapdh mrna levels and calculated as n-fold of the levels of untreated cells that were arbitrarily set as . in (c) wild type mef and knock out mef were infected with pr (moi = ) or left uninfected. supernatants were taken p.i. and used for stimulation of wild type mef for minutes. as control wild type mef were stimulated with u/ml mouse ifnb for minutes. cells were harvested and analyzed for the amount of stat and phospho-stat in western blot analysis by specific antibodies. in (b) and (d) the relative band intensities of phospho-stat of the blots in (a) and (c) were quantified as described. doi: . /journal.ppat. .g other stat -activating cytokines in socs- deficient cells, we performed conditioned medium experiments ( figure c ). mef wild type and mef socs- deficient cells were infected for h and supernatants were subsequently harvested. stimulation of mef wild type cells with these different supernatants for min. revealed no differences in stat phosphorylation, indicating that both infected cell types secrete similar amounts of ifnb and other stat activating cytokines. this is a strong indication that the observed differences in virus-induced stat phosphorylation are directly due to the presence or absence of socs- in wild type and knock out mef, respectively. to answer the question whether enhanced stat phosphorylation in socs- deficient cells would also lead to enhanced expression of isgs, total rna was isolated at different time points p.i. from infected wild type and knock out cells and monitored for induction of sp , irf- and oas ( figure e, f and g ). these genes are described as type i ifn-induced genes [ ] . indeed mrna levels of all three representative isgs were elevated in socs- knock out versus wild type cells at almost every time point during the course of infection. this indicates that enhanced stat phosphorylation and activation in socs- deficient cells results in elevated expression of isgs. the remaining question was, whether the elevated ifn-induced gene response in knock out cells might also affect propagation of influenza a viruses. thus, both wild type and knock out cells were infected with pr ( figure a ) or the strain a/victoria/ / (h n ) ( figure b ). virus titers were assessed at different time points post infection. progeny virus titers from socs- knock out cells were significantly reduced compared to titers from infected wild type cells. to independently confirm these results and to verify that the observed effects are really due to the lack of socs- , we used an sirna approach to specifically knock down socs- mrna in a cells. cells were transfected with nm sirna for h and socs- protein levels were compared to control transfected samples ( figure c, right) . subsequently, cells were infected and progeny virus titers were determined by plaque assay (figure c, left) . similar to the results gained from infected knock out cells, knock down of socs- resulted in decreased virus titers. on the contrary, over-expression of socs- resulted in elevated virus titers ( figure d ) concomitant with an inhibition of ifnb-or virus-induced stat phosphorylation ( figure e) . taken together the data indicate that in the absence of socs- , infection leads to a stronger activation of stat , resulting in enhanced expression of isgs and reduced virus titers. vice versa, over-expression of socs- leads to an inhibition of stat activation and elevated virus titers, probably due to inhibited expression of isgs. this highlights the important role of virus induced socs- to limit the type i ifn-induced antiviral response program. the type i interferon (ifn) system is one of the most powerful innate defenses of vertebrate cells, which limits replication and spread of viral pathogens including avian and human influenza viruses. influenza virus propagation is sensitive to ifn activities and therefore, like other viral pathogens, these viruses do not only induce type i ifn but also antagonize the production and effects of these cytokines at the same time [ ] . for influenza a and b viruses, this is accomplished through their non-structural ns proteins that are structurally related polypeptides of kda (a/ ns ) and kda (b/ns ), which are abundantly expressed in infected cells [ ] . ns proteins predominantly act on the level of ifn gene induction in infected cells by obstructing rig-idependent signaling through interaction with cellular factor(s) and/or sequestration of rnas generated during virus replication [ , , ] . some ns proteins were also described to inhibit the maturation of cellular pre-mrnas raising the possibility that this activity additionally reduces production of ifna/b in infected cells [ , ] . while ns also interferes with the activity of some isgs, such as the dsrna dependent kinase pkr [ , ] , so far no type i ifn antagonistic mechanism was described for influenza viruses that act on the level of ifn signaling rather than gene induction. here we present data, showing that rna-induced expression of socs- in early phases of infection leads to a functional inhibition of ifn-induced stat activation and gene expression. this is a novel mechanism by which influenza virus suppresses the antiviral response of the host and paves the path to efficient virus replication. while it was reported in the literature that expression of socs proteins can be induced upon stimulation with ifn [ ] we could not detect any significant gene induction by ifnb in a cells. instead we observed a significant up-regulation of socs- by viral triphosphate rna, indicating that gene induction occurs via accumulation of vrna during infection. this appears to occur through the rna-mediated activation of the ikk/nf-kb pathway, most likely activated through engagement of the rna sensor rig-i. at a first sight, this might appear controversial since nf-kb activation is among the rig-i-induced signaling responses and ns was reported to inhibit this signaling pathway. however, despite the action of ns it is well known that nf-kb is still significantly activated upon influenza virus infection and many nf-kb and ifnb dependent genes are still expressed. we hypothesized previously that the incomplete inhibition conferred by ns is an indication that the virus exploits the remaining signaling activities for efficient replication [ , , ] . the findings described here, namely nf-kb dependent induction of socs- and limitation of type i ifn signaling responses, provide yet another example how influenza viruses take advantage of nf-kb activity. while the data show that nf-kb is required for viral socs- induction, the factor appears not to be sufficient, since prototype inducers of nf-kb, such as il- b or tnf-a would not induce socs- . thus there seems to be the need of additional virus or rna-induced transcription factors. the most likely candidate would be the constitutively expressed interferon regulatory factor (irf- ), that is known to be simultaneously activated with nf-kb upon virus infection directly via the rig-i rna sensing pathway without the need of type i ifn. furthermore irf- is a factor suppressed by the ns protein. recently it was reported that ifn-induced gene expression responses are potentiated in cells, which lack the nf-kb factors p or p [ ] . although these authors described an inhibitory binding of nf-kb transcription factors to some ifn-induced gene, this mechanism might be cell type dependent since we could not observe similar effects in the cell types used here (data not shown). thus, the underlying molecular mechanisms appear to be not fully clear. it is striking that the effects described for p and p knock out cells in these studies fully correlate with our observations in socs- deficient cells. while in the latter case cells lack the ifnb signaling inhibitor socs- , the p and p knock out cells are deficient for the factors required for socs- induction. thus, given the nf-kb dependent induction of socs- described in the present manuscript, we provide an additional molecular mechanism that may explain the phenomenon described by wei et al. [ ] . first indications for beneficial effects of socs- gene expression on viral replication came from studies using the hcv core protein as a replacement for the influenza a viral ns in the context of infections with a ns deficient influenza virus [ ] . one of the hallmark responses of hcv core expression is a rapid induction of socs- expression. given the role of socs- described here, it was not surprising that hcv core could partially rescue growth of the ns deficient virus [ ] . while this manuscript was in preparation it was demonstrated by pothlichet et al. that influenza a virus-induced socs- and socs- upregulation requires a tlr- -independent, rig-i/ mavs-dependent pathway [ ] . moreover, over-expression of socs- and socs- in infected cells revealed that both molecules inhibit antiviral responses. these studies are perfectly complemented by our findings. here we confirm involvement of rig-i/mavs by showing that triphosphate rna, the ligand for rig-i, is a major inducer of socs- . furthermore, the finding that dsrna is only a weak inducer of socs- is also consistent with the independence from the dsrna sensor tlr- . the only discrepancy of this work and the study of pothlichet et al. is that they show a dependence on the type i ifn receptor. this may be due to the different virus-strains and cell types used. it is well known that the capability of type i ifns to induce socs proteins is strongly cell type specific [ ] . while in some cell types socs- expression appears to be type i ifn dependent (e.g. fetal liver cells) [ ] it is clearly independent of ifn in other cell types [ ] . recently it was shown that socs- is not significantly induced by ifna in a cells [ ] , the major cell type used in our study. evidence that cell type specificities may be the cause of discrepancy is additionally provided by the fact that pothlichet et al. show identical induction kinetics of socs- and socs- . in contrast the kinetics of the two proteins differ clearly in the cells we used, with socs- being induced much earlier than socs- on mrna and protein level. finally, it should be stated that regardless whether socs- is additionally induced by type i ifns at a later stage of infection, it is important that it can be induced earlier and in parallel to ifnb directly by vrna accumulation. this is supported by the finding that ifnb and socs- induction occurs in parallel kinetics ( figure a and b) while ifn-induced genes such as oas and mxa are only up-regulated later in a delayed and more sustained fashion ( figure d ). this makes a qualitative difference since the blocking effect of socs- on ifnb signaling already kicks-in during the first wave of ifnb action. taken together we describe here for the first time that at least some influenza a virus strains are able to suppress type i ifn signaling by a mechanism involving nf-kb dependent activation of socs- expression, which negatively affects stat phosphorylation. this adds a new aspect to our knowledge of the strategies used by influenza a virus to antagonize type i ifn responses. human influenza a/puerto-rico/ / (h n ) (pr ) (giessen variant) and a/victora/ / (h n ) (victoria) were originally taken from the strain collection of the institute of virology, giessen, germany. the human ns deficient influenza virus mutant dns and its isogenic wild type variant were propagated and used as described earlier [ , ] . it should be noted that this isogenic wild type strain as described by garcia-sastre et al. [ ] is different from the pr (giessen variant) used in the other experiments and in many previous studies [ , ] . the supernatant was aspirated and cells were incubated with specific medium containing . % bsa and antibiotics. to score for production of viral plaques the overlay was stained for h using ml neutral red in pbs per well [ ] . to trigger jak/stat signaling cells were stimulated using human ifna/b or c as well as mouse ifnb. for stimulation of a cells or huvecs u/ml human ifna or human ifnb was used. for stimulation of the green monkey epithelial cell line vero or hek cells u/ml human ifnb was applied. ifnc was always used in the concentration of u/ml. mouse embryonic fibroblasts (mef) were incubated with u/ml mouse ifnb. the different ifn were diluted in infection medium. for stimulation after infection, viral supernatants were aspirated and diluted cytokine was incubated for minutes at uc. to investigate the potential of other cytokines to induced socs- gene expression a cells were stimulated with u/ml il b or ng/ml tnfa at uc for times indicated. after stimulation cells were lysed and subjected to immune blotting. to block the activity of phosphatases after infection with influenza virus, sodium vanadate was used. dilutions were prepared using infection medium. sodium vanadate was added to the virus-containing infection medium at the time points indicated. after minutes of incubation ifnb, diluted in infection medium, was added to the medium containing virus and sodium vanadate. the cells were stimulated with ifnb for minutes. incubation with sodium vanadate started min before cells were lysed and subjected to western blotting as described. for conditioned medium experiments wild type and socs- knock out mef were infected with pr (moi = ) for h or left uninfected. supernatants were used for stimulation of mef wild type for minutes. cell lysates were subjected to western blot analysis. to investigate the induction of socs- expression by viral rna, rna isolated from infected or uninfected cells (control) was used. a cells were infected with pr (moi = ) or left mock infected. h post infection rna was isolated using the rneasy mini kit from qiagen according to manufacturer's instructions. to dephosphorylate viral triphosphate rna, calf intestine alkaline phosphatase (ciap) (fermentas) was used. briefly, rna was isolated using trizol according to manufacturer's instructions. for dephosphorylation the reaction mix was set up in a ml volume with mg rna, u ciap and u ribolock rnase inhibitor (fermentas) and was incubated for h at uc. thereafter the rna was isolated using the rneasy mini kit from qiagen. rnas used as control were mock-treated replacing ciap by glycerol. for stimulation, the different rna species and analogues were transfected using lipofectamine (l ) according to manufacturer's instruction (invitrogen). in brief, l was incubated with opti-mem for minutes at room temperature; different amounts of rna were added and incubated for additional minutes. for stimulation of cells with cellular or viral rna ml rna-l mix were added to ml serum-free medium. cells were stimulated for hours and subjected to either western blot analysis or quantitative real time pcr. for silencing socs- mrna, a cells were transfected with nm human socs- sirna h before infection using hiperfect (qiagen) according to manufacturer's instructions. in brief, nm sirna was added to a mixture of d-mem without fcs/antibiotics and hiperfect and incubated for min at room temperature. for transfection ml of this mixture were added to the cells. subsequently cells were subjected to plaque assay analysis or western blot analysis. control sirna was purchased from qiagen. the sequences for the human socs- sirna in use are: human socs- sirna sense -cca aga acc ugc gca ucc adtdt- , human socs- sirna anti-sense -ugg aug cgc agg uuc uug gdtdt- ) (see table for accession number of the human socs- gene). to determine whether tyrosine phosphatases become activated upon infection with influenza virus a phosphatase assay using the tyrosine phosphatase assay system (promega) was performed. a cells were infected for h (moi = ) or left uninfected. cells were harvested in assay buffer ( mm tris-hcl ph . , mm cacl , mm mgcl , . % b-mercapto ethanol), cracked by a single freeze/thaw step at uc and disrupted by ultrasonic pulsing. lysates were precleared from cell debris and residual free phosphates according to the manufacturer's instruction. tyrosine phosphatase activity was measured by enzymatic release of free phosphate of two given pseudosubstrates (phosphorylated peptides representing target sequences for the most common tyrosine phosphatases). quantification was performed in comparison to a given standard according to the manufacturer's instruction. for western blot analysis cells were lysed with ripa [ mm tris/hcl, ph . , mm nacl, % glycerol, . % sds, . % nadoc, % igepal, mm edta, ph . , pyrophosphate mg ml aprotinin; mg ml leupeptin; mm sodium vanadate and mm benzamidine] on ice for a minimum of minutes. supernatants were cleared by centrifugation in a standard tabletop centrifuge (eppendorf) at maximum speed. protein concentration was determined by bradford assay. the phosphorylated and unphosphorylated forms of stat were detected using anti-stat (y ) antibody and anti-stat (bd bioscience). an antibody directed against y of stat was used for detection of the phosphorylated form of stat (upstate). antibodies to detect influenza viral proteins were purchased from serotec (np, m ), santa cruz (pb , pb ). the anti-pa antibody was kindly provided by j. ortin (madrid/ spain). a monoclonal antibody directed against the viral ns was generated at the imv, muenster, germany [ ] . a monoclonal anti-myc-tag antibody to detect myc-m was kindly provided by viktor wixler. imv, muenster, germany. all secondary antibodies were purchased from amersham and diluted : in tbs-t. secondary antibodies were incubated for a minimum of minutes at room temperature. to synthesize cdna from cells, rna was isolated using qiagen rneasy mini kit according to manufacturer's instruction. in brief, cells were lysed in the presence of b-mercaptoethanol and lysates were loaded to a column, washed and eluted in rnase-free water. for reverse transcription mg total rna, . mg oligo dt primer in a total volume of ml were heated for minutes at uc. enzyme mix was prepared ( enzyme buffer (fermentas), water and mm dntps) and pre-warmed at uc for minutes before adding u/ ml revertaid h m-mulv (fermentas). reverse transcription was performed at uc for hour. the enzyme was inactivated at uc for minutes. samples were stored at uc or directly used in quantitative real-time pcr. for analysis of gene expression relative quantification of the dna amount was applied. in order to do that gene expression of the housekeeping gene gapdh was determined. to ascertain changes in expression of the gene of interest the differences between expression of gapdh and the gene of interest was calculated using the ddct method [ ] . for quantitative real time brilliant qpcr sybr green mastermix (stratagene) was used according to manufacturer's instructions. the fragment of interest was amplified in cycles. the following primers were used (see table the pcfg -egz retroviral vector used for transfection [ ] as well as the constructs to express dominant negative mkk (mkk ala) or ikk (ikk kd) have been described earlier [ , ] . the phoenix amphotropic retroviral producer cells (a gift from g. nolan, stanford, ca) [ ] were cultured in dulbecco's modified eagle's medium containing % fetal bovine serum, units/ml penicillin and mg/ml streptomycin. generation of mkk ala or ikkkd expressing producer cells as well as transduction of a cells to stably express these transgenes was performed as previously described [ , ] . figure s infection of huvec results in inhibition of stat phosphorylation and ifnb independent socs- gene transcription. huvec were infected with pr (moi = ) (a, b, d) or stimulated with u/ml ifnb (e) for time points indicated. to assess the mrna levels of socs- (a, e), ifnb (b) and mxa (e) rna was reverse transcribed and cdna was subjected to quantitative real time pcr. equivalent mrna amounts were normalized to endogenous gapdh and calculated as n-fold of untreated cells that were arbitrarily set as . to assess the amount of phosphorylated stat (b) a cells were infected with pr (moi = ) for time points indicated. total cells lysate was subjected to western blot analysis using anti-phospho-stat , anti-stat antibodies. to assess effective viral replication viral ns was detected using an anti-ns antibody. (c) quantification of relative band intensities of (b) using aida software and d densitometry (fuji). figure s il b and tnfa do not affect induction of socs- gene transcription. a wt cells were stimulated with u/ml il b (a), ng/ml tnfa (b) or infected with pr (moi = ) (c) for time points indicated. cells were lysed, and rna was subjected to reverse transcription. cdna was analyzed in quantitative real time pcr to assess mrna amounts of socs- and il (a and b) or il b (c). equivalent mrna amounts were normalized to gapdh mrna levels and calculated as n-fold of the levels of untreated cells that were arbitrarily set as . found at: doi: . /journal.ppat. .s ( . mb tif) ifnbeta induction by influenza a virus is mediated by rig-i which is regulated by the viral ns protein inhibition of retinoic acid-inducible gene i-mediated induction of beta interferon by the ns protein of influenza a virus the -end-processing factor cpsf is required for the splicing of single-intron pre-mrnas in vivo influenza virus ns protein interacts with the cellular kda subunit of cpsf and inhibits end formation of cellular pre-mrnas binding of the influenza virus ns protein to double-stranded rna inhibits the activation of the protein kinase that phosphorylates the elf- translation initiation factor biochemical and genetic evidence for complex formation between the influenza a virus ns protein and the interferon-induced pkr protein kinase influenza a virus ns protein activates the pi k/akt pathway to mediate antiapoptotic signaling responses influenza a virus ns protein binds p beta and activates phosphatidylinositol- -kinase signaling interferons at age : past, current and future impact on biomedicine interferons, interferon-like cytokines, and their receptors ifn-lambdas mediate antiviral protection through a distinct class ii cytokine receptor complex activation of ifn-alpha, ifn-gamma, mxa, and ifn regulatory factor genes in influenza a virusinfected human peripheral blood mononuclear cells influenza a virusinduced ifn-alpha/beta and il- synergistically enhance ifn-gamma gene expression in human t cells the jak-stat signaling pathway: input and output integration stats: transcriptional control and biological impact the receptor of the type i interferon family acetylationdependent signal transduction for type i interferon receptor differential gene induction by type i and type ii interferons and their combination interferon signalling network in innate defence the polyoma virus t antigen interferes with interferon-inducible gene expression ebola virus vp binds karyopherin alpha and blocks stat nuclear accumulation suppressors of cytokine signaling and immunity the elongin bc complex interacts with the conserved socs-box motif present in members of the socs, ras, wd- repeat, and ankyrin repeat families vhl-box and socs-box domains determine binding specificity for cul -rbx and cul -rbx modules of ubiquitin ligases the conserved socs box motif in suppressors of cytokine signaling binds to elongins b and c and may couple bound proteins to proteasomal degradation the jak-binding protein jab inhibits janus tyrosine kinase activity through binding in the activation loop a three-dimensional model of suppressor of cytokine signalling (socs- ) three distinct domains of ssi- /socs- /jab protein are required for its suppression of interleukin signaling socs- inhibits il- -induced stat activation by binding through its sh domain to the stat docking site in the il- receptor beta subunit suppressors of cytokine signalling: socs induction of suppressor of cytokine signaling- by herpes simplex virus type confers efficient viral replication induction of suppressor of cytokine signaling- by herpes simplex virus type contributes to inhibition of the interferon signaling pathway ifn-alpha antagonistic activity of hcv core protein involves induction of suppressor of cytokine signaling- cytokine regulation in sars coronavirus infection compared to other respiratory virus infections respiratory syncytial virus inhibits interferon-alpha-inducible signaling in macrophage-like u cells non-response to antiviral therapy is associated with obesity and increased hepatic expression of suppressor of cytokine signalling (socs- ) in patients with chronic hepatitis c, viral genotype defective hepatic response to interferon and activation of suppressor of cytokine signaling in chronic hepatitis c suppressor of cytokine signaling (socs ) expression and hepatitis c virus-related chronic hepatitis: insulin resistance and response to antiviral therapy socs- regulates onset and maintenance of t(h) -mediated allergic responses role of endogenous inhibitors of cytokine signaling in allergic asthma regulation of jak-stat signalling in the immune system mechanism of inhibition of protein-tyrosine phosphatases by vanadate and pervanadate inhibition of membrane phosphotyrosyl-protein phosphatase activity by vanadate the suppressor of cytokine signaling (socs) and socs but not socs proteins inhibit interferon-mediated antiviral and antiproliferative activities socs- and socs- inhibit ifn-alpha-induced expression of the antiviral proteins , -oas and mxa -triphosphate rna is the ligand for rig-i the mitogen-activated protein (map) kinase p and its upstream activator map kinase kinase are involved in the activation of signal transducer and activator of transcription by hyperosmolarity the mkk / p mitogen-activated protein kinase pathway is capable of inducing socs gene expression and inhibits il- -induced transcription ringing the alarm bells: signalling and apoptosis in influenza virus infected cells dual function of interleukin- beta for the regulation of interleukin- -induced suppressor of cytokine signaling expression ikkalpha, ikkbeta, and nemo/ikkgamma are each required for the nf-kappab-mediated inflammatory response program nf-kappab-dependent induction of tumor necrosis factor-related apoptosisinducing ligand (trail) and fas/fasl is crucial for efficient influenza virus propagation activation of nf-kappa b via the ikappa b kinase complex is both essential and sufficient for proinflammatory gene expression in primary endothelial cells multiple signaling pathways regulate nf-kappab-dependent transcription of the monocyte chemoattractant protein- gene in primary endothelial cells type interferons and the virus-host relationship: a lesson in detente il- induces an anti-inflammatory response in the absence of socs in macrophages ns protein of influenza a virus inhibits the function of intracytoplasmic pathogen sensor, rig-i intracellular warfare between human influenza viruses and human cells: the roles of the viral ns protein regulation of a nuclear export signal by an adjacent inhibitory sequence: the effector domain of the influenza virus ns protein binding of the influenza a virus ns protein to pkr mediates the inhibition of its activation by either pact or double-stranded rna suppressors of cytokine signalling (socs) in the immune system influenza viruses and map kinase cascades -novel targets for an antiviral intervention exploited defense: how influenza viruses take advantage of antiviral signaling responses nfkappab negatively regulates interferon-induced gene expression and antiinfluenza activity cutting edge: innate immune response triggered by influenza a virus is negatively regulated by socs and socs through a rig-i/ifnar -dependent pathway interferon-gamma, but not interferon-alpha, induces socs expression in human melanoma cell lines rac and pak are upstream of ikk-epsilon and tbk- in the viral activation of interferon regulatory factor- influenza a virus lacking the ns gene replicates in interferon-deficient systems caspase activation is essential for efficient influenza virus propagation activation of phosphatidylinositol -kinase signaling by the nonstructural ns protein is not conserved among type a and b influenza viruses analysis of relative gene expression data using real-time quantitative pcr and the (-delta delta c(t)) method a expression is stimulated by cd in b cells and rescues wehi cells from anti-igm-induced cell death transcriptional profiling of ikk /nf-kappa b-and p map kinasedependent gene expression in tnf-alpha-stimulated primary human endothelial cells expression vectors and delivery systems we would like to thank akihiko yoshimura, kyushu university, fukuoka, japan, for providing socs / mef via fred schaper, aachen, germany. we also would like to thank viktor wixler, imv, muenster, germany, for providing anti-myc and anti-ns monoclonal antibodies. key: cord- -h upyzb authors: butler, noah s.; theodossis, alex; webb, andrew i.; nastovska, roza; ramarathinam, sri harsha; dunstone, michelle a.; rossjohn, jamie; purcell, anthony w.; perlman, stanley title: prevention of cytotoxic t cell escape using a heteroclitic subdominant viral t cell determinant date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: h upyzb high affinity antigen-specific t cells play a critical role during protective immune responses. epitope enhancement can elicit more potent t cell responses and can subsequently lead to a stronger memory pool; however, the molecular basis of such enhancement is unclear. we used the consensus peptide-binding motif for the major histocompatibility complex molecule h- k(b) to design a heteroclitic version of the mouse hepatitis virus-specific subdominant s determinant. we demonstrate that a single amino acid substitution at a secondary anchor residue (q to y at position ) increased the stability of the engineered determinant in complex with h- k(b). the structural basis for this enhanced stability was associated with local alterations in the pmhc conformation as a result of the q to y substitution. recombinant viruses encoding this engineered determinant primed ctl responses that also reacted to the wildtype epitope with significantly higher functional avidity, and protected against selection of virus mutated at a second ctl determinant and consequent disease progression in persistently infected mice. collectively, our findings provide a basis for the enhanced immunogenicity of an engineered determinant that will serve as a template for guiding the development of heteroclitic t cell determinants with applications in prevention of ctl escape in chronic viral infections as well as in tumor immunity. despite the antigenic complexity of microbes, primary pathogen-specific cytotoxic cd + t lymphocyte (ctl) responses are commonly directed to just one or a few determinants. furthermore, even when multiple epitopes are targeted, distinct patterns of epitope hierarchy often emerge. such immunodominant epitopes commonly elicit high-magnitude ctl responses characterized by potent cytolytic function, whereas subdominant determinants generate responses that are relatively lower in magnitude and often less efficacious. in general, potent anti-viral ctl strongly correlate with control of infection and less clinical disease. viral progeny selected on the basis of ctl surveillance can evolve to evade t cell responses. this selective pressure results in mutations in immunodominant ctl determinants that abrogate recognition. ctl escape virus is commonly observed in humans and nonhuman primates infected with hiv- , hepatitis c virus (hcv) or simian immunodeficiency virus (siv) and its selection often correlates with disease progression [ ] [ ] [ ] [ ] . escape mutations may diminish binding to the restricting mhc class i molecule, interfere with t cell receptor (tcr) recognition or interfere with antigen processing [ ] [ ] [ ] . escape mutations are usually detected in epitopes targeted by ctl that exhibit high functional avidity because the corresponding potent ctl response exerts high selective pressure on the virus [ ] . in some hivinfected patients, ctl escape occurs without an associated enhancement of virus replication, suggesting that the mutations compromised virus fitness or, alternatively, the variant determinant elicited a de novo ctl response [ , ] . virus fitness is sometimes restored, with concomitant increased virus replication, when a second, compensatory mutation is selected [ , ] . collectively these results suggest that given the importance of virus diversification (ctl escape) in disease progression, suppression of selection or outgrowth of ctl escape variants should improve outcomes in persistently infected animals and humans. modulating the immunogenicity of subdominant ctl determinants could potentially lead to the development of more efficacious vaccines that are more broadly protective and prevent or minimize the appearance of variant viruses that have mutated in dominant epitopes targeted by high-avidity ctl responses. enhancement strategies, which result in augmented responses to the native, subdominant epitope, have been described for both mhc class i and class ii-restricted determinants, whereby the most common approaches involve generating a series of conserved and non-conserved mutations at mhc anchor residues, followed by an empiric determination of whether each individual substitution augments t cell effector function [ ] . furthermore, evaluating the effect of epitope enhancement in vivo has generally been achieved via heterologous infection systems [ ] [ ] [ ] . thus, while these results demonstrate proof of principle, direct evidence for enhanced protection against autologous microbial infection in vivo is lacking. the design of heteroclitic determinants in which non-mhc anchor residues are targeted for substitution are also usually determined empirically. studies of heteroclitic tumor epitopes have demonstrated the clinical utility of such determinants [ , ] . notably, however, there are no well defined examples of viral epitopes which demonstrate enhanced immunity and the molecular basis for the enhanced immunogenicity is not well understood. potential interventions to directly manipulate host-pathogen interactions and thereby diminish ctl escape variant selection are often difficult to evaluate since most examples of ctl escape occur in infected humans or non-human primates. by contrast, mice persistently infected with mouse hepatitis virus (mhv) strain jhm (jhmv) serve as a useful system for investigating anti-viral ctl responses and ctl escape [ ] [ ] [ ] . two jhmv-derived ctl epitopes are recognized in c bl/ (b ) mice. the immunodominant h- d b -restricted ctl epitope (s , cslwngphl, spanning residues - of the spike (s) glycoprotein) elicits a highmagnitude, high-avidity ctl response that drives virus diversification during persistent infection [ ] [ ] [ ] [ ] [ ] . a second subdominant ctl epitope, s (h- k b -restricted, rcqifani, spanning residues - of the s glycoprotein) also elicits an appreciable ctl response [ ] ; however, s -specific ctl exhibit , -fold lower functional avidity and do not protect from ctl escape in s . the presence of a readily mutable dominant and a subdominant epitope with high and low functional avidity, respectively, in jhmv-infected mice is useful for investigating both epitope enhancement and approaches to diminishing ctl escape. consistent with this notion, we have previously shown that the introduction of a second dominant cd t cell epitope into the jhmv genome (gp from lymphocytic choriomeningitis virus (lcmv)) protected mice from the development of ctl escape in s and enhanced virus clearance [ ] . here we determined whether the cd t cell response to s could be enhanced so that it now elicited a more potent t cell response that protected mice against the development of ctl escape at s and subsequent clinical disease. we modified epitope s (s q y ) such that it elicited a high-avidity ctl response and using the crystal structures of the h- k b /s and h- k b /s q y complexes, determined the basis of this enhanced immune response. we then introduced this more immunogenic s epitope into a recombinant version of jhmv and showed that these high-avidity s -specific ctls protected against escape variants in the immunodominant s epitope. immunization with the modified peptide resulted in an improved response to the native s epitope, demonstrating a true heteroclitic effect and suggesting that this strategy may have clinical applications for reducing viral titer and preventing ctl escape during chronic infections. as we have previously observed with other mhc/cyscontaining peptide complexes [ , ] , complexes did not readily form unless the cysteine of the s peptide (rcqifani) was modified with l-a-aminobutyric acid (aba, an isostereomer of cysteine). the aba-modified peptides maintained immunogenicity since a higher frequency of splenic cd + t cells from jhmvimmune mice reacted to aba-modified s peptide, relative to unmodified s peptide ( figure s a ). next, we used the consensus h- k b binding motif [ ] to engineer a novel, high-avidity s ctl epitope. importantly, gln- diverges from the consensus h- k b -restricted ligand binding motif, in which a tyrosine is often present at position [ , ] . therefore, we substituted a glutamine residue for tyrosine (q y, caa to tat, rcyifani) at position of the determinant with the aim of creating a peptide that bound more tightly to h- k b . stability of the h- k b /s and h- k b /s q y complexes was assessed by circular dichroism (cd). as shown in figure , h- k b /s q y was considerably more thermostable than the native complex (tm uc vs uc). to probe the biological properties of the q y substitution, we used reverse genetics to engineer a recombinant version of jhmv expressing the s q y epitope (figure a) . recombinant viruses encoding this substitution replicated as efficiently as wild type jhmv (rj) in vitro during one-step growth kinetics analyses and in vivo virus competition assays ( figure b , c). the immunogenicity of s q y was assessed by intracellular expression of ifn-c by central nervous system (cns)-derived lymphocytes. since cys-containing peptides are often diminished in enhancing the immune responses to pathogens is a chief goal of vaccine development. here, we describe the development of an engineered cd + t cell epitope that elicits an immune response to the native epitope that is more potent than the one that occurs during the natural infection. we showed that this ''improved'' (heteroclitic) epitope protects against clinical disease and against cytotoxic t cell escape that frequently occurs in the immunodominant epitope expressed by the virus. we also performed structural analyses and showed that enhanced immunogenicity was associated with changes in the conformations of both the peptide and the region of the mhc class i molecule that is in close association with the peptide. these studies provide a model for designing t cell epitopes with enhanced immunogenicity that will be useful in vaccine development, with particular emphasis on diseases, such as hiv and hepatitis c, in which epitope mutation and escape is common. seven days p.i., total rna was harvested from the brains of mice and relative representation of virus template was determined via rt-pcr and direct sequencing of pcr products. the relative proportion of animals in which only rj, only rj.s q y , or a mixture of the two viruses is shown. (d) high-ability to elicit a ctl response, we included a reducing agent (tcep, tris [ -carboxyethyl] phosphine) in the cultures. tcep enhanced the stimulatory capacity of the s peptides ( figure s b ), indicating that a proportion of the unmodified s peptide stock had undergone oxidation. thus, we stimulated cns-and spleen-derived ctl ex vivo in the presence of mm tcep, a concentration consistent with other work with cys containing peptides [ , ] . the s q y epitope elicited a ctl response in the cns of rj.s q y infected mice, with nearly % of all cd t cells recognizing the determinant ( figure d ). in addition, ctl primed by s q y cross-reacted with the native s determinant. the converse was not true, however, as cells primed by the native epitope failed to produce ifn-c when stimulated with s q y peptide ( figure d ). of note, the ctl response to the dominant d b -restricted s epitope in rj.s q y -infected mice was diminished relative to responses in mice infected with wildtype rj virus ( figure d , e). next, we assessed the relative functional avidity of ctl populations primed by native s and s q y determinants, as a surrogate measure of the potency of the anti-virus ctl response in vivo. cns-derived mononuclear cells were harvested from mice infected with rj or rj.s q y and examined for ifn-c expression after stimulation ex vivo in the presence of -fold dilutions of the appropriate peptide. cells primed to the native s epitope (cells harvested from rj-infected mice) required approximately -fold more peptide than did s q y -primed cells to elicit a half maximal response ( nm vs. nm, figure a ,b). because we observed that a subpopulation of s q y -primed cells cross-reacted with the native determinant, we next determined whether this subpopulation also exhibited high functional avidity. for this purpose, we isolated cells from the cns of rj-and rj.s q y -infected adult b mice and stimulated them with -fold dilutions of native s peptide. s q y -primed cross-reacting cells exhibited -fold higher functional avidity, relative to those primed by the native determinant ( figure c , d) and are therefore distinct from ctl primed by the native s determinant. consistent with the presence of a distinct population of cells, there were modest differences in vb chain utilization when total populations from rj and rj.s q y -infected mice and when total and cross-reacting cell populations from rj.s q y -infected mice ( figure s ) were compared. in all instances, vb . / . expression was relatively over-represented, but some vb elements were preferentially utilized by specific responding populations (vb , vb and vb ). also consistent with this observation, alanine scanning mutagenesis of the two determinants revealed that the ctl response to each cognate peptide following infection was also subtly different reflecting the altered repertoire. the ctl response to each cognate peptide was very sensitive to mutation at every position except and although mutations in the s q y determinant were tolerated slightly better than changes in the s epitope ( figure s c -e). if ctl recognizing s q y exhibit high functional avidity in vivo, they should protect from ctl escape in s and might select for s q y ctl escape variants. diversification at s is observed in pups infected with neurovirulent jhmv at days of age and nursed by jhmv-immune dams [ ] . these mice are largely protected from developing acute lethal encephalitis, but a variable percentage ( - %) later develop a demyelinating encephalomyelitis. infectious virus isolated from these mice with late onset clinical disease is mutated in s , resulting in enhanced virus replication [ ] , with demyelination occurring during the process of virus clearance [ ] . thus, we next infected maternal antibody-protected suckling mice with rj or rj.s q y viruses and monitored persistently infected mice for the development of clinical signs for days post infection (p.i.). the presence of the highly immunogenic s q y epitope did not protect mice from acute encephalitis ( figure e ), perhaps because the presence of the improved s epitope was accompanied by a diminished response to s ( figure d ). however, we found that among survivors (defined as survival past day p.i.) there was a significant reduction in the incidence of clinical disease as well as in the development of ctl escape in s (table ) . additionally, s q y did not undergo ctl escape in mice persistently infected with rj.s q y , even though single nucleotide changes in the region of the s glycoprotein gene encoding the s determinant could potentially result in fifty-one ctl escape mutations. thus, as expected, the ''improved'' s q y epitope was protective in vivo in infected mice, likely because s q yspecific ctl are present in higher numbers and exhibit higher functional avidity than the native s -specific response. since this lack of mutation at s q y might reflect enhanced suppression of virus replication mediated by co-dominant ctl responses directed against s and s q y , we developed a recombinant virus encoding s q y in the context of a common s ctl escape mutation, s w r (rj.s w r+q y , figure a ). the ctl response is predicted to be largely directed at s q y in mice infected with this virus. the w r mutation (position substitution in s epitope, cslrngphl) occurs in % of all ctl escape variants [ , , , , ] , and completely abrogates native s ctl recognition [ ] . we verified that virusspecific ctl responses were focused on s q y in adult b mice infected with rj.s w r+q y ( figure f ). to examine the phenotype of the s q y /s w r double mutant, we infected antibody-protected suckling b mice with this virus and appropriate controls and monitored mice for survival ( figure g ). as expected, . % of mice infected with rj survived the acute infection (day - p.i.). all mice infected with rj.s w r developed fatal encephalitis but, in marked contrast, . % of mice infected with rj.s w r+q y survived. we also found that survival correlated with virus clearance ( figure h ). relative to rj-infected mice, replication was suppressed in mice infected with virus encoding the s q y epitope and greatly elevated in mice infected with rj.s w r . in mice infected with rj.s w r+q y , virus titers were intermediate between rj and rj.s w r . thus, the presence of the heteroclitic s q y determinant contributed to suppression of virus replication and to increased survival, even when the highmagnitude, high-avidity ctl response to s was largely abrogated. surprisingly, s q y still did not undergo sequence diversification in mice that survived the rj.s w r+q y infection ( table ) . this result was unexpected, as the majority of ctl in the rj.s w r+q y -infected cns specifically target the magnitude, unidirectional cross-reactivity. representative dot plots demonstrating the frequency of epitope-specific cd t cells in a mouse infected with rj (top panels) or rj.s q y (bottom panels). numbers represent the frequency of epitope-specific cd t cells among total cd t cells recovered from the brains of mice days p.i. (e) summaries of the frequency (left panel) and absolute number (right panel) of epitope-specific cd t cells recovered from the brains of rj and rj.s q y -infected mice days p.i. data shown in d represent mean sem for independent experiments. doi: . /journal.ppat. .g s q y determinant and exhibit high functional avidity ( figure a-c) . one possible explanation for this result is that s is not as plastic as s , even though both determinants are derived from a region of the spike gene that is hypervariable and even deleted in some strains of mhv [ ] . to determine whether cells primed to s q y that crossreact with the native s determinant were more protective in vivo than s -primed cells, we vaccinated mice with bone marrow-derived dendritic cells (bmdc) alone, or bmdc pulsed with peptides corresponding to s or s q y (figure ) . seven days later, mice were challenged via intranasal inoculation of pfu of wild type, non-recombinant jhmv. similar to results observed following rj.s q y infection (figure ) , ctl primed via dc-s q y vaccination exhibited higher functional avidity when reacted against the native s determinant when compared to those arising after dc-s vaccination ( figure a ). in other experiments, we examined the survival of mice vaccinated with each determinant, but we observed no significant differences between groups (data not shown), probably because mortality is largely cd t cell-mediated in adult mice with acute encephalitis [ , ] . in terms of virus titers, vaccination with either the native or enhanced s determinants resulted in , - % reduction in virus burden compared to mice that received un-pulsed bmdc ( figure b ). when we examined the one-fourth of the pups in an individual litter were infected with each virus. at the indicated day p.i., brains were aseptically harvested, homogenized in sterile pbs and clarified by centrifugation. supernatants were collected and infectious virus was titered on hela-mhvr (hela cells transfected with ceacam , the jhmv receptor [ ] ) as previously described [ ] . symbols on graph represent individual mice assayed from multiple independent litters. the limit of detection (lod) for the assay is pfu/brain. frequency and numbers of virus-specific ctl in the cns of these same mice, we detected markedly fewer s -specific ctl in the cns of mice that received bmdc pulsed with s q y peptides ( figure c) . calculation of the product of virus titers and ctl numbers within individual mice, as an approximate measure of ctl potency, indicated that the s -specific ctl in s q ycoated bmdc vaccinees were , - -fold more efficacious on a per cell basis ( figure d ). thus, s -specific ctl induced by the s q y determinant show similar enhancement in function compared to s -primed cells, whether measured in vitro ( figure c ,d) or in vivo ( figure d ). while these studies clearly demonstrated that s q y is heteroclitic, they did not provide a mechanism for the immune enhancement. to address this, we determined the crystal structures of the h- k b /s (pdbid zsv, protein data bank japan (http://www.pdbj.org/)) and h- k b /s q y (pdbid zsw) complexes to . Å and . Å resolution respectively. the structure of h- k b /s consists of two heterodimers in the asymmetric unit (r.m.s.d. of . Å for ca atoms), with the s peptide clearly bound in the antigen binding cleft of the heavy chains (hc, figure s a ). the two peptide copies display a virtually identical configuration with root mean square deviation (rmsd) values of only . Å for all peptide atoms ( . Å for ca atoms). the mode of s and s q y binding within the agbinding cleft is unambiguous, with the exception of arg- , whose side chain is partially disordered (figure a,d; figure s c ). the s peptide adopts an extended conformation, with the side chains of arg- , ile- and asn- extending prominently out of the cleft ( figure c ). ala- is also largely solvent exposed with its side chain pointing towards the a helix, while the side chains of cys (aba)- , gln- , phe- and ile- are buried within the cleft. while the cysteine analogue's side chain is not involved in any hydrophilic interactions, there are a number of suitably positioned hydrogen bonding partners (glu- , tyr- and asn- ), with which the original thiol side chain could potentially interact ( figure s b ; table s ). in addition to main chain interactions across the length of the peptide, s is anchored to the mhc via the side chains of gln- , phe- and ile- . gln- forms hydrogen bonds with the ser- and gln- of the hc (figure b ), while phe- and ile- are buried within hydrophobic pockets. in addition, the side chains of phe- and gln- pack against each other and between the aromatic rings of hc residues tyr- and phe- , constraining the peptide's backbone conformation at those positions. the structure of h- k b /s q y consists of four heterodimers in the asymmetric unit (rmsd of . Å for heavy chain (hc) ca's), the four copies of the peptide adopting virtually identical conformations (rmsd of . Å for all peptide atoms and . Å for ca atoms). s q y displays the same conformation as the s determinant (the average rmsd for peptide ca atoms between the two structures is . Å ) and forms equivalent interactions with the mhc. the only prominent structural difference is observed at the mutated position (q ry ) ( figure b,e, a,b) . in contrast to gln- , the tyr- side chain is oriented towards the a- helix rather figure e ). the side chain of tyr- also forms a number of close contacts with the residues in its immediate environment, specifically hc residues gln- , arg- , leu- and tyr- , as well as intramolecular interactions with phe- (table s ). in addition to tyr- , deviations of potential significance (. . Å ) in the peptide structures that are attributable to the q y mutation are observed at ile- and phe- . overall the s determinant displays greater complementarity for the antigen binding cleft of h- k b in its n-terminal region, with few stabilizing interactions observed between the hc and positions and . (figure s b, d; table s ). nevertheless, a pocket is observed between the a- helix and the peptide near position in the index structure that is filled by the steric bulk of tyr- in the q y structure ( figure c,f) . this increase in surface complementarity and the greater number of observed interactions resulting from the q ry mutation would account for the enhanced thermostability (, uc) measured for the h- k b /s q y -aba complex by circular dichroism (figure ). this increase in complementarity of the mhc for s q y and the greater stability of the resulting complex were predicted from comparisons of the wt determinant complex with existing structures of h- k b bound with peptides similar to s and possessing the consensus tyrosine anchor residue at position , as well as another aromatic residue at position . with respect to the hc, the a- and a- domains of the two structures superimpose well with an rmsd of . Å for ca atoms (residues - ). nevertheless, significant deviations (. . Å ) are observed between the two structures at a number of positions in the region of the antigen binding cleft. in particular, changes in the conformation of ser- , gln- , and gly- -glu- are associated with the bound peptides. changes in the side chain conformations of ser- and gln- in the q y complex structure are consistent with the loss of hydrogen bonding interactions with position . in the wild type peptide structure, glu- forms a salt bridge with arg- , the guanadinium group of which also forms a hydrogen bond with the main chain carbonyl group of the peptide's ile- . while these interactions are maintained in the q y complex, the side chain of glu- displays a conformational shift consistent with the formation of a hydrogen bond with the peptide's tyr- ( figure ). interestingly the region of the a- helix around glu- (gly- -glu- ) also displays significant main chain and side chain deviations between the two structures (rmsd of . Å for ca atoms; figure ). thus, consistent with the functional analyses, the structure of the heteroclitic variant of the s epitope displays relatively small changes in conformation yet the combination of these subtle changes in the tcr accessible residues and the structural landscape of the mhcp in addition to the enhanced stability of the complex lead to more efficacious ctl responses. we describe the identification of a heteroclitic determinant that enhances recognition by virus-specific cd t cells, and use the crystal structure of the new determinant (s q y ) to provide a basis why it elicits an enhanced ctl response. comparison of s to the consensus binding motif suggested a suboptimal interaction with the h- k b molecule at the secondary anchor position (gln- ) and, consequently, an approach to enhance the immunogenicity of the determinant. replacement of the gln- with tyr- (q y) did, indeed, result in an determinant with increased thermostability without diminishing the cd t cell response. most strikingly, the q y change resulted in subtle changes in the conformation of the a- helix locally in the vicinity of glu- . these subtle changes are likely critical for the enhanced tcr recognition that we detected. s q y elicited a response with higher functional avidity to both the cognate and native determinants than s , and this was not reflected in differential vb usage. the t cell response to s in rj-infected mice is very diverse [ ] . as assessed by vb usage, the response to s and s q y in rj.s q y was similarly diverse with only modest differences noted when cells from mice primed by s and s q y were compared. while we cannot exclude the possibility that cross-reacting s -specific cells primed by s q y are biased for vb chains not analyzed in this study, it is more likely that the fine specificity of the complementarity-determining region (cdr ) determines their greater affinity for h- k b /s . although an increase in stability of the mhc class i/peptide complex is not generally expected to enhance tcr affinity for the complex, similar results have been observed in mice immunized with analogues to a common tumor antigen [ ] . one unexpected result was that s exhibited both low mhc class i and low tcr avidity. previous studies showed that this determinant exhibited low functional avidity [ ] , but it was not known whether this reflected low binding to the mhc class i or to the tcr. assuming that low affinity for mhc class i results in a low effective concentration of h- k b /s complex on the cell surface, the responding t cells should be high avidity, based, primarily, on in vitro studies [ ] . while the relationship between level of surface peptide and avidity of the responding t cells generated in vivo is not as clearcut [ ] , there is no obvious explanation for how a peptide with low mhc class i binding also elicits a low avidity t cell response. this selection of only a subset of cd t cells capable of responding to s may partly explain why s -primed cells do not recognize the q y determinant. the biological significance of the heteroclitic q y determinant was shown by its ability to protect jhmv-infected mice from ctl escape at s and to diminish clinical disease. this was important to demonstrate because other studies, using tumor models, have shown that immunogenicity and tumor recognition are not necessarily concordant [ ] . mutations resulting in ctl escape occur most commonly in determinants that are exposed to high selective pressure [ ] [ ] [ ] and outgrowth of ctl escape variants is efficiently suppressed by effective and rapid virus clearance [ ] [ ] [ ] [ ] , as occurs in mice infected with rj.s q y . thus, even though ctl escape is not detected in normal mice infected with lcmv or influenza, escape does occur when mice transgenic for a single lcmv-specific tcr are infected with high amounts of virus [ , ] . under these conditions, the immune response is highly focused on a single cd t cell determinant and virus replication continues for extended periods of time, facilitating mutation at the targeted determinant. in mice infected with wild type jhmv, the ctl response is functionally focused on s [ ] ; the q y substitution effectively prevents ctl escape at either s or s q y by the induction of a second high avidity ctl response. mutations in s q y were not detected even when the ctl response was directed primarily at this determinant (e.g. mice infected with rj.s w r + q y ). consistent with this inability to readily tolerate mutations, we were unable to generate recombinant virus mutated in position ile- (i d,e,k,r,t) and recombinant virus mutated at phe- (f a) was highly attenuated (data not shown). the combination of induction of high avidity ctl and inability to tolerate mutation without adversely affecting virus fitness make s q y an ideal target for the anti-jhmv ctl response. further, the ala scanning results suggest that s q y -specific ctls may more readily tolerate changes in the h- k b /peptide complex, and this plasticity would also minimize the likelihood of ctl escape. in contrast, we have previously shown that introduction of the lcmv-specific gp determinant, which also elicits ctl with high functional avidity, into jhmv greatly diminished clinical disease but did not prevent ctl escape [ ] . the gp determinant was introduced at a site in the genome that tolerated mutation and deletion and intact determinant was no longer present in virus by day p.i. collectively these results suggest that a response to a second determinant that elicits ctl exhibiting high functional avidity at early times p.i. results in enhanced suppression of virus replication, but its presence throughout the infection is required to protect against ctl escape. in conclusion, we have demonstrated that crystal structures are useful in gaining an understanding of the basis of heteroclitic epitopes and can also prove valuable in guiding the rational design of ''better'' ctl epitopes. in our mouse system, immunization with the heteroclitic determinant resulted in the generation of unique populations of ctl that respond with high functional avidity to an otherwise modestly immunogenic viral epitope. the generation of unique populations of ctl that respond with high functional avidity to weakly immunogenic epitopes will be useful for the treatment and prevention of human infectious diseases. our proposed structure-guided approach has direct application to hiv, hcv and other chronic infections in which virus persistence and ctl escape occurs. by modulating t cell immunity through prophylactic or therapeutic peptide-based vaccination, virus titers may be reduced and ctl escape and other consequences of viral persistence circumvented. specific pathogen-free b and balb/c mice were obtained from national cancer institute (bethesda, md). to obtain infected mice in which ctl escape at s was detected, suckling b mice were infected intranasally with - pfu of recombinant jhmv at days of age and nursed by dams that were immunized with jhmv, as described previously [ ] . for experiments comparing multiple jhmv variants, each litter served as an internal control: equal numbers of pups were infected with rj and one to three recombinant variant viruses, depending on litter size. all animal studies were approved by the university of iowa animal care and use committee. mononuclear cells were harvested from the brains of acutely ill mice days p.i. and analyzed for expression of ifn-c by an intracellular cytokine staining protocol as previously described [ ] . unless otherwise noted, peptides corresponding to epitopes were used at a final concentration of mm and cells were stimulated in the presence of mm tcep (sigma, st. louis, mo). cells were analyzed using a facscan flow cytometer (bd biosciences, mountain view, ca). data sets were analyzed using flowjo software (tree star, inc, ashland, or). all antibodies and reagents were purchased from bd pharmingen (san diego, ca). recombinant wild-type and s and s variants of jhmv were generated as previously described [ , ] . briefly, overlapping extension polymerase chain reaction (pcr) was used to generate the s q y and s w r variants. primers that overlapped the glutamine at residue of the spike glycoprotein were ( to ) q y fwd, atgatcgctgctatatttttgctaacatat-tg; q y rev, aatatgttagcaaaaatatagcagcgat-cat. primers that overlapped the tryptophan at residue were ( to ) w r fwd, gtgagtgttctcttcggaatgggc-cccatttgcgctcggc; w r rev, agcgcaaatgg-ggcccattccgaagagaacactcac. the outer primers for each targeted change were fwd, tgttgattgcgccag-cagctacattag; and rev, acctacggattgaacgctat-cattgac. underlined nucleotides correspond to the nucleotides encoding the gln to tyr and trp to arg substitutions within s and s , respectively. recombinant viruses encoding the variant epitope(s) were selected, propagated and titered as previously described [ ] . at least two independent isolates of each recombinant virus were analyzed. virus was inoculated onto confluent cl- monolayers in a well plate at a multiplicity of infection (moi) of . . groups of cells were harvested at the indicated time points and total virus (cellassociated and cell-free) was titered as previously described [ ] . equal pfu ( - ) of rj and rj.s q y were combined and inoculated intranasally into -week-old b and balb/c mice. total rna was harvested from the brains of mice days p.i. and the relative representation of wt (wild type) versus variant template was determined by rt-pcr and sequencing. this assay can specifically detect a given species of template when that species comprises at least % of a heterogeneous pool [ ] . primers used were : forward, aacccctcgtcttggaataggagg-tatgg; and reverse, cctacggattgaacgctatcatt-gactaac. pcr products were sequenced directly by the university of iowa dna core. for alanine scanning, cells were stimulated ex vivo with the indicated concentration of native or variant peptide and stained for cd and ifn-c as described above. data were normalized to the frequency of cells that reacted to the unmodified s or s q y peptides. for functional avidity determination, mononuclear cells were harvested from the brains of rj or rj.s q y -infected mice days p.i. and stimulated ex vivo in the presence of el- cells pulsed with tenfold dilutions of peptide corresponding to native s or s q y epitopes. after . hours, cells were stained for intracellular ifn-c as described above. for each epitope-specific population, data were normalized to the frequency of antigenspecific ctl detected using the highest titration of peptide ( mm). cd spectra were measured on a jasco spectropolarimeter using a thermostatically controlled cuvette at temperatures between and uc. far-uv spectra were collected and analyzed as described [ ] . cells were harvested from the cns of mice days p.i. and stimulated ex vivo with s or s q y peptides. cell aliquots were subsequently stained for cd (pe-cy -anti-cd a) and vb (fitc-anti-vb , , , . / . , , , , , b , , , or , bd-pharmingen) followed by intracellular staining for ifn-c (peanti-ifn-c). data were collected using a becton dickinson lsr ii instrument at the university of iowa flow cytometry facility. data are expressed as the proportion of antigen-specific cd t cells that express each vb chain. total rna was purified with trizol (invitrogen, carlsbad, ca) from the cns of mice. the base pair region of the spike glycoprotein encompassing both s and s was amplified by rt-pcr and sequenced directly as previously described [ ] . bone marrow-derived dc were prepared, pulsed with peptides and injected into mice as previously described [ ] . briefly, lps-matured dc were left uncoated, coated with s or s q y peptides and injected via tail vein into groups of week-old mice. seven days following dc-vaccination, mice were infected intranasally with pfu of jhmv. seven days following virus infection, brains were harvested from mice and the frequencies of epitope-specific cd t cells were determined by ex vivo stimulation and intracellular cytokine staining as described above. in independent studies, dc-s and dc-s q y priming was verified by harvesting spleens from several mice seven days following dc-vaccination. crystal structure of h- k b /s and h- k b /s q y complexes crystals of h- k b s -aba were grown at uc in . m cacodylate ph . , % peg (polyethylene glycol) , , . m ca(oac) , using a protein concentration of mg/ml. crystals were cryoprotected by stepwise equilibration against mother liquor containing , and % glycerol and flash frozen by placing in a nitrogen stream. a . Å resolution dataset was collected on an inhouse x-ray source. crystals of h- k b s q y -aba were grown at uc in . m cacodylate ph . , % peg , , . m ca(oac) , using a protein concentration of mg/ml. crystals were cryoprotected by gradual equilibration against mother liquor containing % peg , and % glycerol before flash freezing. a . Å resolution dataset was collected on an in-house source. the wt data were integrated in mosflm [ ] and scaled/merged using scala [ ] . the q y variant data were processed using hkl . both structures were solved by molecular replacement in phaser [ ] , against previously solved h- k b complexes (pdbid's: g q and rjy, respectively). the resultant models underwent iterative cycles of refinement in phenix [ ] and, refmac [ ] (restrained and tls refinement) followed by model building/ correction in coot [ ] . the solvent structures were built using arp/warp [ ] and coot. a summary of the processing and refinement statistics is presented in table s . statistical significance was determined by nonpaired, two-sided student's t test or chi-squared test, where indicated. (a) total mononuclear cells were harvested from mice infected with rj or rj.s q y and stimulated ex vivo with s and s q y peptides, respectively. following stimulation, aliquots of cells were surface stained for cd and the indicated vb chain followed by intracellular cytokine staining for ifn-c. (b) cnsderived cells from rj.s q y -infected mice were stimulated ex vivo with peptides corresponding to native s or s q y epitopes. following stimulation, cells were analyzed as described for (a). data represent the fraction of ifn-c+cd + t cells expressing each vb chain and are derived from cells pooled from - individual mice. (c) alanine scanning of the native s determinant. cns-derived mononuclear cells were recovered from rj-infected mice days p.i. and stimulated ex vivo in the presence of mm tcep and mm of the indicated peptide then stained for cd and intracellular ifn-c. data are normalized to the frequency of epitope-specific cells detected when stimulated with the native s determinant. (d) alanine scanning of the s q y determinant. cells were harvested and tested as described for b except in this case the cells originated from the rj.s q y -infected cns and were stimulated with nm s q y peptide. (e) alanine scanning of the s determinant recognized by s q y -primed, cross-reactive ctl. as in c, but cells were stimulated with nm s peptide. for b-d, concentrations of peptide equivalent to that required for half maximal stimulation were used; data are mean sem from four independent experiments. note that the differential responses to rcaifani in panels c and d reflect the differing amounts of peptide used in the two assays. found at: doi: . /journal.ppat. .s ( . mb tif) figure s refined structures of wt and q y s -aba bound to h- k b . (a) view of the h- k b antigen binding cleft from above. the hc is shown as a cartoon representation and coloured slate. the peptide is in stick format with carbon atoms coloured yellow. the unbiased f o -f c map density for the peptide contoured at . s is shown as a magenta mesh. (b) the same view as in a displaying key interactions (dashed lines) between h- k b and s -aba. selected residues of the hc are drawn in stick format (slate carbon atoms) and ordered water molecules are shown as red spheres. peptide residues are labelled in italics. c and d, equivalencies to a and b, respectively, for the h- k b / s q y -aba structure. in these panels the hc is drawn in green and the peptide in cyan. mutational escape from cd + t cell immunity: hcv evolution, from chimpanzees to man hiv and siv ctl escape: implications for vaccine design escape of human immunodeficiency virus from immune control loss of viral control in early hiv- infection is temporally associated with sequential escape from cd + t cell responses and decrease in hiv- -specific cd + and cd + t cell frequencies immune selection for altered antigen processing leads to cytotoxic t lymphocyte escape in chronic hiv- infection hepatitis c virus mutation affects proteasomal epitope processing cd epitope escape and reversion in acute hcv infection de novo generation of escape variant-specific cd + t-cell responses following cytotoxic t-lymphocyte escape in chronic human immunodeficiency virus type infection hiv- epitope-specific cd + t cell responses strongly associated with delayed disease progression cross-recognize epitope variants efficiently extraepitopic compensatory substitutions partially restore fitness to simian immunodeficiency virus variants that escape from an immunodominant cytotoxic-t-lymphocyte response clustered mutations in hiv- gag are consistently required for escape from hla-b restricted ctl responses more than one reason to rethink the use of peptides in vaccine design epitope-enhanced conserved hiv- peptide protects hla-a -transgenic mice against virus expressing hiv- antigen epitope enhancement of a cd hiv epitope toward the development of the next generation hiv vaccine structural features of peptide analogs of human histocompatibility leukocyte antigen class i epitopes that are more potent and immunogenic than wild-type peptide heteroclitic immunization induces tumor immunity design of multi-epitope, analogue-based cancer vaccines antiviral antibodies are necessary to prevent cytotoxic t-lymphocyte escape in mice infected with a coronavirus structural and biological basis of ctl escape in coronavirus-infected mice cytotoxic t cellresistant variants are selected in a virus-induced demyelinating disease cd + t-cell epitopes within the surface glycoprotein of a neurotropic coronavirus and correlation with pathogenicity protection against ctl escape and clinical disease in a murine model of virus persistence functional and structural characteristics of ny-eso- -related hla a -restricted epitopes and the design of a novel immunogenic analogue allelespecific motifs revealed by sequencing of self-peptides eluted from mhc molecules the structure of the antigen-binding groove of major histocompatibility complex class i molecules determines specific selection of self-peptides the insulin a-chain epitope recognized by human t cells is posttranslationally modified late onset, symptomatic, demyelinating encephalomyelitis in mice infected with mhv-jhm in the presence of maternal antibody cd and cd t cells have redundant but not identical roles in virus-induced demyelination antibodymediated protection against cytotoxic t-cell escape in coronavirus-induced demyelination cytotoxic t-cell-resistant variants arise at early times after infection in c bl/ but not in scid mice infected with a neurotropic coronavirus immune response to the immunodominant epitope of mouse hepatitis virus is polyclonal, but functionally monospecific in c bl/ mice coronavirus pathogenesis and the emerging pathogen severe acute respiratory syndrome coronavirus pathogenic role for virus-specific cd t cells in mice with coronavirus-induced acute encephalitis cd t cells contribute to virus control and pathology following cns infection by neurotropic mouse hepatitis virus very diverse cd t cell clonotypic responses after virus infections structural and kinetic basis for heightened immunogenicity of t cell vaccines selective expansion of high-or low-avidity cytotoxic t lymphocytes and efficacy for adoptive immunotherapy antigen density presented by dendritic cells in vivo differentially affects the number and avidity of primary, memory, and recall cd (+) t cells ex vivo identification, isolation and analysis of tumor-cytolytic t cells the hypervariable immunodominant np - epitope from the influenza a virus nucleoprotein is recognized by cytotoxic t lymphocytes with high functional avidity comprehensive analysis of cd (+)-t-cell responses against hepatitis c virus reveals multiple unpredicted specificities differential selection pressure exerted on hiv by ctl targeting identical epitopes but restricted by distinct hla alleles from the same hla supertype the outcome of hepatitis c virus infection is predicted by escape mutations in epitopes targeted by cytotoxic t lymphocytes immunobiology of cytotoxic t-cell escape mutants of lymphocytic choriomeningitis virus viral escape by selection of cytotoxic t cell-resistant variants in influenza a virus pneumonia immune evasion versus recovery after acute hepatitis c virus infection from a shared source viral escape by selection of cytotoxic t cell-resistant virus variants in vivo retargeting of coronavirus by substitution of the spike glycoprotein ectodomain: crossing the host cell species barrier inactivation of expression of gene of mouse hepatitis virus strain jhm does not affect virulence in the murine cns sensitive sequencing method for kras mutation detection by pyrosequencing quantitation of cd + t cell expansion, memory, and protective immunity after immunization with peptide-coated dendritic cells recent changes to the mosflm package for processing film and image plate data data reduction likelihood-enhanced fast rotation functions a robust bulk-solvent correction and anisotropic scaling procedure refinement of macromolecular structures by the maximum-likelihood method coot: model-building tools for molecular graphics automated refinement of protein models receptor for mouse hepatitis virus is a member of the carcinoembryonic antigen family of glycoproteins we thank james mccluskey, frank carbone and stephen turner for critical reading of the manuscript. key: cord- -zt i o authors: bischof, larry j.; kao, cheng-yuan; los, ferdinand c. o.; gonzalez, manuel r.; shen, zhouxin; briggs, steven p.; van der goot, f. gisou; aroian, raffi v. title: activation of the unfolded protein response is required for defenses against bacterial pore-forming toxin in vivo date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: zt i o pore-forming toxins (pfts) constitute the single largest class of proteinaceous bacterial virulence factors and are made by many of the most important bacterial pathogens. host responses to these toxins are complex and poorly understood. we find that the endoplasmic reticulum unfolded protein response (upr) is activated upon exposure to pfts both in caenorhabditis elegans and in mammalian cells. activation of the upr is protective in vivo against pfts since animals that lack either the ire- -xbp- or the atf- arms of the upr are more sensitive to pft than wild-type animals. the upr acts directly in the cells targeted by the pft. loss of the upr leads to a normal response against unrelated toxins or a pathogenic bacterium, indicating its pft-protective role is specific. the p mitogen-activated protein (mapk) kinase pathway has been previously shown to be important for cellular defenses against pfts. we find here that the upr is one of the key downstream targets of the p mapk pathway in response to pft since loss of a functional p mapk pathway leads to a failure of pft to properly activate the ire- -xbp- arm of the upr. the upr-mediated activation and response to pfts is distinct from the canonical upr-mediated response to unfolded proteins both in terms of its activation and functional sensitivities. these data demonstrate that the upr, a fundamental intracellular pathway, can operate in intrinsic cellular defenses against bacterial attack. pore-forming toxins (pfts) are the single most prevalent protein virulence factor made by disease-causing bacteria and are important for the virulence of many important human pathogens including staphylococcus aureus, streptococcus pyogenes, clostridium perfringens, and aeromonas hydrophilia [ , ] . crystal (cry) toxins produced by the invertebrate pathogen bacillus thuringiensis (bt) are a large family of pfts that target the intestinal cells of insects and nematodes [ , , ] . the fact that some cry proteins target nematodes, in particular c. elegans, has been exploited to provide the only in vivo genetic model for studying pfts. this system led to the discovery of the first signal transduction pathway that protects cells against pfts, the p mitogen-activated protein kinase (mapk) pathway, which has been confirmed in mammalian cells [ , ] . there is growing evidence that the response of cells to pfts is, however, complex and there is a great deal yet to learn [ ] . the unfolded protein response (upr) of the endoplasmic reticulum (er) is a fundamental stress response used by eukaryotic cells to match protein synthesis demand to its capability to fold proteins within the er to maintain cellular homeostasis [ ] . in c. elegans and other animals there are three transducers that signal from the er to activate this response. these three distinct arms of the upr are mediated by irei, atf , and perk in mammals [ ] , which correspond to the genes ire- , atf- , and pek- in c. elegans [ , , ] . all three pathways are regulated by the er chaperone bip in response to an increase in unfolded proteins [ ] . here we demonstrate that the er stress response, in particular the ire- arm, is activated upon exposure of c. elegans and mammalian cells to pfts. we demonstrate for the first time that the ire- -xbp- arm of the upr (and to a lesser extent the atf- arm) is functionally important for defense against a pathogenic attack since loss of this pathway leads to animals hypersensitive to pft, but not to other toxic insults. furthermore, we demonstrate that activation of the ire- -xbp- pathway by pft requires p mapk and its associated mapk kinase and that the in vivo response of the upr to a pft can be separated from its response to unfolded proteins. these results indicate that activation of the upr plays an important role in cellular defenses against pathogens. in a genetic screen for genes involved in the cellular response of c. elegans to the pft cry b, we found a mutant predicted to be defective in protein n-glycosylation in the er (l.j.b. and r.v.a., manuscript in preparation). since defects in protein glycosylation induce the upr, this result suggested that perhaps the upr might play a role in protection against pfts. to test this hypothesis, we first investigated whether or not the upr was activated by a pft. the xbp- gene is spliced upon activation of the ire- branch of the upr, and its splicing is one marker for ire- (and upr) activation [ ] . in c. elegans, the xbp- intron spliced by ire- is nucleotides and the induction of this splicing event can be detected by rt-pcr [ ] . to analyze xbp- mrna transcript splicing, animals were fed escherichia coli expressing cry b and compared to worms fed control e. coli ( figure a ). while there is abundant unspliced xbp- mrna transcript in both samples, there is an increase in the spliced xbp- transcript from worms ingesting cry b, indicating activation of the ire- pathway. quantitative analyses indicate that the xbp- spliced transcript increases . , . , and . fold at the , , and h time points respectively. to independently test this result, we analyzed the in vivo expression of an ire- regulated gene, hsp- , a bip homolog. in vivo analysis of the hsp- promoter coupled to green fluorescent protein (gfp) demonstrated expression of this gene requires ire- and xbp- [ ] . a c. elegans strain containing hsp- ::gfp was fed either control e. coli or cry b expressing e. coli for hours at uc. as shown, a strong and specific increase in gfp expression in the intestine can be seen in the presence of the pft ( figure b , middle panel), consistent with activation of the ire- -xbp- pathway by cry b. heat shock of this strain in the absence of cry b confirms gfp could be induced in other cell types in addition to the intestine ( figure b, right panel) , as was demonstrated with the n-glycosylation inhibitor tunicamycin [ ] . the fact that cry b only induced expression in intestinal cells suggests the pft is only targeting these cells (see below). to address whether the ire- -xbp- pathway is also activated in mammalian cells in response to a pft, activation of the pathway was ascertained in hela cells exposed to the aeromonas hydrophila pft, aerolysin. as detected by the presence of the spliced protein isoform of xbp- , treatment of mammalian cells with a pft also results in robust activation of the ire- -xbp- pathway ( figure c ). the er stress response is required for defense of c. elegans against cry b to determine whether the er stress response played a role in the defense of c. elegans against the pft, c. elegans mutants in the er stress response pathway were qualitatively compared to wildtype n animals in their susceptibilities to cry b. the mutants that were tested included those encoding the three er stress transducer genes, atf- (ok ), pek- (ok ), and ire- (v ), as well as xbp- (zc ); these mutations are predicted or known to be loss of function mutations in their respective genes [ , , ] . in the absence of cry b, the wild type and mutant worms are healthy adults with similar appearance, except ire- (v ), which is clearly smaller than the other strains ( figure a ). in the presence of lowmoderate levels of the pft cry b, wild-type worms are slightly intoxicated compared to those found on control no-toxin plates, as evidenced by their smaller sizes and paler appearances (figure a ). to the same extent seen with wild-type worms, atf- (ok ) and pek- (ok ) mutant animals are also slightly intoxicated on lowmoderate levels of the pft cry b, indicating lack of either of these genes does not result in overt hypersensitivity or hyperresistance to cry b (figure a) . however under the same conditions, the ire- (v ) and xbp- (zc ) mutant worms are more severely intoxicated than wild-type worms as they are relatively smaller and considerably paler compared to their corresponding no toxin controls. the hypersensitivity to cry b resulting from lack of ire- and xbp- was also seen using rna interference (rnai; data not shown), confirming the phenotype is caused by loss of function in these genes. we call this hypersensitivity phenotype ''hpo'' for hypersensitive to pore-forming toxin. the sensitivity to cry b of animals mutant for the three er stress response pathways was quantitatively assessed using a dosedependent lethality assay ( figure b ). from these data, an lc s s (lethal concentrations at which % of the animals die) were obtained (table ) . our quantitative results confirm that ire- (v ) and xbp- (zc ) mutant animals are statistically more sensitive to pft than wild type animals (table ) and thus are hpo relative to wild type (caution is called for in interpreting the ire- (v ) data since many of these animals also have significant overt defects, e.g., developmental delays which prevents them from being as well synchronized at the start of the assay compared to the other strains [ ] ). our results indicate that atf- (ok ) mutant animals are also hpo, albeit to a lesser extent ( . vs. . fold increase in sensitivity for atf- vs. xbp- ). although atf- (ok ) hypersensitivity was not discerned with the plate assay, it is likely that the quantitative lethality assay is a more sensitive test for cry b hypersensitivity than the qualitative plate assay. in contrast to xbp- and atf- mutant animals, the sensitivity of pek- (ok ) mutant animals is not statistically different from that of wild-type animals ( table ) . to independently confirm these results, we used a developmental assay to assess the relative sensitivity of the four er stress response mutants to cry b. this experiment was performed by placing newly hatched l stage worms on plates containing different percentages of cry b expressing e. coli and then counting the worms that developed to either the l stage or adulthood ( figure c ). in the absence of cry b, nearly all worms developed to the l stage or adulthood for all strains with the exception of ire- (v ). this result confirms developmental defects previously seen with this mutant [ ] , and it was therefore excluded from subsequent analyses. wild type n and pek- (ok ) were both similarly inhibited in their development by increasing percentages of cry b. compared to n and pek- (ok ) animals, though, both atf- (ok ) and xbp- (zc ) were hpo, i.e., each is more developmentally inhibited by cry b than wild-type animals ( figure c ). because the ire- -xbp- pathway has a more discernible effect on protection against cry b than atf- , further experiments were focused on this arm of the er stress response. pore-forming toxins (pfts) are bacterial toxins that form holes at the plasma membrane of cells and play an important role in the pathogenesis of many important human pathogens. although pfts comprise an important and the single largest class of bacterial protein virulence factors, how cells respond to these toxins has been understudied. we describe here the surprising discovery that a fundamental pathway of eukaryotic cell biology, the endoplasmic reticulum unfolded protein response (upr), is activated by pore-forming toxins in caenorhabditis elegans and mammalian cells. we find that this activation is functionally important since loss of either of two of the three arms of upr leads to hypersensitivity of the nematode to attack by pfts. the response of the upr to pfts can be separated from its response to unfolded proteins both at the level of activation and functional relevance. the response of the upr to pfts is dependent on a central pathway of cellular immunity, the p mapk pathway. our data show that the response of cells to bacterial attack can reveal unanticipated uses and connections between fundamental cell biological pathways. taken together, the above results suggest that the ire- -xbp- pathway functions to protect the host against the pft cry b. however, an alternative explanation for our results is that animals mutant in this pathway (e.g., xbp- mutant animals) are sickly and have compromised health and therefore would respond poorly to any toxic insult. to address this alternative hypothesis, we tested whether xbp- (zc ) animals are hypersensitive to two toxic chemical compounds, the heavy metal cuso (a toxic insult that kills with kinetics similar to cry b) and the oxidative stress agent h o (a toxic insult that kills rapidly). the mutant xbp- (zc ) has the same sensitivity as wild type to killing by either cuso or h o ( figure d and e; table ). these data argue against the supposition that this mutant is hypersensitive to the pft merely because it is generally unhealthy. rather, the protective response is somewhat specific against the pft. these conclusions are strengthened by the finding that c. elegans lacking the upr respond normally to attack by the pathogenic bacteria pseudomonas aeruginosa, which does not make a pft ( figure f and table ). the xbp- pathway functions in the intestine to protect against cry b pft mosaic and expression analyses have shown that the targeting of intestinal cells by the pft cry b is both necessary and sufficient to intoxicate worms [ , ] . if the ire- -xbp- pathway is functioning directly to protect against the effects of the pft, then we would predict that the ire- -xbp- pathway should function in the target cells of the toxin, the intestinal epithelial cells. alternatively, the pathway might be functioning indirectly to protect against the effects of the pft (e.g., it might hypothetically function in neurons that then sends protective signals to the intestine). consistent with the first hypothesis, that the pathway is functioning directly in the target cells to protect against the pft, we previously noted that a marker for downstream activation of the pathway, hsp- , is turned on exclusively in intestinal cells ( figure b , middle panel), although the pathway is capable of being activated throughout the worm by a more general stress, such as heat shock ( figure b, right panel) . elegans fed e. coli expressing cry b compared to control e. coli not expressing cry b. the time the worms were allowed to feed on the e. coli before total rna was prepared for rt-pcr is indicated at the top, and the positions of the nucleotide size markers are indicated at the left. (b) compared to worms fed control non-cry b expressing e. coli, in vivo activation of hsp- ::gfp occurs specifically in the intestines of worms fed cry b expressing e. coli at uc for hours. as a comparison for gfp induction, separate worms on control bacteria were heat shocked at uc for hours to induce the er stress response by causing unfolded proteins. the heat shock worms have a strong increase in gfp throughout the body including the head, intestine and hypodermis. thus, although the entire worm is capable of activating the ire- -xbp- pathway as judged by hsp- induction, activation in cry b-fed animals is occurring only in those cells targeted by the pft. images taken by light microscopy are compared to images with fluorescence microscopy. scale bar is . mm. the experiment was performed three times, and representative worms are shown. (c) aerolysin induces activation of ire in mammalians cells. exposure of hela cells to proaerolysin ( ng/ml) leads to increased production of spliced xbp protein as shown on this immunoblot (upper) and quantitated relative to no toxin control (lower). dtt ( mg/ml for h) was used as a positive control. positions of molecular weight markers (kda) are indicated on right side of the figure. a nonspecific antibody-reacting band was used as a loading control and normalization of the xbp signal in each lane. doi: . /journal.ppat. .g figure . loss of specific upr pathways cause hypersensitivity to pft but not other toxins or a pathogenic bacteria. (a) comparison of er stress response mutants to wild-type n on % cry b-expressing e. coli plates indicate ire- (v ) and xbp- (zc ) are hypersensitive to cry b intoxication. two representative worms are shown for each strain hours after feeding either on e. coli without cry b or on e. coli of which % expressed cry b. scale bar is . mm. (b) a lethal concentration assay was performed using purified cry b toxin to quantitatively compare sensitivities of wild-type n and the er stress mutants. lethality was determined after days. this semi-log graph represents three independent experiments, and each data point is the mean and standard deviations of the experiments. (c) a cry b developmental inhibition assay was performed beginning with synchronized worms at the first larval stage. worms were grown on plates containing different percentages of cry b-expressing e. coli (% cry b as indicated under the figure), and the percent of worms reaching the l stage or adulthood hours later is indicated. ire- (v ) was included only on the plates with % cry b. data are presented as mean and standard deviation. (d) a lethal concentration assay comparing sensitivity to cuso revealed xbp- (zc ) is not hypersensitive compared to wild-type n . lethality was determined after days of cuso exposure, the same time frame as the cry b lethality assay. data, plotted semi-log, are the mean and standard deviation of three independent experiments. (e) a lethal concentration assay comparing sensitivity to h o revealed xbp- (zc ) is not hypersensitive compared to wild-type n . lethality was determined after hours of h o exposure. data, plotted semi-log, are the mean and standard deviation of three independent experiments. (f) a lifespan assay was used to compare the er stress mutants to slow killing by p. aeruginosa pa . this graph represents combined data from three experiments. doi: . /journal.ppat. .g to directly demonstrate the role of xbp- in protecting intestinal cells against cry b, the intestinal specific app- promoter [ ] was used to drive expression of xbp- in xbp- (zc ) mutant animals to determine if expression in the intestine is sufficient to rescue the hpo phenotype. as a negative control, gfp was similarly expressed under control of the app- promoter in xbp- (zc ) mutant animals. in control animals, expression of the gfp solely in intestinal cells was confirmed (data not shown). as expected, the majority of wild-type n animals showed only a low-modest degree of intoxication upon exposure to % cry b-expressing e. coli ( figure a , b; they were smaller and somewhat paler than the wild-type worms on control plates but were still quite active). also as predicted, both xbp- (zc ) mutant animals and xbp- (zc ) mutant animals transformed with app- ::gfp were hpo and intoxicated to similar extents ( figure a , b; most animals were very pale, small, and inactive). in contrast, xbp- (zc ) worms expressing xbp- under the app- promoter were significantly healthier than either untransformed or app- ::gfp transformed xbp- (zc ) animals fed with cry b ( figure a, b) . however, these app- ::xbp- -transformed xbp- (z ) worms were not as healthy as wild-type n under the same conditions. this partial rescue could indicate the expression of the artificial xbp- transgenes did not fully recapitulate wild-type xbp- expression levels and/or that there is some role for the ire- -xbp- pathway in other cell types. nonetheless, our results support a significant protective function for xbp- within the cells targeted by cry b. induction of ire- -xbp- pathway's role in response to pft but not unfolded proteins is regulated by the p mapk pathway er stress responses have been studied extensively for their role in protecting cells against unfolded proteins [ , ] . one way to assess the role of the er stress pathways in protecting against unfolded proteins is with the drug tunicamycin (a natural compound that leads to the accumulation of unfolded proteins in the er due to its inhibitory effect on n-linked protein glycosylation [ ] ). previous data in c. elegans have indicated different sensitivities of the three er stress response pathways for tunicamycin [ , ] . using a different toxicity assay, we have confirmed these observations: atf- (ok ) mutant animals have a similar sensitivity to tunicamycin as wild-type animals whereas both xbp- (zc ) and pek- (ok ) mutant animals are more readily killed by tunicamycin (figure ) . these results are in contrast to the response of these different er stress pathways to cry b, to which atf- mutant animals are more sensitive than pek- mutant animals. these data suggest that there are differences in how er stress pathways are activated in response to unfolded proteins and to the pft cry b. it is known that pfts trigger the activation of p mapk, which promotes cell survival and cellular defenses and which seems to play a central role in cellular responses to pfts [ , , ] . we therefore investigated whether pft-mediated activation of the upr and the p mapk pathway might be connected. we first investigated whether the ire- -xbp- pathway plays a role in the pft-induced activation of p by comparing the activation of the p mapk in wild-type and xbp (zc ) animals. we find that addition of cry b to wild-type c. elegans results in an increase in phosphorylated p , indicating the p pathway is activated by a pft in c. elegans just as it is in mammalian cells [ ] ( figure a ). we find that p activation occurs normally in xbp- (zc ) mutant animals ( figure a ), indicating that the upr is not required for activation of p mapk pathway in response to pft. we extended this result using ttm- , a downstream transcriptional target of the p mapk pathway in response to cry b and a gene required for normal defense against cry b pft [ ] . upregulation of ttm- mrna was dependent on the p mapk pathway but not dependent on xbp- ( figure f ). we next analyzed the reverse relationship between the ire- -xbp- and the p mapk pathways, namely whether the p mapk pathway is required for pft-induced activation of the ire- -xbp- pathway. we find that activation of the ire- -xbp- pathway in response to pft is dependent on the p mapk pathway, namely on sek- , the mapk kinase (mapkk) gene upstream of p , and on pmk- , the p mapk downstream of sek- ( figure ). we find that increased splicing (activation) of xbp- in response to cry b does not occur in sek- (km ) mapkk mutant animals ( figure b) . quantitatively, at the h time point the spliced form of xbp- is induced . fold in animals with an intact p mapk pathway and depressed . fold in sek- (km ) mapkk mutant animals relative to untreated controls. however, sek- is not absolutely required for splicing of xbp- since, in response to tunicamycin, splicing of xbp- is normal in sek- (km ) mutant animals ( figure c ). in agreement with these results, we find that in vivo activation of the downstream target of the ire- -xbp- pathway, hsp- ::gfp, by cry b within intestinal cells does not occur in pmk- (km ) p mapk mutant animals ( figure d ), whereas activation of hsp- ::gfp by tunicamycin does occur normally in pmk- (km ) mutant animals ( figure e ). to independently confirm and extend these results, we analyzed a different downstream target of the ire- -xbp- pathway. using proteomics, we identified a protein, y c a. (a homolog of the beta-prime subunit of the coatomer complex), that increased . fold in c. elegans animals exposed to cry b and whose increase was completely dependent on xbp- (see materials and methods and protocol s ). the gene encoding this protein was previously demonstrated to be transcriptionally regulated by tunicamycin in an ire- and xbp- dependent manner [ ] . using real time pcr, we find that both hsp- mrna and y c a. mrna are induced by either cry b or tunicamycin ( figure f ). consistent with activation of the ire- -xbp- pathway by p mapk in response to pft but not unfolded proteins, the full induction of both mrnas by cry b, but not tunicamycin, is dependent on sek- mapkk. interestingly, whereas induction of both mrnas by cry b is lacking in xbp- (zc ) mutant animals (confirming that activation of hsp- and y c a. by pft is via the upr), both mrnas are still somewhat induced by cry b in a sek- (km ) mutant, albeit at lower levels than in wild-type animals. these data suggest that some of the upr-mediated transcriptional response is p pathway independent. based on these data, we predicted that animals mutant in the p pathway should be more sensitive to pft than animals mutant in the upr pathway. this hypothesis is based on the fact that the p pathway is upstream of the upr, is required for full activation of the upr in response to pft, and is involved in uprindependent pft defense pathways (e.g., ttm- ). comparison of sek- (km ) and xbp- (zc ) mutant animals on cry b indicates sek- (km ) animals are more severely intoxicated than xbp- (zc ) animals at the same dose of cry b ( figure g ). this conclusion was quantitatively confirmed by performing lc experiments on n and sek- (km ) animals (table ) . whereas the lc of xbp- (zc ) animals on cry b is . fold lower than n , the lc of sek- (km ) animals on cry b is fold lower than n . here we demonstrate that er stress response pathways play a central but heretofore unknown role in innate defenses in vivo. specifically, we find that bacterial pore-forming toxins (pfts) activate the ire- -xbp- branch of the er unfolded protein response (upr) in c. elegans and mammalian cells and that the ire- -xbp- and atf- , but not the pek- , branches of the upr are important for c. elegans cellular defenses against a pft since elimination of either of these two branches leads to hypersensitivity to the pft cry b. the er stress response has been previously associated with pathogenic attack, mostly in the opposite direction shown here, e.g., aiding viral replication and pathogenesis ( [ ] and references therein). in a few cases, the er stress response has been linked with innate immunity since induction of er stress can activate creb-h, which in turn promotes the acute inflammatory response [ ] . it has also been suggested that ire- could influence immunity via its association with traf- , which in turn can regulate nf-kb [ ] . data from studies in plants suggest that in response to pathogens, signals can be produced that lead to an ''anticipatory'' upr to handle the massive synthesis of new secretory proteins required [ ] . here we definitively demonstrate a functional role of the upr in defense against a pathogen in vivo. loss of xbp- leads to animals nearly fold more susceptible to pft whereas loss of atf- leads to animals nearly fold more susceptible. our data suggest that cells have adapted the upr pathway for a specific response to pfts in order to promote cellular defense against this common form of pathogenic attack. first, we found that loss of the xbp- arm of the upr does not lead to hypersensitivity to a heavy metal or hydrogen peroxide nor does loss of either xbp- or atf- lead to decreased protection against a bacterial pathogen that lacks a pft. second, the ire- -xbp- and atf- arms of the upr are involved in the defense but the pek- arm is not. third, the activation and function of the upr in pft defenses can be separated from the role of the upr in dealing with unfolded proteins (here tested using the drug tunicamycin) in two ways: ) the relative importance of the various arms of the upr for defense against pft is different than their importance for protection against unfolded proteins and ) the activation of the ire- -xbp- pathway by pft, but not unfolded proteins, requires p mapk (see below). a link between the p and upr pathways has been shown in previous studies, although not with the level of functional relevance demonstrated here. various arms of the upr have been shown as both upstream or downstream of the p pathway, depending on the circumstances [ , , , , , ] . the p pathway itself is implicated extensively in innate immune protection of many organisms against pathogens [ ] and against pfts in worms and mammals [ , ] . our data presented here for the first time functionally link the upr to this major innate immune signal transduction pathway. our findings on the activation and role of the upr and p pathways in defense against pft are summarized in figure . why would induction of the er stress response play a protective role against pfts? it is possible that pfts somehow lead to the accumulation of unfolded proteins in a cell. for example, pfts are known to perturb calcium homeostasis and changes in calcium homeostasis are known to affect protein folding [ , ] . in this model, cells would respond to the toxin via p mapk and turn on the upr to anticipate and ameliorate the detrimental effects of unfolded proteins. arguing against this model, however, is our data showing that sensitivity of the three arms of the upr to cry b is different than their sensitivity to a global unfolder of er proteins, tunicamycin. a second model is that activation of the er stress response by cry b in a p mapk dependent manner may prepare the cell to handle an altered biosynthetic load in the er to defend against a toxin. for example, transcriptional array analysis indicate that over genes are differentially regulated in c. elegans by cry b ingestion [ ] , which could in turn lead to significant changes in the protein load of the er. a third model is (bn ) and glp- (bn );sek- (km ) after hours of exposure to either control (dmso) or tunicamycin ( mg/ml). this is a representative experiment of three independent experiments. (d) in vivo induction of hsp- ::gfp by cry b requires pmk- (p mapk). the strains hsp- ::gfp and hsp- ::gfp;pmk- (km ) were fed either control e. coli or e. coli expressing cry b for hours and the expression of gfp was then analyzed. cry b induces gfp within the intestinal cells of the strain hsp- ::gfp but not in the strain containing the pmk- (km ) mutant. the experiment was performed three times and representative worms are shown. scale bar is . mm. (e) in vivo induction of hsp- ::gfp by tunicamycin does not require pmk- (p mapk). the strains hsp- ::gfp and hsp- ::gfp;pmk- (km ) were exposed to either control (dmso) or tunicamycin ( mg/ml) for hours and the expression of gfp was then analyzed. tunicamycin induces gfp throughout both the strains hsp- ::gfp and hsp- ::gfp;pmk- (km ), including within the intestinal cells. the experiment was performed three times and representative worms are shown. scale bar is . mm. (f) downstream targets of the upr require the p mapk pathway for induction by pft but not unfolded proteins. the fold change in the levels of hsp- and y c a. mrna transcripts by cry b and tunicamycin were determined for glp- (bn ), glp- (bn );xbp- (zc ) and glp- (bn );sek- (km ) using real-time pcr. in addition, the fold change in ttm- transcripts was determined in response to cry b. data are mean and standard deviation of three independent experiments. (g) animals lacking sek- mapkk are more sensitive to cry b than animals lacking xbp- . wild-type n , sek- (km ), and xbp- (zc ) animals were placed on plates spread with e. coli transformed with empty vector ( %) or spread with empty vector e. coli diluted : ( %) or : ( %) with cry b-expressing e. coli (% thus gives toxin dose on a plate relative to undiluted cry b-expressing e. coli). the assay was initiated with l stage worms and photographs were taken hours later. in the absence of cry b, the worms developed into dark, gravid, active, healthy adults. on % cry b-expressing e. coli, xbp- (zc ) were slightly smaller than n but healthier than sek- (km ), which were as small, pale, inactive, and severely intoxicated. on % cry b-expressing e. coli, xbp- (zc ) was more intoxicated than n but not as intoxicated as sek- (km ) animals. scale bar is . mm. doi: . /journal.ppat. .g based on the fact that activation of the ire- -xbp- pathway leads to increased phospholipid biogenesis [ ] . it is possible that the defensive role of the ire- -xbp- pathway is to produce phospholipids that play a protective role against pfts. consistent with this, it has been shown that inhibiting the activation of srebps, the central regulators of membrane biogenesis, leads to hypersensitivity of mammalian cells to the pft aerolysin [ ] . in summary, we have identified specifically the ire- -xbp- and atf- er stress transducer pathways as components of cellular defenses against a pft. while p mapk was previously demonstrated to function in this regard [ ] , we have discovered a major and unexpected downstream target of this pathway for pft defenses, namely the upr. these results demonstrate the fundamental requirement for specific cell responses to bacterial pfts and support the notion of intrinsic cellular defenses (or inced, formerly, cellular non-immune defenses), a budding concept in immunity that emphasizes the intrinsic ability of epithelial cells to defend against bacterial toxins and the importance of these defenses as a supplement to the innate immune and adaptive immune systems [ ] . additionally, the differential importance of the three er stress transducer pathways in response to cry b versus tunicamycin, the differential activation of ire- -xbp- by p mapk in response to cry b versus tunicamycin, and the divergent pathways regulated by p mapk in protective responses reveal how studying pathogenesis can uncover a wonderful complexity and new connections among intracellular pathways. c. elegans strains were maintained at uc on ng plates using escherichia coli strain op as the food source [ ] . strains used in this study were wild-type bristol strain n [ ] , atf- (ok ), glp- (bn ), ire- (v ), pek- (ok ), pmk- (km ), sek- (km ), sj (zcis [hsp- ::gfp]) and xbp- (zc ). atf- (ok ) and pek- (ok ) were each outcrossed a total of times. sj was outcrossed an additional times as it had been outcrossed twice upon receipt from the caenorhabditis genetics center. xbp- (zc ) was created by outcrossing strain sj (xbp- (zc ); zcis [hsp- ::gfp]) four times and removing the integrated hsp- ::gfp during the outcrosses. images were acquired with an olympus bx microscope with the objective linked to a . camera mount and a dvc camera. worms were placed on % agarose pads containing . % sodium azide for photography. all assays were performed at uc unless indicated elsewhere. qualitative toxicity assays based on visual comparison of worm intoxication were performed on plates with e. coli-expressed cry b as described [ , ] . beginning with the th larval (l ) stage worms, worms were fed for hours either on control plates with e. coli jm that did not express cry b (empty vector) or plates prepared with e. coli jm expressing cry b diluted : with empty vector transformed jm . this amount of cry b ( %) mildly intoxicates wild-type c. elegans, which allows for identification of strains that are hypersensitive to cry b as these strains will be more severely intoxicated than wild type. quantitative lethal concentration assays were performed as described [ ] except the worms were scored after days for cry b, cuso , and tunicamycin. lethal concentration assays with h o did not include e. coli or -fluoro- -deoxy-uridine, and worms were scored after hours. concentrations of each toxin were set-up in triplicate for each assay, and each assay was performed independently three times. purified cry b was prepared as described [ ] and dissolved in mm hepes (ph . ) prior to use. approximately worms were scored for each strain in the calculation of the lc values for each toxin. for tunicamycin assays, the set up was identical to the lethality assay with cry b. for the developmental inhibition assay, cry b plates were prepared as described [ , ] . approximately l stage worms (from bleached embryos hatched off overnight) were placed on each plate ( mm) and the number of worms at the l or adult stage days later was determined. this assay was performed independently three times. the p. aeruginosa lifespan assay was performed on slow-killing plates as described [ ] , with the following modifications: pa was cultured overnight in tryptic soy broth instead of king's broth and then spread on slow-killing plates complemented with um mm -fluoro- -deoxy-uridine. the experiment was performed three times with approximately - worms total per strain, at uc. to determine if there was rescue of the hypersensitivity phenotype in the intestinal-specific promoter studies, % e. coli-expressing cry b plates were used to compare cry b sensitivities of wild-type n , xbp- (zc ), and xbp- (zc ) that were transformed with constructs to express either green fluorescent protein (gfp) or xbp- mrna within intestinal cells using the app- promoter (plasmids are described in protocol s ). transgenic l stage worms were placed on the % e. coli expressing cry b plates and their health status was assessed hours later. specifically, the relative health of each worm was determined qualitatively by comparing body size, darkness of the intestine as an indicator of feeding, and activity, including whether the worm demonstrated spontaneous movement. for scoring of the transgenic worms, comparisons were made using both n as a reference for healthy worms, as they demonstrated dark intestines and continuous spontaneous movement, and xbp- (zc ) as a reference for intoxicated worms that had pale intestines and demonstrated rare or no spontaneous movement. the glp- (bn ) strain was used for these experiments (including the double mutants glp- (bn );xbp- (zc ) an glp- (bn ); sek- (km )) since it has a greatly reduced number of germ cells when grown at figure . schematic illustrating relationship between p mapk, ire- -xbp- , and pft defense pathways. pfts at the cell surface of epithelial cells activate p mapk that activates ire- that induces splicing of xbp- , which then turns on defense against pfts. residual activation of xbp- targets in the absence of the p mapk pathway suggests there might be p -independent activation of the ire- -xbp- pathway in response to pft as well (not shown). independent of ire- activation, p mapk can also activate ttm- and other pft defenses. tunicamycin, which causes the accumulation of unfolded proteins in the er, activates ire- via a mechanism independent of the pft and p mapk. doi: . /journal.ppat. .g uc. this helps remove the background of macromolecules not isolated from the intestine. the response to cry b is not altered in this strain compared to wild type [ ] . primers used for these experiments are described in protocol s . approximately , l stage worms were used per mm dish for each treatment group. worms were exposed to cry b for the indicated period of time on either e. coli jm containing empty vector or e. coli jm expressing cry b as described [ , ] . after exposure to each treatment, worms were rinsed from plates with water, centrifuged at g for seconds, and washed two additional times with water. rna was prepared from worms using trizol (invitrogen) and further purified with rneasy columns (qiagen). cdna was prepared by reverse transcription using oligo-dt. standard pcr was used to detect xbp- splicing, and products were analyzed on % agarose gel. unspliced xbp- transcript is nucleotides and spliced transcript is nucleotides. to quantitate the amount of xbp- splicing, loading was normalized by quantitating cdna levels using real time pcr and eft- primers [ ] . equal amounts of cdna were used for the xbp- splicing pcr experiments and microliters of each reaction were loaded onto a % agaose gel and stained with ethidium bromide. nih imagej was then used to quantitate the intensities of xbp- spliced forms in cry b treated samples relative to untreated samples at the same time point. real time pcr was performed on an abi instrument using sybr green detection (applied biosystems). eft- was used as the real time pcr normalization control [ ] . experiments with cry b used either a control plate (e. coli not expressing cry b) or a cry b plate on which % of the e. coli expressed cry b. tunicamycin experiments used e. coli op as a food source and either dmso as the control or tunicamycin at mg/ml incorporated into the plates. three independent experiments for the splicing and real time pcr were performed for each treatment. hela cells were cultured in mem media supplemented with % fetal calf serum, % penicillin-streptomycin, % glutamine and % non-essential amino acids, in a humidified incubator with % co at uc. aerolysin was purified as described [ ] . cells were continuously treated with ng/ml ( . nm) of proaerolysin. at different time points, cells were washed with pbs and lysed at uc in . m sucrose supplemented with proteases inhibitor (roche, germany) using a needle. the whole cell extracts were subjected to sds-page and western blotting. xbp (r- ) antibody was from santa cruz inc. band intensities were quantified, after background removal, using imagej software (nih). the loading in each lane was normalized relative to the intensity of a nonspecific antibody-reacting band on the blot. approximately l stage worms were grown in a single well of a well plate containing ml s media [ ] and e. coli op . when worms had reached the l to young adult stage, glucose was added to mm and either mm hepes (ph . ) or cry b dissolved in mm hepes (ph . ) to give a final concentration of mg/ml was added. after one hour, worms were removed, centrifuged, and ml of media was removed. twenty five ml of sodium dodecyl sulfate loading buffer was added, and worms were boiled for minutes. ten microliters of lysate were used for immunoblotting. monoclonal antibody to phospho p mapk (cell signaling technology cat. no. ) was used at : and monoclonal antibody to a-tubulin (sigma-aldrich cat. no. t ) was used at : . l stage glp- (bn ) and glp- (bn );xbp- (zc ) worms were used for this experiment. approximately , worms of each strain were used for both control and cry b treatments. control plates consisted of mm plates spread with e. coli that did not express cry b, while cry b treatments consisted of plates in which % of the e. coli expressed cry b. approximately , worms were used per plate. worms were fed on the bacteria for hours at uc. for details of mass spectrometry, please see protocol s . all experiments were performed a minimum of three times. lc values were determined by probit analysis [ ] . the lethal concentration assays are represented graphically using nonlinear regression performed with the software graphpad prism. statistical analysis between two values was compared with a paired t-test. statistical analysis among three or more values was compared with matched one way anova using the tukey post test. lifespan data was analyzed with kaplan-meier survival curves. statistical significance was set at p, . . protocol s found at: doi: . /journal.ppat. .s ( . mb doc) molecular features of the cytolytic pore-forming bacterial protein toxins membrane-damaging toxins: pore formation structure, diversity, and evolution of protein toxins from spore-forming entomopathogenic bacteria using worms to better understand how bacillus thuringiensis kills insects mode of action of bacillus thuringiensis cry and cyt toxins and their potential for insect control mitogen-activated protein kinase pathways defend against bacterial poreforming toxins differential role of p mitogen activated protein kinase for cellular recovery from attack by pore-forming s. aureus a-toxin or streptolysin o bacterial pore-forming toxins: the (w)hole story? signaling the unfolded protein response from the endoplasmic reticulum er stress and the unfolded protein response complementary signaling pathways regulate the unfolded protein response and are required for c. elegans development genetic interactions due to constitutive and inducible gene regulation mediated by the unfolded protein response in c. elegans ire couples endoplasmic reticulum load to secretory capacity by processing the xbp- mrna er stress signaling by regulated splicing: ire /hac /xbp bt toxin resistance from loss of a putative carbohydrate-modifying enzyme resistance to a bacterial toxin is mediated by removal of a conserved glycosylation pathway required for toxin-host interactions functional expression and characterization of the cytoplasmic aminopeptidase p of caenorhabditis elegans that which does not kill me makes me stronger: adapting to chronic er stress stress signaling from the lumen of the endoplasmic reticulum: coordination of gene transcriptional and translational controls epithelial cells are sensitive detectors of bacterial pore-forming toxins coronavirus infection modulates the unfolded protein response and mediates sustained translational repression endoplasmic reticulum stress activates cleavage of crebh to induce a systemic inflammatory response endoplasmic reticulum stress: cell life and death decisions endoplasmic reticulum quality control and the unfolded protein response: insights from plants suppressive effects of fr , an inhibitor of p mitogen-activated kinase, on calreticulin mrna expression induced by endoplasmic reticulum stresses functional coupling of p -induced up-regulation of bip and activation of rna-dependent protein kinase-like endoplasmic reticulum kinase to drug resistance of dormant carcinoma cells nckdependent activation of extracellular signal-regulated kinase- and regulation of cell survival during endoplasmic reticulum stress up-regulation of grp and antiapoptotic signaling in murine peritoneal macrophages exposed to insulin requirement of the p mitogen-activated protein kinase signalling pathway for the induction of the kda glucose-regulated protein/ immunoglobulin heavy-chain binding protein by azetidine stress: activating transcription factor as a target for stress-induced phosphorylation cholesterolinduced macrophage apoptosis requires er stress pathways and engagement of the type a scavenger receptor map kinases in the immune response pore worms: using caenorhabditis elegans to study how bacterial toxins interact with their target host endoplasmic reticulum stress response and neurodegeneration signal integration in the endoplasmic reticulum unfolded protein response caspase- activation of lipid metabolic pathways in response to bacterial pore-forming toxins promotes cell survival pore-forming toxins and cellular nonimmune defenses (cnids) the genetics of caenorhabditis elegans assays for toxicity studies in c. elegans with bt crystal proteins a purified bacillus thuringiensis crystal protein with therapeutic activity against the hookworm parasite ancylostoma ceylanicum pseudomonas aeruginosa killing of caenorhabditis elegans used to identify p. aeruginosa virulence factors purification and some properties of the hemolytic toxin aerolysin the nematode caenorhabditis elegans we are grateful to danielle huffman for help with the sek- lc experiment. strains used within this publication were provided by the caenorhabditis genetics center. the plasmid ppd . was obtained by courtesy of andrew fire through addgene. key: cord- - zrv k authors: chachu, karen a.; lobue, anna d.; strong, david w.; baric, ralph s.; virgin, herbert w. title: immune mechanisms responsible for vaccination against and clearance of mucosal and lymphatic norovirus infection date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: zrv k two cardinal manifestations of viral immunity are efficient clearance of acute infection and the capacity to vaccinate against secondary viral exposure. for noroviruses, the contributions of t cells to viral clearance and vaccination have not been elucidated. we report here that both cd and cd t cells are required for efficient clearance of primary murine norovirus (mnv) infection from the intestine and intestinal lymph nodes. further, long-lasting protective immunity was generated by oral live virus vaccination. systemic vaccination with the mnv capsid protein also effectively protected against mucosal challenge, while vaccination with the capsid protein of the distantly related human lordsdale virus provided partial protection. fully effective vaccination required a broad immune response including cd t cells, cd t cells, and b cells, but the importance of specific immune cell types varied between the intestine and intestinal lymph nodes. perforin, but not interferon gamma, was required for clearance of mnv infection by adoptively transferred t lymphocytes from vaccinated hosts. these studies prove the feasibility of both mucosal and systemic vaccination against mucosal norovirus infection, demonstrate tissue specificity of norovirus immune cells, and indicate that efficient vaccination strategies should induce potent cd and cd t cell responses. more than % of epidemic nonbacterial gastroenteritis worldwide can be attributed to human noroviruses (hunv) [ ] [ ] [ ] . infection is transmitted fecal-orally, and symptomatic infection is characterized by nausea, vomiting and/or diarrhea lasting - hours within hours of exposure [ ] . despite the significant costs and morbidity of hunv infections, no vaccine is currently available. the elderly and individuals in long-term care facilities may be more susceptible to either norovirus infection or norovirusinduced disease [ ] and would be an important target population for a norovirus vaccine. the reasons for increased incidence and/ or susceptibility to hunv disease are unknown. this is due in part to our incomplete understanding of norovirus immunity. the potential to vaccinate against these and related viruses has been demonstrated in gnotobiotic piglets, cats and rabbits [ ] [ ] [ ] , but the immune mechanisms responsible have not been identified. the challenges for vaccine efficacy may be very different between different caliciviruses. for example, variation in mnv strains is significantly less than between hunv strains [ ] . human volunteer studies demonstrate short-term, but not long-term, protection against homologous, but not heterologous, viral challenge [ ] [ ] [ ] . since hunv belong to genogroups (gi, gii and giv) with many strains in each genogroup [ ] , this lack of cross-protection is a challenge for vaccine development. frequent exposure to noroviruses within short time periods stimulates sustained immunity and resistance to norovirus induced illness [ , ] . serum antibody levels in adults reflect susceptibility to infection and do not always correlate with protection [ , ] . in children, however, serum antibody levels correlate with protection, likely reflecting short-term immunity and recent exposure [ ] [ ] [ ] . a nonfunctional fucosyl transferase gene (fut ) accounts for a significant proportion, though not all, of resistance to norwalk virus infection, suggesting that other factors, yet undiscovered, may contribute to norovirus resistance [ , ] . in the absence of a cell culture system for hunv, virus like particles (vlps) that assemble when the viral capsid protein is expressed have been important for evaluating norovirus immune responses [ ] [ ] [ ] [ ] . studies using norwalk virus (gi), snow mountain virus (gii) and hunov-hs (gii) vlps to evaluate immunity after infection with live virus or immunization with vlps orally show production of t cell effector cytokines such as il- and interferon c (ifn-c) and proliferation of norovirus specific t cells after in vitro restimulation with vlps [ ] [ ] [ ] . these studies show that t cell responses develop, but do not define their role in either clearance of primary infection or resistance to re-challenge. together, they suggest the potential for vaccination, but leave open important questions about the effectiveness and longevity of vaccine immune responses, mechanisms of vaccination, the viral protein targets for protective responses, and the potential for cross-protection between distantly related noroviruses. the identification of the first murine norovirus, mnv, and its propagation in cultured cells provides a facile animal model for studies of norovirus immunity and pathogenesis [ , ] . mnv, an enteric virus that infects tissues of the gastrointestinal tract, is spread by the fecal-oral route ( [ ] and unpublished studies). the mnv genome encodes four open reading frames. orf encodes a polyprotein that is cleaved into individual non-structural proteins similar to the polyprotein of hunv [ ] . orf encodes the major capsid protein vp and orf encodes a minor capsid protein. the existence of a protein product for orf has not been confirmed. in the mnv virion structure, the capsid, like that of human noroviruses, consists of dimers of vp [ ] . there are differences between the mnv virion and previously reported vlp structures. the mnv protruding domain is lifted off the shell domain by approximately angstroms and rotated approximately degrees in a clockwise fashion, forming interactions at the p base in an infectious virion that have not been observed previously. the existence of these novel aspects of the structure are consistent with the hypothesis that mnv may undergo a capsid maturation process [ ] . studies of mnv pathogenesis reveal an important role for interferon (ifn) and stat- mediated innate immunity in resistance to infection and mnv induced lethality [ , ] . the importance of adaptive immunity in control of mnv infection is indicated by the observation that rag -/-mice develop persistent mnv infection while wild type (wt) mice can clear infection with some strains of mnv [ , , ] . while mnv is an efficient enteric virus that infects many mice in research mouse colonies around the world, diarrhea has not been reported after mnv infection. thus, mnv provides an infection only model for hunv infection. viral titers in tissues of infected mice have not been reported to exceed pfu/ml, and this highest level of viral titer is obtained after infection of highly susceptible stat -/-mice [ ] . in rag -/-mice and wt mice, viral titers of to pfu/ml are routinely observed [ , ] . the availability of a plaque assay for mnv allows the analysis of mnv infection despite these low titers. some mnv strains persist at a low level in wt mice, while others are cleared from intestine, spleen, liver, mesenteric lymph nodes (mln) and feces within days of infection [ , , ] . additionally, in wild type c bl /j mice mnv replicates maximally in the distal ileum [ ] , in comparison to wild type s /svevtac mice where replication occurs in the proximal intestine [ ] . the significance of these differences is not known. studies of norovirus infection in human volunteers have not specifically investigated whether the infection spreads beyond the intestine to the local lymph nodes, however, it is possible that systemic invasion occurs in humans with chronic conditions or immunosuppressed hosts [ ] [ ] [ ] [ ] [ ] . additionally, viremia has been reported in infections of gnotobiotic pigs and calves [ , , ] . thus, the ability of mnv to spread to tissues other than the intestine after oral infection may not be unique, but the relationship of this aspect of mnv pathogenesis to human infection is not clear. the availability of strains that can be cleared from wt mice, such as mnv .cw , provides an opportunity to define the mechanisms responsible for two cardinal aspects of viral immunity: the capacity to effectively clear acute infection and the immune mechanisms responsible for effective vaccination. b cells and mnv specific antibody are important in the clearance of primary mnv infection [ ] , but the role of t cells in clearance and the potential and mechanisms of vaccination against mucosal norovirus challenge are unknown. we show here that vaccination with either live mnv or venezuelan equine encephalitis replicon particles (vrps) expressing the mnv capsid protein vp protect the intestine against re-challenge for at least six months. live virus was more effective than vrp-mediated vaccination. there was partial cross protection against mnv infection after vaccination with a hunv capsid protein. we found that both the clearance of primary infection and vaccination require the concerted efforts of cd t cells, cd t cells, b cells, and that t cells required the effector molecule perforin for maximal impact on mnv infection. the effects of specific immune cell types were tissue specific, differing between ileum and mesenteric lymph nodes. these are the first studies to demonstrate immune mechanisms responsible for norovirus clearance and vaccination. we first determined whether we could detect short-term immunity to homologous mnv challenge and whether proteins encoded by specific mnv orfs could elicit effective immunity. vrps expressing orf , orf and orf of mnv .cw and orf of the hunv lordsdale (genogroup gii. ) and chiba (genogroup gi. ) were produced for vaccination experiments. western blots of vrp-infected cell lysates revealed proteins of appropriate sizes [ , ] and additionally showed that hyperimmune polyclonal rabbit antisera to mnv [ ] cross-reacted at low levels with vlps from chiba virus and lordsdale virus ( figure s a ). wt mice were vaccinated and boosted three weeks later. two weeks after boosting, mice were challenged with mnv .cw and organs titered for mnv three days later ( figure a ). in these wt mice, maximal mnv replication in the intestinal tract occurs in the distal ileum [ ] and viral titers could not be detected in duodenum/ jejunum (data not shown). after oral inoculation with mnv .cw , wt mice exhibit detectable viral titers in the distal ileum and the mln three to five days post-infection [ , ] . prior infection with human noroviruses are the most common cause of epidemic nonbacterial gastroenteritis in the world. despite their importance as human pathogens, little is known about how the immune system controls and clears norovirus infection, and the potential and mechanisms of vaccination remain unclear. here, we used norovirus infection of mice to show that vaccination can provide long-lasting immunity against mucosal norovirus challenge and to identify the types of immune cells that are important in vaccination against norovirus infection. similarly, we identified the types of immune t cells that are important for clearance of acute infection. efficient vaccination required all three major arms of adaptive immunity: cd t cells, cd t cell, and b cells. importantly, protective vaccination against mucosal challenge was observed after either mucosal or systemic norovirus antigen exposure. the pore-forming molecule perforin was important for t cell-mediated control of norovirus infection. our study has important implications for understanding adaptive immunity to norovirus infection, and may provide insight into the directions to take in developing a human norovirus vaccine. either mnv .cw (p = . ) or mnv .cw (p = . ) significantly decreased mnv .cw replication in the distal ileum compared to control mice infected with reovirus ( figure b ). similar decreases were observed in the mln after vaccination with mnv .cw (p = . ) or mnv .cw (p = . ) ( figure c ) compared to the reovirus controls. similar results were observed in the spleen (data not shown). there was no statistically significant difference between vaccination with mnv .cw or mnv .cw . this demonstrates that a protective secondary immune response develops after clearance of primary mnv infection. orf vrps protected against mnv .cw in both distal ileum (p = . ) and mln (p = . ) compared to control vrps expressing hemagglutinin (ha) from a mouse adapted influenza a virus [ ] (ha vrp control group). controls for vrp vaccination also included pbs. ha vrp controls were not significantly different from pbs controls across all experiments and statistical comparisons for vrp vaccination are therefore shown to ha vrp controls. orf vrps alone in the distal ileum, or in both the distal ileum and mln when combined with orf vrps, had a small but statistically significant effect on mnv .cw levels ( figure b and c). orf vrps alone did not confer significant protection ( figure b and c). together these data show that vaccination with either live virus or orf vrps can confer shortterm protection against mnv challenge. we next assessed vaccination with heterologous orf proteins. mice were vaccinated and boosted with vrps expressing orf from chiba virus or lordsdale virus and challenged with mnv .cw . vaccination with lordsdale virus capsid led to statistically significant protection against mnv infection in the distal ileum, (p = . , figure b ) but not the mln ( figure c ). no significant reduction in mnv titers was seen after immunization with chiba virus capsid ( figure b and c ). protection after lordsdale orf vrp vaccination did not correlate with generation of cross-reactive serum igg in these mice, measured by elisa, despite the potential for such cross-reactivity revealed by western blot ( figure s b ). fecal extracts from immunized mice yielded no measurable homotypic or heterotypic igg or iga (data not shown). taken together, these data show that there is measurable functional immunologic cross protection between lordsdale virus and mnv in the distal ileum. the lack of a correlation between serum or fecal antibody responses and protection suggested that protection may be t cell mediated. since older adults may be more susceptible than younger adults to norovirus infection or disease [ ] , we determined whether increased age altered vaccine efficacy. prior work has shown that mice older than year of age have diminished vaccine responses to sars virus antigens [ ] . we therefore compared vaccine efficacy in adult ( week old) and aged ( month old) mice. adult and aged mice were vaccinated and challenged as before. in contrast to studies using sars virus antigens [ ] , aged mice responded as well as adult mice to mnv orf vaccination in both the distal ileum and mln ( figure d and e). despite this protective effect, sera from vaccinated aged mice had significantly lower anti-mnv orf igg compared to adult mice ( figure s c ). these data indicated that protection against mnv infection occurred in the absence of robust serologic responses, again raising the possibility that t cells play a fundamentally important role in vaccination against mnv. we next determined whether protection conferred by mnv .cw or mnv orf vrps was long lived. wt mice were primed and boosted as shown in figure a with mnv .cw or mnv orf vrps. mice were then challenged with mnv .cw two, four, , or weeks later and mnv titers measured three days post-challenge. two weeks post-boost, we observed complete protection against ileal mnv .cw infection after vaccination with either mnv .cw (p = . ) or orf vrps (p, . ) compared to reovirus or ha vrp controls ( figure b ). at two weeks, while vaccination with either mnv .cw or orf vrps limited mnv .cw replication in mln, live virus vaccination was more effective (p, . ) ( figure c ). live virus vaccination conferred full protection against mnv .cw replication in both the distal ileum and the mln at four, and weeks after vaccine boost. vaccination with orf vrps was also protective, albeit less effective than vaccination with mnv .cw ( figure b and c). thus both live virus and subunit vaccine induce long-term protection against mnv infection, with live virus vaccination providing more complete protection. we next determined the mechanism(s) responsible for effective vaccination. we vaccinated mice lacking both major histocompatibility complex (mhc) class i and b microglobulin (b m) [ ] (cd t cells deficiency [ ] ), mhc class ii (cd t cells deficiency [ ] ), or b cell deficient mice [ ] ( figure a) . these experiments were conducted concurrently with the experiments in figure above, as such the data from wt mice are repeated in the figure for comparison. live mnv vaccination induced significant protection against mnv challenge in both the distal ileum and the mln of b cell-/-, mhc class ii-/-and mhc class i b m-/-mice (p, . in all cases, figure b and c). however, there was considerable variation in the efficacy of vaccination in distal ileum and mln between different immunodeficient strains. in b cell-/-mice, after vaccination with live virus, only out of mice had any titer (and those two mice had less than pfu of mnv) and in mhc class i b m-/-mice, similar vaccination led to undetectable viral titers in the distal ileum ( figure b ) but detectable titers in the mln ( figure c ). in mhc class ii-/-mice, there were detectable titers in both tissues ( figure b and c). results for orf vaccination showed that protection required the activity of all major aspects of the adaptive immune response ( figure b and c). moreover, there was no protection elicited by orf vaccination in either intestine or mln tissue after vaccination of mhc class i b m-/-mice with orf vrps (figure b and c) indicating that protection by vrps critically depends on cd t cells. these data demonstrated that complete protection in all tissues after vaccination with live virus required the concerted actions of b cells, mhc class ii, mhc class i and b m. further, the results were consistent with tissue specific roles for b cells, cd t cells and cd t cells in the development of complete protection against mnv infection. we next determined whether the same cell types that were required for vaccination were also required for efficient clearance of acute infection. we focused on the role of t cells in clearance since the role of b cells in clearance has already been demonstrated [ ] . to determine the role of t cells in clearance of acute mnv infection we inoculated wt, mhc class ii-/-, and mhc class i b m-/-mice orally with mnv .cw and measured viral titers in the distal ileum and mln three, five, seven and days post-infection ( figure b- e) . there was no significant difference in viral titer between mhc class i b m-/-mice and wt mice at three and five days postinfection, indicating that mhc class i and b m were not required in mnv infection at early time points ( figure b ii-/-compared to wt mice at seven days post-infection (p = . , figure e ). by eight days post-infection, mln infection was cleared. together these data indicated that mhc class ii, and by inference cd t cells, were necessary for control of acute mnv infection but are not required for eventual clearance of mnv infection. to exclude the possibility that the phenotypes we observed in mhc class i b m-/-and mhc class ii-/-mice were due to abnormal immune ontogeny in knockout mice, we determined the requirement for cd and cd t cells in the clearance of primary mnv infection in wt mice depleted of cd and cd t cells. depletion of cd and cd t cells was at least % effective as assessed by flow cytometry of isolated splenocytes ( figure a ) and this depletion protocol is effective at depleting t cells in secondary lymphoid organs and the intestine [ , ] . in comparison to control antibody, depletion of cd t cells, led to a significant increase in mnv titers in the distal ileum (p = . , figure b ), but not the mln ( figure c ). in contrast, depletion of cd t cells led to an increase in mnv titers in both the distal ileum (p = . , figure b ), and the mln (p = . , figure c ). together, these data from primary challenges of non-immune mice lacking antigen presenting molecules or depleted of specific t cell subsets demonstrated that cd t cells are important for efficient mnv clearance in the distal ileum especially at days three we next determined whether cd and cd t cells from vaccinated mice can, alone or in combination, clear mnv infection from mucosal sites. we have previously shown that mnv infected rag -/-mice have high levels of viral rna present in multiple tissues up to days post-infection [ ] . we therefore determined mnv viral titers in rag -/-mice. by days postinfection, all rag -/-mice had consistent, high levels of mnv in both duodenum/jejunum and distal ileum ( figure a and b) , as well as several other tissues (data not shown). these data confirmed that mice lacking adaptive immunity fail to clear mnv infection [ ] . the availability of persistently infected rag -/-mice allowed us to determine the role of cd and cd t cells in clearance of mnv infection using adoptive transfer of splenocytes from mnv immune wt mice into persistently infected rag -/-mice. transfer of immune, but not non-immune, splenocytes signifi- to define which cells were required for mnv clearance, cd or cd t cells were depleted from splenocytes transferred into rag -/-recipients. anti-t cell antibodies effectively depleted the appropriate t cell populations, as measured six days post-transfer by flow cytometry (figure a ). depletion of either cd or cd t cells individually led to a significant increase in mnv titers in duodenum/jejunum compared to control depletion ( figure b , cd depletion p = . ; cd depletion p = . ). depletion of both cd and cd t cells from transferred immune splenocytes caused a significant additional increase in mnv titers when compared to either cd depletion alone (p = . ) or cd depletion alone (p = . ). in the distal ileum, depletion of either cd t cells (p = . ) or cd t cells (p, . ) led to a significant increase in mnv titers ( figure c ). these data demonstrated that both immune cd and cd t cells were necessary for clearance of persistent mnv infection from the intestine. two major effector mechanisms for the antiviral effects of t cells are the production of ifnc and perforin mediated cytolysis [ ] . we therefore adoptively transferred immune splenocytes from ifnc-/-or perforin-/-mice into persistently infected rag -/-mice and determined their capacity to clear intestinal mnv infection. immune splenocytes from ifnc-/-mice were as effective as those from wt mice ( figure b and c) . however, immune splenocytes from perforin-/-mice were less effective at clearing mnv infection from the duodenum/jejunum (p = . , figure c ) or distal ileum (p = . , figure b ) than cells from either wt or ifnc-/-mice, but more effective compared to transfer of non-immune cells in duodenum/jejunum (p = . ) or distal ileum (p = . ). thus, while perforin was critical for efficient clearance of mnv infection from the intestine, it was not the only relevant effector mechanism. in this paper we define the mechanisms of immunity to norovirus infection as measured by both vaccination against, and clearance of, mucosal infection. we found that it is possible to generate highly effective, and remarkably long lasting, immunity to norovirus infection by oral exposure to live virus. further, systemic exposure to the viral capsid protein expressed in a vaccine vector resulted in effective immunity, albeit not as effective as that observed after live virus vaccination. importantly, this shows that the mnv vp protein contains relevant b cell, cd t cell and cd t cell epitopes. vaccination was effective in aged mice. additionally, vaccination in adult mice required the concerted action of cd t cells, cd t cell, and b cells to be completely protective in the tissues surveyed. interestingly, the activities of different components of the adaptive immune system in clearance and vaccination were tissue specific, with different cells playing roles in the intestine itself compared to the draining lymph nodes. perforin was an important effector molecule. these data have important implications for understanding adaptive immunity to an animal norovirus, representative of a genus that causes significant disease in humans. hunv infection and disease is rapid, with symptoms developing within - hours of infection and lasting for a few days. thus, we selected three days after challenge as a readout for infection in our studies, since relevant vaccine-generated immune responses would have to act very early after challenge. lack of any of the three components of the adaptive response: b cells, cd t cells, or cd t cells significantly diminished vaccine effects generated by either live virus or vp capsid protein immunization, and delayed viral clearance during primary infection. this indicates that vp has antibody epitopes as well as mhc h- b restricted cd and cd t cell epitopes. these data suggest that it may be necessary to engage the concerted actions of an intact immune response including both mhc class i and mhc class ii restricted t cells and antibody responses to efficiently vaccinate against hunv infection. the protection against mnv infection in aged mice in the absence of robust generation of anti-mnv antibodies raised the possibility that an important component of the vaccine response is t cell dependent, a hypothesis borne out in adoptive transfer studies. importantly, the antiviral effector perforin is important in the clearance of mnv from the intestine, suggesting that the cytotoxic t cell response is a key effector mechanism. it is possible that other cell types such as nk cells might also use perforin as a mechanism to control mnv infection. our data do not rule out a role for ifnc in clearance of mnv infection since nk cells in recipient rag -/-mice can make ifnc, but do suggest that t cell derived ifnc plays at most a minor role in effector t cell function in the ileum. this argues that classical ctl assays may be a good surrogate for the development of effective vaccine-generated immune responses to hunv. live virus vaccination was more effective than vrp based vaccination. the lower level of protection that we observed with orf vrps in contrast to mnv .cw may be due to many factors, and this study does not provide mechanistic insights into this difference. in comparison to vlps, vrps may have advantages in systemic vaccination including targeting dendritic cells and intrinsic adjuvant activities [ ] . these properties of vrps may be responsible for the effectiveness of systemic single protein subunit vaccination against mucosal viral challenge in this case. however, it may be that because vrps undergo a single round of replication at the site of inoculation they cannot generate the same breadth of immunity that is generated by live replicating virus. while vrp vaccination clearly induces some relevant effector and memory cell responses, vaccination with capsid alone may not sufficient to generate the complete antigenic repertoire required for effective immunity. interestingly, we found some protection with the non-structural orf polyprotein, suggesting that protective epitopes exist outside of the capsid protein. as the orf polyprotein is expressed early after infection, it may be that these epitopes would be valuable targets for generating an efficient immune response. of note, vaccination with vp via the subcutaneous route provided significant protection despite the fact that the vaccination occurred systemically, while protection was read out at a mucosal site. this indicates that an active systemic immune response can provide protection against norovirus infection, and a mucosal vaccine may not be necessary to vaccinate against norovirus infection. importantly, systemic vaccination was dependent on t cells, indicating that the relevant cells can traffic to the intestine after peripheral vrp-based vaccination. these studies leave several important questions unanswered. firstly, we used a homologous virus challenge. in nature, it is likely that hosts are repeatedly challenged with antigenically distinct noroviruses. however, the mouse norovirus strains identified so far fall into a single genogroup, gv, which likely represents a single serotype [ ] . in this way murine noroviruses identified to date may present less of a challenge for the immune system than hunv, which are distributed across genogroups and appear to evolve under antibody selection [ ] . in addition, we selected a strain of mnv that is cleared by wt mice. other strains persist for prolonged periods of up to days [ ] . it remains to be determined whether vaccination will be effective against persistent mnv strains. it is interesting that human noroviruses can persist beyond the time frame of usual clinical symptoms [ ] [ ] [ ] [ ] . longterm persistence might contribute to explaining the sporadic epidemics of infection in the absence of an animal reservoir. antigenic and pathogenetic complexity will likely be a major issue for the development of norovirus vaccines. the lack of comparable variation in mnv strains limits the utility of the mnv model for assessing immunity to antigenically distinct strains. perhaps this limitation will be overcome as additional strains of mnv are identified, sequenced, and studied. however, the fact that we observed partial cross protection between mnv and one hunv, and the demonstration that vaccination with many different vlps can enhance generation of cross reactive antibodies [ ] provide some encouragement. there are two ways in which murine norovirus infection may not represent the same biology as hunv infection. the first is the lack of diarrhea in mice infected with the strains of mnv used here. it is possible that the adaptive responses that clear mnv from the intestine demonstrated here are irrelevant to the responses that may prevent human disease. in this regard, it is important to note that studies of adult mice with rotaviruses (also an infection only model), have been important to our consider-ations of rotavirus vaccines [ ] . importantly, human studies may not reveal the mechanisms of effective immunity and are based on surrogate assays of immunity, since invasive sampling of tissues may be technically difficult. studies in piglets may be revealing since piglets develop diarrhea when infected with the hunv strain, hunov-hs [ ] . however, it is more difficult to study immune mechanisms in this system. thus, we are left with several imperfect systems for considering what one should seek in a hunv vaccine. our studies in mice argue for a vaccine that induces all aspects of the adaptive immune response, and that assays for cytotoxic lymphocyte responses to hunv infection may be an important surrogate assay for protection. the second aspect of murine norovirus infection that is of unknown relevance to human infection is the impressive capacity of mnv to infect lymph nodes draining the intestine (this paper and [ , , ] ). this may be related to the tropism of mnv for dendritic cells and macrophages [ , ] and likely reflects spread of mnv directly from the intestine, but may also reflect seeding of the mln from systemic sites. considering the distal ileum alone, b cells and mhc class i and b m were not required for live virus vaccination, and there was significant, but incomplete, protection in mhc class ii-/-mice ( figure b and c). consistent with this, studies of primary clearance showed that any single arm of the adaptive response was dispensable for ultimate control of primary infection in the intestine. however, vaccination-mediated control of infection in the mln, and clearance of primary infection from the mln [ ] , required b cells. this differential requirement for components of the immune response in different organs raises an important question about norovirus pathogenesis and lymphoid infection: are the cells infected in intestine and mln the same? differences in viral tropism in the two tissues might explain the differential requirement for b cells between ileum and mln, indicating the importance of future studies on the role of immunity in norovirus cell and organ tropism. viruses, viral stocks, vrps, plaque assays mnv strains mnv .cw or mnv .cw were used in all virus infections [ , , ] . two mutations (that result in changes in the encoded amino acids) distinguish the genomes of mnv .cw and mnv .cw [ ] . to generate a concentrated virus stock, raw . cells (atcc, manassas, va) were infected in vp-sfm media (gibco, carlsbad, ca) for days at a multiplicity of infection (moi) of . . supernatants were clarified by low-speed centrifugation for min at , rpm. virus was concentrated by centrifugation at uc for h at , rpm ( , g) in a sw rotor. viral pellets were resuspended in pbs and titered on raw . cells as previously described [ ] . type i lang reovirus was kindly provided by dr. terrence s. dermody (vanderbilt university, nashville, tn). plaque assays were performed as previously described [ ] with the following modifications. tissues were harvested into sterile, screw-top -ml tubes containing ml of -mm zirconia/silica beads (biospec products, bartlesville, ok) and stored at uc. to obtain viral titers in these tissues ml of complete dmem was added to each sample on ice and homogenized using a magna lyser (roche applied science, hague road, in) prior to plaque assay. the limit of detection was plaque forming units (pfu)/ml. all vrps were produced as previously described [ ] . briefly, orfs , and from mnv .cw and orf from lordsdale virus and chiba virus were each cloned into vrp expression vectors. following infection of bhk cells with vrps for h, culture supernatants were harvested and cells lysed. proteins were separated by sds-page and analyzed by western blot with polyclonal rabbit anti-mnv serum [ ] . vrp titers and efficient expression of recombinant protein were determined by immunofluorescence assay using mouse antisera generated from inoculation with respective antigens. cell lysates from mnv orf , chiba virus and lordsdale virus vrp-infected cell cultures were further purified to obtain vlps [ ] . raw . cells were maintained as previously described [ ] . monoclonal antibodies (mabs) specific to cd (yts . [ ] ), cd (h [ ] ) and sfr -dr (atcc hb- [ ] ) were produced from hybridoma cell lines in integra celline cl flasks (integra biosciences, ijamsville, md) using cd hybridoma media (gibco, carlsbad, ca) as previously described [ ] . all mice (or cage sentinel mice for mice deficient in antibody production) were tested by elisa for the presence of mnv antibody prior to experiments [ ] . all mice used in these studies were seronegative at the initiation of experiments. mice used in vaccination studies were immunized with pfu of mnv .cw [ ] , mnv .cw [ ] , or control type i lang reovirus per orally (p.o.) in ml of dmem containing % fetal bovine serum (hyclone, logan, ut) (cdmem). vrp immunizations were with . infectious units (iu) of each vrp expressing mnv .cw orf , orf , or orf individually or in groups of - vrps; chiba virus orf or lordsdale virus orf in ml or ml volume by footpad inoculation (into the subcutaneous space) [ ] on day and boosted on day . ha vrp and pbs immunizations in ml or ml volume by footpad inoculation [ ] on day and boosted on day served as controls for all vrp immunizations. mice were challenged with pfu of mnv .cw at specified times after boost and tissues harvested three days post-challenge. controls for vrp vaccination included pbs or vrps expressing hemagglutinin (ha) from a mouse adapted influenza a virus [ ] . pbs served as a control for ha vrp in these experiments in the event that ha vrp had a significant effect on mnv replication. ha vrp controls were not significantly different from pbs controls in all experiments and both are presented in all figures for completeness. rag -/-and all splenocyte donor mice were infected with pfu of mnv .cw p.o. in ml of cdmem. all other mice were infected with pfu mnv .cw p.o. in rag -/-mice two segments of the small intestine were harvested: a one inch section of the small intestine immediately distal to the pylorus of the stomach, (designated the duodenum/jejunum), and a one inch section of the small intestine immediately proximal to the cecum (designated the distal ileum). in all other mice the distal ileum and three mesenteric lymph nodes (mln) were harvested. with the exception of rag -/-mice (inoculated at - weeks of age) and aged wt mice (inoculated at months of age), all other mice were inoculated at - weeks of age. spleens were harvested from mice and single cell suspensions were generated [ ] . cells were counted and diluted in rpmi- media (sigma, saint louis, mo) supplemented with % fetal calf serum (hyclone, logan, ut), u penicillin/ml, mg/ml streptomycin, mm hepes (n- -hydroxyethylpiperazine-n - -ethanesulfonic acid), mm sodium pyruvate, mm -mercaptoethanol and mm l-glutamine (crpmi). in all adoptive transfer experiments, cells were injected into persistently infected rag -/-mice by intraperitoneal (i.p.) injection in . ml crpmi. for depletions in wt mice, mg of lymphocyte-depleting antibody or an isotype-matched control antibody [sfr -dr , igg b] was administered i.p. one day prior and one day after infection. for depletions in adoptive transfer experiments, depleting antibodies were administered to rag -/-recipients as described above with one dose one day prior to splenocyte transfer and a second dose on the day of trnsfer. the efficacy of lymphocyte depletion in both sets of depletion experiments was monitored by flow cytometric analysis of splenocytes at the end of the experiment. elisa to detect binding of polyclonal anti-serum or fecal extract-derived antibody to purified mnv virions or mnv vlps was performed as previously described [ , ] . all data were analyzed using graphpad prism software (graphpad software, san diego, ca). viral titer data were analyzed with the nonparametric mann-whitney test. all differences not specifically stated to be significant were insignificant (p. . ). noroviruses everywhere: has something changed? molecular epidemiology of ''norwalk-like viruses'' in outbreaks of gastroenteritis in the united states epidemiologic and molecular trends of ''norwalk-like viruses'' associated with outbreaks of gastroenteritis in the united states human caliciviruses norovirus activity-united states immunogenic properties of rabbit haemorrhagic disease virus structural protein vp expressed by a recombinant baculovirus: an efficient vaccine a human norovirus-like particle vaccine adjuvanted with iscom or mlt induces cytokine and antibody responses and protection to the homologous gii. human norovirus in a gnotobiotic pig disease model feline calicivirus murine noroviruses comprising a single genogroup exhibit biological diversity despite limited sequence divergence biological properties of norwalk agent of acute infectious nonbacterial gastroenteritis clinical immunity in acute gastroenteritis caused by norwalk agent comparison of three agents of acute infectious nonbacterial gastroenteritis by cross-challenge in volunteers multiplechallenge study of host susceptibility to norwalk gastroenteritis in us adults viral shedding and fecal iga response after norwalk virus infection detection of norwalk virus or norwalk-like virus infections in finnish infants and young children immunity to calicivirus infection evidence of immunity induced by naturally acquired rotavirus and norwalk virus infection on two remote panamanian islands human susceptibility and resistance to norwalk virus infection norwalk virus infection and disease is associated with abo histo-blood group type expression, self-assembly, and antigenicity of the norwalk virus capsid protein human enteric caliciviridae: the complete genome sequence and expression of virus-like particles from a genetic group ii small round structured virus expression and self-assembly of recombinant capsid protein from the antigenically distinct hawaii human calicivirus characterization of toronto virus capsid protein expressed in baculovirus cellular and humoral immunity following snow mountain virus challenge humoral, mucosal, and cellular immune responses to oral norwalk virus-like particles in volunteers cytokine and antibody responses in gnotobiotic pigs after infection with human norovirus genogroup ii. (hs strain) stat -dependent innate immunity to a norwalk-like virus replication of a norovirus in cell culture reveals a tropism for dendritic cells and macrophages cleavage map and proteolytic processing of the murine norovirus nonstructural polyprotein in infected cells the structure of antibody neutralized murine norovirus and unexpected differences to virus like particles murine norovirus infection is associated with histopathological changes in immunocompetent hosts, but clinical disease is prevented by stat -dependent interferon responses chronic excretion of a norovirus in a child with cartilage hair hypoplasia (chh) characteristics of human calicivirus enteritis in intestinal transplant recipients risk groups for clinical complications of norovirus infections: an outbreak investigation evolution of human calicivirus rna in vivo: accumulation of mutations in the protruding p domain of the capsid leads to structural changes and possibly a new phenotype norovirus outbreak in a pediatric oncology unit pathogenesis of a genogroup ii human norovirus in gnotobiotic pigs pathogenesis and immune responses in gnotobiotic calves after infection with human norovirus (hunov) genogroup ii. -hs strain antibody is critical for the clearance of murine norovirus infection processing map and essential cleavage sites of the nonstructural polyprotein encoded by orf of the feline calicivirus genome replicon-helper systems from attenuated venezuelan equine encephalitis virus: expression of heterologous genes in vitro and immunization against heterologous pathogens in vivo vaccine efficacy in senescent mice challenged with recombinant sars-cov bearing epidemic and zoonotic spike variants virus subversion of the mhc class i peptide-loading complex normal development of mice deficient in beta m, mhc class i proteins, and cd + t cells depletion of cd t cells in major histocompatibility complex class ii-deficient mice a b cell-deficient mouse by targeted disruption of the membrane exon of the immunoglobulin mu chain gene role of t lymphocytes in rat , , -trinitrobenzene sulphonic acid (tnbs) induced colitis: increased mortality after gammadelta t cell depletion and no effect of alphabeta t cell depletion treatment with either anti-cd or anti-cd monoclonal antibodies blocks alphabeta t cell-mediated rejection of intestinal allografts in mice perforin-mediated target-cell death and immune homeostasis mucosal and systemic adjuvant activity of alphavirus replicon particles mechanisms of gii. norovirus persistence in human populations prolonged norovirus shedding in infants ,or = months of age with gastroenteritis natural history of human calicivirus infection: a prospective cohort study norwalk virus infection of volunteers: new insights based on improved assays norwalk virus shedding after experimental human infection multivalent norovirus vaccines induce strong mucosal and systemic blocking antibodies against multiple strains immunity and correlates of protection for rotavirus vaccines persistent infection with and serologic cross-reactivity of three novel murine noroviruses pathology of immunodeficient mice with naturally occurring murine norovirus infection systemic, mucosal, and heterotypic immune induction in mice inoculated with venezuelan equine encephalitis replicons expressing norwalk virus-like particles therapy with monoclonal antibodies by elimination of t-cell subsets in vivo myosin-induced acute myocarditis is a t cellmediated disease sfr -dr , a monoclonal antibody with hla-dr specificity critical role of cd t cells in an antibody-independent mechanism of vaccination against gamma-herpesvirus latency we thank martha collier for assistance in packaging vrps, lindsay droit for technical assistance in screening mice for mnv status prior to the initiation of experiments at washington university, darren kreamalmayer for his truly outstanding expertise in the breeding of animals at washington university, and dr. larissa thackray and dr. christiane wobus for critical reading of this manuscript. key: cord- -q eijunp authors: casadevall, arturo; pirofski, liise-anne title: the ebola epidemic crystallizes the potential of passive antibody therapy for infectious diseases date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: q eijunp nan in , an ebola virus epidemic began in west africa and it has affected over , individuals [ ] . unlike earlier ebola outbreaks that were largely confined to isolated villages, this one struck in populated cities and was sustained through person-to-person transmission. at the end of , the epidemic remained uncontrolled [ ] . currently, there are no drugs to treat ebola, but, in the urgency and emergency triggered by the epidemic, two types of ab-based therapies have been used: convalescent sera from patients who have recovered and a mab cocktail known as zmapp produced in plants [ ] . although at the time of this writing the news releases on the efficacy of ab-based therapies have been largely favorable, the evidence is anecdotal and firm conclusions cannot be made until formal clinical trials are done, such as those encouraged by the world health organization [ ] . therefore, we will refrain from commenting on the efficacy of ab therapies against ebola virus and will focus, instead, on how this concurrent epidemic brings into focus the promise of this therapy while also highlighting the difficulties involved in reintroducing antibodies for the therapy of infectious diseases. antibodies are molecules that can exert antimicrobial activity through different mechanisms that include promoting phagocytosis and ab-dependent cellular cytotoxicity (adcc), activating complement, neutralizing viruses and toxins, modulating inflammation, and affecting microbial metabolism (reviewed in [ ] ). this diversity of function makes it possible to tailor ab therapies to specific diseases depending on what might be needed for protection. for example, antibodies overcome the anti-phagocytic properties of encapsulated microbes, such as streptococcus pneumoniae and cryptococcus neoformans, by promoting phagocytosis through fc receptors while the toxin-neutralizing properties inhibit the deleterious effects of toxigenic bacteria, such as bacillus anthracis. the versatility associated with ab therapies is further amplified by the availability of different isotypes that differ in serum half-life and effector function, with the latter partially dependent on fc receptors (fcr) that can amplify or reduce inflammatory responses. in this regard, the efficacy of broadly neutralizing antibodies to hiv is dependent on fcrs [ ] and vaccine-elicited subclass selection, which could drive optimal fcr effector function of non-neutralizing antibodies against hiv [ ] . although several types of ab therapies have been highly successful, it is noteworthy that, to date, available therapies have not taken full advantage of either ab or fcr structural and functional diversity. historically, ab preparations for therapy were obtained by immunizing animals or from recovered individuals as convalescent sera. serum therapy for pneumococcus relied largely on horse immune serum. animal sera were effective, but they could also elicit allergic reactions or the phenomenon of "serum sickness." human convalescent sera were used for viral diseases specific to humans, but such preparations were in short supply, and, in retrospect, carried the risk of inadvertent transmission of blood borne diseases. nevertheless, in the latter half of the th century, immune gamma globulin preparations were recommended in certain clinical settings for the prevention and therapy of diseases caused by hepatis b virus, cytomegalovirus, rabies virus, tetanus toxin, and botulinum toxin, among others. since the mid- s, monoclonal antibodies have been available and tools now exist for reducing the antigenicity of animal antibodies through humanization and for the generation of fully human abs. given that abs are natural products, they have little inherent toxicity and the ability to reduce or eliminate their antigenicity means that side effects associated with serum therapy are significantly diminished. perhaps the greatest difference between ab and conventional antimicrobial therapies is the exquisite specificity of most immunoglobulin molecules for their targets. unlike most antimicrobial drugs that function indiscriminately against multiple species, antibodies target a single species and, often, a single serotype or variant within a species. this is a double-edged sword for the development of ab therapies. great specificity has the advantage that it targets only the offending microbe. for example, given that there is increasing evidence that antibiotic-induced disruptions of the microbiota are associated with deleterious effects in the treatment of bacterial diseases, the high specificity of ab-based therapies offers a tremendous advantage. regarding the limitation that highly specific abs can fail to bind highly related variants, particularly for viruses, there are two strategies to overcome this problem: the development of abs that target broadly neutralizing epitopes, as has now been demonstrated for hiv [ ] , dengue [ ] , and influenza viruses [ ] , and the generation of ab cocktails composed of abs against multiple serotypes or variants, as has been done for rabies virus [ ] and clostridium difficile toxin [ ] . similarly, although sera from patients who have recovered from ebola virus disease can exhibit persistent neutralizing activity [ ] , antibodies against different ebola virus strains often do not cross-react with other strains [ ] . in the pre-antibiotic era, this was addressed by using serotype-specific sera that required isolating and typing the strain before instituting therapy. the problem is especially acute for mabs, which recognize a single epitope, but this limitation can be bypassed by creating cocktails targeting various subtypes, although this increases the cost of research and development. mab cocktails can also be designed to neutralize different targets with the goal of achieving higher efficacy through synergy. it is noteworthy that the experimental mab treatment of ebola, zmapp, consists of a cocktail of three mouse-human chimeric abs directed to the viral glycoprotein [ ] . for reasons that are not fully understood, ab therapies work best when given in a prophylactic mode (e.g., before infection) or early in the course of disease. for example, serum therapy for the treatment of pneumococcal pneumonia was effective only when given within the first three days of symptoms. in contrast, antimicrobial agents are often effective in established infection and disease. one proposed explanation for this limitation is that abs work best in neutralizing the infective inoculum and cannot cope with the high microbial burdens of established infection [ ] . an alternative explanation is that abs work by altering the inflammatory response and once inflammation is established that it is difficult for abs to exert their protective functions [ ] . for viral diseases, the reduced efficacy of abs in treatment mode could reflect a molar imbalance between ab molecules and increasing numbers of viral particles, as well as the requirement for cell-mediated immunity to eradicate established infection. whatever the explanation, the need for early administration is a limitation for therapy since this means a potential lack of efficacy in the setting of advanced disease. however, in contrast to antimicrobial therapy, which mediates protection only while the drug is pharmacologically available, the administration of ab results in a state of immediate immunity that, combined with the long half-life of certain immunological molecules, can confer a long-standing state of reduced susceptibility. in contrast to conventional antimicrobial therapies, ab therapies can be developed extremely quickly and, sometimes, in the midst of an epidemic. for example, a potentially clinically useful mab against the coronavirus responsible for the severe acute respiratory syndrome (sars) was rapidly generated, in months [ ] , but was not used because the epidemic was contained. an even more expedient strategy is to use convalescent serum from survivors in an epidemic as a source of antibodies to treat those at risk and with concurrent disease. in the past, convalescent sera was used to treat influenza and ebola virus disease [ , ] . today, convalescent sera from survivors of ebola virus disease has reportedly been used to treat cases, although details of how the sera have been used and evidence of their efficacy is anecdotal. nonetheless, we note that there are at least three established mechanisms of antibody function that could benefit patients with ebola: direct neutralization of ebola virus, enhancement of ebola virus uptake and/or killing by phagocytes, and modulation of ebola-virus-induced inflammatory response. regarding the latter, it is of interest that immunomodulation has been proposed as an intervention for ebola virus [ ] and anecdotal reports suggest that control of ebola-virus-induced cytokine storm might be beneficial therapeutically. despite having pioneered the use of ab therapy, the use of ab-based therapies in the field of infectious diseases remains a work in progress despite enormous technological progress in ab engineering and production. in contrast to oncology and rheumatology, where ab therapies are now common, there are only two licensed mabs for infectious diseases: one for the prevention of respiratory syncytial virus infection and the other for the therapy of anthrax. hence, ab-based therapies are severely underdeveloped for the treatment of infectious diseases. although the causes for this are complex, we identify five factors that are, in some cases, interrelated and, yet, all work in synergy to hinder the widespread reintroduction of ab therapies to this field. i. cost. ab therapies are generally significantly more expensive than small molecules because they must be produced in animals or cell culture. immunoglobulins are proteins that require refrigeration for storage, which, in turn, increases their cost. however, when one considers the costs of antibiotic-associated colitis, damage to the microbiota, resistance in non-targeted organisms, and superinfections resulting from non-specific therapy, the cost accounting may be more favorable for ab therapy. ii. specificity. as alluded to above, the specificity of ab therapies means that they are pathogen specific and often target a subgroup of organisms within a pathogenic species. this limitation can be bypassed by creating mab cocktails, but that, in turn, increases the complexity of production and cost. in addition, cocktails could face more complicated regulatory hurdles than therapies composed of a single active agent. despite this, we note that mab cocktails are being developed against ebola virus, c. difficile colitis, and for the prevention of rabies. iii. market size and profitability. the antimicrobial spectrum of a drug combined with the prevalence of disease caused by the organisms for which it has efficacy determines its market size. this law of pharmaceutical economics has favored the development of broadspectrum therapies that are responsible for widespread resistance and deleterious effects of ab therapies on host microbiota. hence, the exquisite specificity of mab-based drugs de facto means smaller market sizes, which, in turn, increases costs, reduces profitability, and makes these reagents suitable for treating specific diseases unattractive to industry. iv. the availability of existing therapies. for many infectious diseases, the current availability of effective therapy means that any attempt to develop ab therapy involves establishing the advantage of such therapies either alone or in combination with existing therapies, and this greatly complicates clinical development and marketing. in other fields, where no therapy was available, the development of ab therapies was easier because it provided something new. however, this situation may change since the declining efficacy of conventional antimicrobial therapies due to widespread resistance could create windows for the development of ab-based therapies. v. underdevelopment in diagnostics. the specificity of ab therapies means that they will be effective only in situations where a precise microbial diagnosis is available. for bacterial diseases, the widespread availability of broad-spectrum antimicrobial drugs with relatively little toxicity created a culture of empiricism that has translated into underdevelopment in diagnostics. consequently, microbial culture has remained the gold standard for the diagnosis of many infectious diseases for decades despite the availability of new technologies, such as nucleic acid amplification, that could have led to more rapid diagnosis. fortunately, the situation is changing. an increasing recognition of the problems associated with broadspectrum therapy combined with declining efficacy of such drugs due to widespread resistance is leading to the development and use of rapid diagnostic tools that could support the use of ab-based therapies. the ongoing ebola epidemic provides a special lens for understanding the promise and roadblocks to the development of ab-based therapies for infectious diseases, as well as ethical and cultural considerations that pertain to conducting clinical trials in the midst of an epidemic in under-resourced countries. precedent for the use of convalescent sera during epidemics signals promise that trials of sera can be conducted even as the current ebola epidemic rages [ ] . we note that if a stockpile of effective ab preparations against ebola had been available early in the current outbreak, the administration of these preparations to contacts might have provided them with immediate immunity, possibly resulting in early containment of the epidemic and prevention of thousands of deaths. the fact that such preparations were not available in early reflects many factors, including cost, uncertainty about efficacy, lack of available markets, and the erroneous assessment, based on prior epidemics, that the threat would be small and localized. the suggestion has also been made that such therapies remain underdeveloped because they are unattractive to the pharmaceutical industry and to academic investigators, who may view them as not fitting with the current drug development model or not being cutting-edge science, respectively [ ] . however, the fact that ab-based therapies for ebola are now in development, clinical trials of sera are being designed, and compassionate use of these therapies has been employed in the midst of the current emergency suggests that clinically useful preparations may be identified and available shortly. for the near horizon, it is likely that ab-based therapies will continue to make incremental advances in the repertoire of anti-infective strategies. such areas include infectious diseases caused by drug-resistant organisms for which conventional therapies have lost efficacy and diseases for which there is no available therapy, such as ebola. for example, the widespread use of broad-spectrum therapy has been associated with an increase in c. difficile colitis creating an opportunity for the development of toxin-neutralizing ab therapy [ ] . for the far horizon, we are optimistic that ab-based therapies will be widely reintroduced as anti-infective agents given their inherent advantages in being natural products with low toxicity, high specificity, and established efficacy. continued technological advances in the form of more efficient production strategies and alternative production sources, such as expression in plants, that could lower costs, combined with new therapeutic needs and better diagnostics, will make their use more attractive. return to the past: the case for antibody-based therapies in infectious diseases update: ebola virus disease epidemic-west africa plant-based vaccines against viruses hyperimmune serum from healthy vaccinated individuals for ebola virus disease? a new synthesis for antibody-mediated immunity broadly neutralizing anti-hiv- antibodies require fc effector functions for in vivo activity polyfunctional fceffector profiles mediated by igg subclass selection distinguish rv and vax vaccines broadly neutralizing antibodies present new prospects to counter highly antigenically diverse viruses recognition determinants of broadly neutralizing human antibodies against dengue viruses broadly protective monoclonal antibodies against h influenza viruses following sequential immunization with different hemagglutinins first administration to humans of a monoclonal antibody cocktail against rabies virus: safety, tolerability, and neutralizing activity treatment with monoclonal antibodies against clostridium difficile toxins profile and persistence of the virus-specific neutralizing humoral immune response in human survivors of sudan ebolavirus (gulu) serologic cross-reactivity of human igm and igg antibodies to five species of ebola virus perspective: hypothesis: serum igg antibody is sufficient to confer protection against infectious diseases by inactivating the inoculum antibody-mediated regulation of cellular immunity and the inflammatory response development and characterization of a severe acute respiratory syndrome-associated coronavirusneutralizing human monoclonal antibody that provides effective immunoprophylaxis in mice hark back: passive immunotherapy for influenza and other serious infections treatment of ebola hemorrhagic fever with blood transfusions from convalescent patients. international scientific and technical committee a practical treatment for patients with ebola virus disease ebola raises profile of blood-based therapy which are the antibodies to watch in key: cord- -boto h x authors: danthi, pranav; pruijssers, andrea j.; berger, angela k.; holm, geoffrey h.; zinkel, sandra s.; dermody, terence s. title: bid regulates the pathogenesis of neurotropic reovirus date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: boto h x reovirus infection leads to apoptosis in both cultured cells and the murine central nervous system (cns). nf-κb-driven transcription of proapoptotic cellular genes is required for the effector phase of the apoptotic response. although both extrinsic death-receptor signaling pathways and intrinsic pathways involving mitochondrial injury are implicated in reovirus-induced apoptosis, mechanisms by which either of these pathways are activated and their relationship to nf-κb signaling following reovirus infection are unknown. the proapoptotic bcl- family member, bid, is activated by proteolytic cleavage following reovirus infection. to understand how reovirus integrates host signaling circuits to induce apoptosis, we examined proapoptotic signaling following infection of bid-deficient cells. although reovirus growth was not affected by the absence of bid, cells lacking bid failed to undergo apoptosis. furthermore, we found that nf-κb activation is required for bid cleavage and subsequent proapoptotic signaling. to examine the functional significance of bid-dependent apoptosis in reovirus disease, we monitored fatal encephalitis caused by reovirus in the presence and absence of bid. survival of bid-deficient mice was significantly enhanced in comparison to wild-type mice following either peroral or intracranial inoculation of reovirus. decreased reovirus virulence in bid-null mice was accompanied by a reduction in viral yield. these findings define a role for nf-κb-dependent cleavage of bid in the cell death program initiated by viral infection and link bid to viral virulence. tissue injury in response to infections by many viruses occurs as a consequence of apoptosis. multiple studies using animal models of viral disease demonstrate a correlation between apoptotic potential and disease severity [ , , , ] . these observations highlight proapoptotic signaling following virus infection as an attractive target for antiviral therapy. however, despite its central importance in viral pathogenesis, gaps in knowledge about the identity of death signaling pathways that modulate virus-induced apoptosis in vivo, along with an incomplete understanding of how these signaling cascades are activated during virus infection, have hampered the deployment of this strategy for treatment of viral disease. mammalian reoviruses injure infected cells via apoptosis both in culture and in tissues of infected animals. as such, studies of these viruses have contributed to an understanding of how virus infection culminates in apoptotic cell death. unlike other viruses in which virulence correlates with cell-death capacity, the identity of viral and cellular factors that regulate reovirus-induced apoptosis in cell culture are for the most part known [ , , , , , , ] . moreover, many of these intermediaries also modulate reovirus-induced apoptosis in vivo [ , , , ] . studies using reassortant reoviruses [ , ] , ectopically expressed proteins [ ] , and genetically engineered reovirus mutants [ , ] highlight a critical role for reovirus outer-capsid protein m in apoptosis induction. collectively, these studies indicate that prodeath signaling evoked by m occurs subsequent to membrane penetration but prior to synthesis of viral rna or protein [ , , , ] . classical death-receptor-mediated extrinsic apoptotic pathways stimulated by reovirus infection execute the death response [ ] . treatment of cells with soluble trail receptors or expression of a dominant-negative form of fas-associated death domain (fadd) protein blocks apoptosis, demonstrating that signaling via death receptors is required for execution of the apoptotic program [ ] . in keeping with the function of extrinsic apoptotic signaling in reovirus infection, caspase- activation [ ] and bid cleavage [ ] are observed in cells infected with reovirus [ ] . reovirus infection also stimulates intrinsic apoptotic pathways, as evidenced by release of cytochrome c and smac/diablo from the mitochondria and activation of caspase- [ , , , , ] . concordantly, reovirus-induced apoptosis is dampened by over-expression of bcl- [ ] , which inhibits mitochondrial apoptotic pathway activation [ ] . bid is a proapoptotic bh -only member of the bcl- family that functions to link the extrinsic apoptotic pathway and the mitochondrial amplification loop of the intrinsic pathway. following death-receptor signaling, cytoplasmically resident bid is cleaved by activated caspase- to generate a truncated form of bid known as tbid [ ] . tbid translocates to the mitochondria and triggers the release of cytochrome c and activation of the core mitochondrial apoptotic machinery [ , ] . it is not known whether bid plays a functional role in apoptosis induction by reovirus. moreover, the relationship between apoptosis effector pathways and early events in viral replication are not understood. in addition to these classical apoptotic pathways, the innate immune response transcription factor, nf-kb, is activated following reovirus infection [ ] . nf-kb activation by reovirus depends on the viral m protein and can be accomplished by genome-deficient reovirus particles [ , , ] . blockade of nf-kb signaling using chemical inhibitors or cell lines genetically deficient in nf-kb p , nf-kb p /rela, ikb kinase (ikk)-a, or ikk adaptor ikkc/nemo significantly diminishes reovirus-induced apoptosis [ , ] . consistent with these findings, activation of nf-kb occurs within the first few hours of reovirus infection and precedes the biochemical and morphological hallmarks of apoptotic cell death [ , ] . these observations suggest that nf-kb couples m -mediated events to the cellular apoptotic machinery. although regulation and function of nf-kb has been extensively studied, the precise relationship between nf-kb and the cell-death machinery remains undefined. in this study, we examined the function of cellular apoptosis regulator bid using genetically deficient murine embryo fibroblasts (mefs) and mice. we found that while bid is dispensable for reovirus replication in cell culture, its function is required for reovirus-induced apoptosis. blockade of nf-kb signaling, which diminishes apoptosis induction by reovirus [ , ] , prevents cleavage of bid. in comparison to wild-type mice, bid-deficient mice display diminished susceptibility to reovirus-induced cns disease following either peroral (po) or intracranial (ic) inoculation. attenuated reovirus virulence in the absence of bid is associated with decreased reovirus replication in the murine cns. these results define an important role for bid in virusinduced apoptosis and disease and illuminate bid-dependent prodeath signaling as a viable target for antiviral therapy. reovirus infection of hek epithelial cells leads to a biphasic loss of full-length (fl) bid [ ] . since a mitochondrial amplification loop through bid is required for apoptosis only in some cell types, such as hepatocytes [ , ] , it is not known if bid is cleaved in all cell types infected by reovirus. in addition, although calpains [ , ] , caspases [ , , , ] , and cathepsins [ , , , ] can mediate bid cleavage and have been implicated in apoptosis induction by reovirus [ , , ] , the precise identity of the protease that generates tbid following reovirus infection is not known. to determine whether tbid is generated following reovirus infection of fibroblasts, and to define the mechanism of bid cleavage following reovirus infection, we infected murine l fibroblasts with reovirus strain type dearing (t d) and monitored levels of fl bid and tbid over h ( figure a ). while levels of fl bid remained unchanged in mock-infected cells, we observed loss of fl bid between and h post-infection. decreased levels of fl bid correlated with a corresponding increase in levels of tbid. to determine whether the generation of tbid results in activation of the mitochondrial loop of the intrinsic apoptotic pathway, we assessed viruses injure host tissues by activating signaling pathways that trigger cell death by a process called apoptosis. hence, blockade of apoptosis may serve as a useful strategy to dampen the severity of viral disease. however, deployment of such a strategy requires identification of host signaling networks that control cell death and a detailed molecular blueprint of how these pathways are activated by a virus. in this study, we used mammalian reovirus, an important experimental model for studies of viral encephalitis, to elucidate how cell death pathways are activated following viral infection and whether these signaling cascades influence the capacity of a virus to produce lethal cns disease. we found that bid, a host regulator of cell death, influences apoptosis induction by reovirus. moreover, bid is required for efficient reovirus replication in the cns and modulates reovirus neurological disease. these findings highlight bid as a critical regulator of viral pathogenesis and illuminate a potential new target for development of antiviral therapeutics. (a) l cells were adsorbed with pbs (mock) or reovirus t d at an moi of pfu/cell. following incubation at uc for the indicated intervals, whole cell extracts were prepared, resolved by sds-page, and immunoblotted using antisera specific for bid, actin, or procasapse- . (b) l cells were adsorbed with reovirus t d at an moi of pfu/cell. following incubation at uc for the indicated intervals in the presence of or mm of z-ietd-fmk, whole cell extracts were prepared, resolved by sds-page, and immunoblotted using antisera specific for bid, actin, or procasapse- . protein bands are indicated on the right. a non-specific band is indicated by an asterisk (*). doi: . /journal.ppat. .g levels of procaspase- as a surrogate for the formation of the caspase- -containing apoptosome ( figure a ). in a time frame consistent with cleavage-induced generation of tbid, we observed a decrease in procaspase- levels in reovirus-infected cells. these findings suggest that following reovirus infection of murine fibroblasts, bid serves to activate the mitochondrial apoptotic pathway. the adaptor molecule fadd is required for cleavage of bid following reovirus infection of hek cells [ ] . based on these data, we hypothesized that caspase- activity as a consequence of extrinsic prodeath signaling, is required for cleavage and activation of bid. to test this hypothesis, we assessed the capacity of reovirus to mediate bid cleavage in l cells treated with caspase- inhibitor z-ietd-fmk ( figure b ). as anticipated, bid cleavage was not observed in mock-infected cells or mock-infected cells treated with z-ietd-fmk (data not shown). although tbid was generated at - h following reovirus infection of vehicle-treated cells, reovirus failed to efficiently induce activation of bid in z-ietd-fmk-treated cells until h post-infection, providing evidence that reovirus evokes cleavage of bid via caspase- . in response to a variety of death agonists, bid amplifies death signaling by linking the extrinsic (caspase- ) and intrinsic (caspase- ) apoptotic pathways [ ] . since our findings with reovirus parallel this pattern, our results suggest that bid functions similarly following reovirus infection by linking the death-receptor and mitochondrial apoptotic pathways. signaling via the intrinsic pathway is essential for reovirusinduced apoptosis [ ] . this observation, along with the dependence of mitochondrial apoptotic signaling on cleavage of bid, suggests that bid serves an essential function in reovirusinduced apoptosis. to directly test whether bid is required for apoptosis induction following reovirus infection, we compared reovirus-induced apoptosis in wild-type and bid-deficient mefs. for these experiments, mefs were infected with t d, and apoptosis was assessed by chemiluminescent measurement of the activity of caspase- and caspase- , which serve as effector caspases for both the extrinsic and intrinsic apoptotic pathways ( figure a ). in comparison to mock-infected cells, infection of wild-type cells resulted in a significant increase in caspase- / activity at h post-infection. since mefs are poorly permissive for reovirus infection [ ] , staining of infected cells by indirect immunofluorescence indicated that adsorption with pfu/cell of t d resulted in infection of only , % of cells at h post infection (data not shown). despite a low frequency of infection, this moi resulted in an , -fold increase in caspase- / activity. when infection was initiated at pfu/cell, , % cells were infected (data not shown), and caspase- / activity increased , fold. in contrast, infection of bid-deficient cells resulted in minimal caspase- / activity following infection at either moi. increase in caspase- / activity following treatment of each cell type with a broad-spectrum protein kinase inhibitor, staurosporine, was equivalent (, -fold), demonstrating that although bid-deficient cells possess functional death-signaling pathways, they resist apoptosis induction by reovirus. as an alternative means to quantify apoptosis, we compared wild-type and bid-deficient mefs for the onset of morphological characteristics of apoptosis following reovirus infection using an acridine orange (ao) staining assay ( figure b ). infection of wildtype cells resulted in a significant increase in the fraction of apoptotic cells at h post-infection with % and % of the cells exhibiting apoptotic features at mois of and pfu per cell, respectively. in contrast, bid-deficient cells infected with t d at either moi displayed levels of apoptosis equivalent to mock-infected cells, , %. similar results were obtained following infection with another apoptosis-proficient reovirus strain, t sa+ (data not shown). these data indicate that bid is required for apoptosis induction following reovirus infection. to determine whether decreased apoptosis in bid-deficient cells is attributable to alterations in reovirus infection in the absence of bid, we compared reovirus infectivity in wild-type and biddeficient cells using an indirect immunofluorescence staining assay ( figure c ). an equivalent proportion of reovirus antigen-positive cells was detected at h post-adsorption of wild-type and biddeficient cells. these data indicate that reovirus is capable of initiating infection in bid-deficient cells. to determine whether reovirus completes a full infectious cycle in bid-deficient cells, wild-type and bid-deficient cells were adsorbed with t d, and viral titers were determined by plaque assay at , , , and h after infection ( figure d ). reovirus replicated with similar kinetics and produced equivalent yields in wild-type and biddeficient cells. thus, the failure of bid-deficient cells to undergo apoptosis in response to reovirus is not a consequence of diminished reovirus infection of these cells. we conclude that bid is a key regulator of reovirus-induced apoptotic cell death. the identification of an essential role for bid in apoptosis induction following reovirus infection allowed us to examine the relationship between nf-kb activation and bid cleavage. to determine whether bid is required for activation of nf-kb following reovirus infection, we compared reovirus-induced nf-kb activation in wild-type and bid-deficient cells using a reporter assay. wild-type and bid-deficient mefs were transfected with an nf-kb-luciferase reporter plasmid and infected with reovirus. analogous to treatment with tnfa, a control nf-kb agonist, reovirus infection resulted in equivalent (, -to -fold) activation of nf-kb-driven gene expression in wild-type and bid-deficient cells ( figure a ). these results indicate that bid is dispensable for nf-kb activation following reovirus infection and suggest that either reovirus-induced nf-kb activation occurs prior to bid cleavage or that nf-kb activation and bid cleavage occur in parallel but independent pathways that both function in apoptosis induction by reovirus. to determine whether cleavage-induced bid activation is dependent on nf-kb, we examined bid cleavage in cells lacking p /rela, an nf-kb subunit required for apoptosis induction following reovirus infection [ ] . infection of wild-type mefs with reovirus results in generation of tbid at - h after infection ( figure b ). in contrast, infection of p /rela-deficient mefs with reovirus did not lead to tbid generation even though efficient viral replication is observed in these cells [ ] . treatment of both wild-type and p /rela-deficient mefs with apoptotic agonists tnfa and cycloheximide resulted in efficient cleavage of bid, indicating that cell-death pathways leading to bid cleavage are intact in both cell types. these findings suggest that cleavage and activation of bid following reovirus infection requires nf-kb and place bid cleavage subsequent to nf-kb signaling in response to reovirus infection. moreover, since bid amplifies death responses from the extrinsic apoptosis pathway by activating the mitochondrial loop, these findings suggest that death-receptor signaling during reovirus infection occurs in an nf-kb-dependent manner. apoptosis-signaling pathways involving death receptors dr and dr and death ligand trail, as well as fas and fasl, have been implicated in apoptosis induction by reovirus [ , , ] . however, it is not known which of these pathways mediates cleavage-induced activation of bid. it is also not understood whether nf-kb regulates the activation of these pathways. since upregulation of fas following reovirus infection is dependent on prodeath signaling via c-jun n terminal kinase (jnk) [ ] , and because jnk is activated via a mechanism distinct from nf-kb following reovirus infection [ ] , we focused our efforts on assessing the regulation and function of death-receptor signaling via trail following reovirus infection. for these studies, we assessed the capacity of reovirus to induce apoptosis in mefs lacking trail-r, the only known receptor for trail on murine cells [ , , ] (figure ). in comparison to mock infection, t d infection of wild-type cells resulted in an moi-dependent , -to -fold increase in caspase- / activity at h post-infection ( figure a ). although t d infection of trail-r-deficient cells also resulted in an increase in caspase- / activity in comparison to mock-infection, the magnitude of this increase was only , -to -fold. assessment of apoptosis in wild-type and trail-rdeficient mefs using ao staining also showed an increase in apoptosis both in wild-type and trail-r-deficient cells in comparison to mock-infected cells ( figure b ). however, a substantially greater fraction of wild-type cells showed morphologic features of apoptosis in comparison to trail-r-deficient cells infected at equivalent moi, suggesting that efficient induction of apoptosis by reovirus requires trail-r. t d displayed comparable replication kinetics and produced equivalent yields in wild-type and trail-r-deficient cells ( figure c ). thus, figure . bid is required for apoptosis induction following reovirus infection. (a) wild-type or bid-deficient mefs were adsorbed with t d at the mois shown. after incubation at uc for h, caspase- / activity in cell lysates was determined. results are expressed as the mean ratio of caspase- / activity from infected cell lysates to that from mock-infected cells for triplicate samples. error bars indicate sd. *, p, . as determined by student's t-test relative to wild-type mefs infected at an equivalent moi. cells were treated with mm staurosporine (sts) for h as a control. (b) wild-type or bid-deficient mefs were adsorbed with t d at the mois shown. after incubation at uc for h, cells were stained with ao. results are expressed as the mean percentage of cells undergoing apoptosis for three independent experiments. error bars indicate sd. *, p, . as determined by student's t-test relative to wild-type mefs infected at an equivalent moi. (c) wild-type and bid-deficient mefs were adsorbed with particles/ cell of t d. after incubation at uc for h, cells were visualized by immunostaining with polyclonal reovirus-specific antiserum, followed by incubation with alexa -labeled anti-rabbit igg. reovirus-infected cells were quantified by counting fluorescent cells. results are expressed as mean fluorescent focus units (ffu) per field for triplicate samples. error bars indicate sd. (d) wild-type and bid-deficient mefs were adsorbed with t d at an moi of pfu/cell. the inoculum was removed, and cells were incubated at uc for the times shown. viral titers were determined after two cycles of freeze-thaw by plaque assay using l cells. results are presented as mean titers from three independent experiments. error bars indicate sd. doi: . /journal.ppat. .g differences in the apoptotic potential of reovirus in wild-type and trail-r-deficient cells are not associated with differences in reovirus growth in these cells. to determine whether reovirus-induced cleavage of bid is dependent on signaling via trail-r, we monitored bid cleavage following infection of trail-r-deficient cells ( figure d ). at h post-infection of wild-type cells with t d, fl bid was cleaved to generate tbid. in contrast, fl bid was not cleaved in t d-infected trail-r-deficient cells. while the apparent difference in the levels of fl bid in wild-type and trail-rdeficient cells was not reproducible, we consistently observed that levels of fl bid remained unchanged in trail-r-deficient cells following reovirus infection. these data indicate that trail-r contributes to the induction of apoptosis by reovirus and suggest that cleavage of bid following reovirus infection is dependent on trail-r signaling. reovirus virulence correlates with its capacity to cause apoptosis [ , , , , , ] . given the central role of bid in apoptosis induction by reovirus in cell culture, we hypothesized that reovirus apoptosis and virulence would be diminished in the absence of bid. to test this hypothesis, we inoculated two-day-old wild-type and bid-deficient mice perorally with a highly virulent, enteric, neurotropic reovirus strain, t sa+ [ ] , and monitored infected animals for signs of neurological disease and infection-induced morbidity over a period of days ( figure a ). following inoculation with pfu of t sa+, most wild-type mice developed paralysis and respiratory distress. in contrast, the majority of bid-deficient mice were asymptomatic. consistent with this observation, , % of wild-type mice succumbed to reovirus infection with a median survival time of days, whereas only , % of bid-deficient mice died. due to the relative resistance of bid-deficient mice to reovirus-induced encephalitis, a median survival time could not be determined. thus, the cellular apoptotic regulator bid modulates reovirus-induced encephalitis. to determine whether the enhanced survival of bid-deficient mice in comparison to wild-type mice following t sa+ infection results from reduced reovirus replication, we compared titers of reovirus at sites of primary and secondary replication at , , and d post-inoculation ( figure b -e). peak titers of reovirus were comparable or slightly higher (, -to -fold) in the intestine, liver, and heart of wild-type mice in comparison to bid-deficient animals. in contrast, substantially greater differences in peak reovirus titers were observed in the brain, with wild-type animals showing , -to -fold higher titers in comparison to those in bid-deficient mice at d post-inoculation. however, by d postinoculation, titers of reovirus in wild-type and bid-deficient mouse brains were equivalent. these findings suggest that reovirus infection is inefficient in the absence of bid, especially in the cns. although titers of reovirus in the cns were decreased in bidnull mice following po inoculation, it was not clear whether reduced reovirus titer in the cns was a consequence of diminished reovirus dissemination to the cns or diminished reovirus replication at that site. to distinguish between these possibilities, we inoculated wild-type and bid-deficient mice intracranially with pfu of t sa+ and monitored infected animals for signs of cns disease and mortality for days (figure a ). at this dose of t sa+, most wild-type and biddeficient mice displayed symptoms of neurological disease. concordantly, both strains of mice succumbed to reovirus-induced disease with equivalent frequency and a median survival time of days. reovirus titers in the brains of wild-type and bid-deficient mice also were comparable at , , and d post-inoculation ( figure b ). these results indicate that following a high-dose whole cell lysates were prepared at the indicated times after infection or h following tnfa/chx treatment and resolved in % polyacrylamide gels and transferred to nitrocellulose membranes. the membranes were probed with polyclonal antisera specific for bid or actin and appropriate hrp-conjugated secondary antibodies and visualized using chemiluminescence. doi: . /journal.ppat. .g inoculation, bid is dispensable for reovirus growth in the murine cns and attendant encephalitis. peak titers of reovirus in the brains of intracranially-inoculated wild-type mice were , -fold higher than those in perorallyinoculated wild-type animals (compare figures e and b) . we thought it possible that this difference in viral load might contribute to the dramatic difference in the requirement for bid in the pathogenesis of reovirus-induced cns disease following po and ic inoculation. to test this hypothesis, we inoculated wild-type and bid-deficient animals intracranially with a considerably lower but still lethal dose of t sa+, pfu, and monitored infected animals for signs of reovirus encephalitis ( figure c ). in comparison to wild-type mice in which , % succumbed to disease, , % of bid-deficient mice developed lethal encephalitis. moreover, the median survival time of wildtype mice infected with t sa+ was significantly less ( days) than that of bid-deficient mice ( days). to determine whether this difference in survival correlates with the efficiency of reovirus replication in the cns, we compared titers of reovirus in brains resected from infected mice at , , and d post-inoculation ( figure d ). titers of reovirus in brains of wild-type mice were substantially higher (, -to -fold) at each interval in results are expressed as the mean ratio of caspase- / activity from infected cell lysates to that from mock-infected cell lysates for triplicate samples. error bars indicate sd. *, p, . as determined by student's t-test relative to wild-type mefs infected at an equivalent moi. (b) wild-type or trail-r-deficient mefs were adsorbed with t d at the mois shown. after incubation at uc for h, cells were stained with ao. results are expressed as the mean percentage of cells undergoing apoptosis for three independent experiments. error bars indicate sd. *, p, . as determined by student's t-test relative to wild-type mefs infected at an equivalent moi. (c) wild-type and trail-r-deficient mefs were adsorbed with t d at an moi of pfu/cell. the inoculum was removed, and cells were incubated at uc for , , and h. viral titers were determined after two cycles of freeze-thaw by plaque assay using l cells. results are presented as mean titers from three independent experiments. error bars indicate sd. (d) wild-type and trail-r-deficient mefs were mock-infected or infected with t d at an moi of pfu/cell. whole cell lysates were prepared at h after infection and resolved in % polyacrylamide gels and transferred to nitrocellulose membranes. the membranes were probed with polyclonal antisera specific for bid or actin and appropriate hrp-conjugated secondary antibodies and visualized using chemiluminescence. doi: . /journal.ppat. .g figure . bid modulates reovirus replication and pathogenesis following po inoculation. (a) two-day-old wild-type and bid-deficient mice were inoculated perorally with pfu of t sa+. mice (n = to ) were monitored for survival for days. ***, p, . as determined by logrank test in comparison to wild-type mice. (b-e) two-day-old wild-type and bid-deficient mice were inoculated perorally with pfu of t sa+ and euthanized at the times shown. intestines (b), liver (c), heart (d), and brain (e) were resected and homogenized by freeze-thaw and sonication. viral titers in organ homogenates were determined by plaque assay. results are expressed as viral titer in organs of single infected animals as indicated by comparison to those in bid-deficient mice, with those at and d post-inoculation reaching statistical significance. these findings indicate that at a lower viral inoculum, bid promotes efficient replication of reovirus in the cns. collectively, these data suggest that bid influences reovirus virulence by regulating the growth of reovirus in the brain. to assess the capacity of t sa+ to produce neurological injury in the presence and absence of bid, we examined hematoxylin and eosin (h&e)-stained coronal brain sections prepared from wildtype and bid-deficient mice euthanized d following ic inoculation with pfu of t sa+ (figure ) . this time point was chosen to coincide with the presence of maximal viral titers following inoculation by this route. since the inoculum used for these experiments was at least , -fold lower than that used for most other studies of reovirus cns pathogenesis [ , , , , , , ] , the extent of injury following infection of wild-type mice was not as extensive. nonetheless, inoculation of wild-type mice with t sa+ resulted in neuronal death in the cerebral cortex, hippocampus, thalamus, and hypothalamus, consistent with previous reports [ , , , , , , ] . while the majority of infected wild-type mouse brains showed signs of injury, tissue damage was minimal in all of the brains examined from similarly infected bid-deficient animals. examination of the hippocampal region of a representative wild-type mouse brain at higher magnification showed damage to the ca region, with the pyramidal cells showing condensed nuclei characteristic of apoptosis ( figure b ). in contrast, little damage was detected in an equivalent region of a bid-deficient mouse brain ( figure b ). these findings indicate that bid is required for neurological injury produced by reovirus in mice. to determine whether these differences in neurological injury are attributable to alterations in tropism of reovirus in the absence of bid, sections of mouse brain were stained for reovirus antigen. reovirus displayed similar tissue distribution in wild-type and biddeficient mouse brains, indicating that bid expression does not influence reovirus tropism (data not shown). the ca region of a wild-type mouse brain showed reovirus antigen in areas coincident with extensive neuronal damage ( figure b ). regions positive for reovirus antigen also stained with an antibody for activated caspase- . although similar regions of bid-deficient mouse brains contained reovirus antigen, staining was of diminished intensity and frequency ( figure b) , consistent with the decreased efficiency of reovirus replication in the cns ( figure b) . accordingly, few cells showing intense caspase- staining were observed in regions that contained reovirus. these data suggest that neuronal apoptosis following reovirus infection is diminished in the absence of bid. thus, bid links reovirus replication and apoptosis induction in the production of fatal encephalitis. early steps in reovirus replication elicit apoptosis via a signaling pathway dependent on nf-kb [ , , , , ] . it is not understood how virus-induced nf-kb activation leads to cell death. in this study, we evaluated the function and regulation of bid in apoptosis caused by reovirus. we found that although bid is dispensable for reovirus replication in cell culture, it is required for the induction of apoptotic cell death following reovirus infection. in this context, bid is converted to its active, proapoptotic form, tbid, in an nf-kb-dependent manner. generation of tbid in reovirus-infected cells requires signaling via trail-r and caspase- . these findings indicate that nf-kb signaling following reovirus infection results in activation of the extrinsic apoptotic pathway. in turn, the extrinsic apoptotic pathway evokes the mitochondrial apoptotic cascade via cleavage-induced activation of bid. together, these events culminate in the induction of apoptotic cell death. many viruses induce apoptosis via activation of host-encoded apoptosis-regulating factors. for example, the vsv m protein induces apoptosis by inhibiting the transcription of antiapoptotic factors such as bcl-xl [ ] . in other cases, virus-encoded polypeptides insert into mitochondrial membranes and trigger cytochrome c release, leading to activation of the mitochondrial apoptotic pathway. for example, influenza a virus pb -f is thought to directly activate proapoptotic signaling by interaction with the mitochondrial membrane-associated factors ant and vdac [ ] . although one model for apoptosis induction by reovirus suggests that the w fragment of reovirus m protein induces apoptosis by directly targeting mitochondria analogous to pb -f [ ] , our studies using apoptosis-defective reovirus mutants [ , ] , coupled with data presented here, support the idea that w-mediated nf-kb signaling activates the mitochondrial apoptotic pathway indirectly via death-receptor signaling and bid cleavage. this indirect mechanism of mitochondrial pathway activation by a proximal signal transducer also explains the timing of prodeath signaling in the reovirus replication cycle. we think that events associated with viral entry into cells, which are mediated by the m w fragment subsequent to membrane penetration, activate nf-kb within h of infection [ , ] . unlike other nf-kb agonists such as tnfa which rapidly and transiently activate nf-kb, activation of nf-kb following reovirus infection is gradual and sustained and occurs maximally at - h post infection [ ] . activated nf-kb complexes lead to expression of genes that promote cleavage-induced activation of bid at - h post infection and elicit characteristic features of apoptosis, including effector caspase activation and dna fragmentation. these changes occur subsequent to completion of viral replication, and, therefore, apoptosis appears to have little detectable effect on viral growth in cell culture [ , , , ] . although unusual, nf-kbdependent apoptotic pathways are also utilized by other viruses such as dengue virus [ ] , hiv [ ] , infectious bursal disease virus [ ] , and sindbis virus [ ] . thus, our studies may have uncovered a potentially conserved signaling pathway utilized by viruses to induce apoptosis via nf-kb. it is not known how activation of nf-kb by reovirus culminates in cell death. three previous studies have attempted to identify proapoptotic host genes that serve as effectors of the death response following reovirus infection. in the first, gene-expression profiles following infection with reovirus strains type lang (t l) and type abney (t a), which differ in the capacity to induce apoptosis [ ] , were compared by microarray analysis [ ] . these experiments did not demonstrate differences in expression of death ligands or their respective receptors following infection by either strain. thus, it was concluded that expression of these death mediators by reovirus is unlikely to contribute to apoptosis induction by reovirus. however, some differences were observed in expression of regulators of death-receptor signaling [ ] . but since t l and t a display significant genetic diversity and vary in the modulation of multiple signaling pathways [ , ] , the contribution of nf-kb to the expression of prodeath genes could not be established. in the second study, gene-expression profiles following t d infection in the presence and absence of functional nf-kb were compared [ ] . although this study identified several nf-kb-dependent genes that coordinate the cellular antiviral immune response, including numerous interferon-stimulated genes (isgs), no classical components of death receptor-mediated signaling pathways or proapoptotic bcl- family members were significantly upregulated in response to reovirus infection. in the third study, gene-expression profiles of reovirus strains that differ in the capacity to elicit translational shutoff were compared [ ] . this study also demonstrated an increase in isg expression but did not identify obvious nf-kb-dependent candidates that could serve to activate death receptor signaling. we think there are three possibilities to explain why apoptosisregulating, transcriptional targets of nf-kb, such as death ligands (e.g., fasl and trail) [ , ] , death receptors (e.g., fas and dr) [ , ] , and death-signaling regulators (e.g., bax and bcl-x s ,) [ ] , were not identified in these studies. first, changes in the expression of prodeath genes activated by reovirus infection may be too transient to have been detected in the intervals selected for analysis. second, the transformed nature of the cell lines used in these studies may not have been amenable to detection of alterations in gene expression induced by reovirus infection. third, nf-kb activation following reovirus infection may regulate death signaling at a post-transcriptional level by an as yet unknown mechanism. in support of this idea, levels of dr protein increase following reovirus infection [ ] but not its mrna [ ] . additional studies using primary, non-transformed cell lines and genetically engineered viruses that differ only in the capacity to activate nf-kb are required to define how reovirus activates extrinsic apoptotic pathways to evoke cell death. in addition to enhancing an understanding of mechanisms by which virus-induced signaling leads to activation of bid, our studies highlight a critical role for bid in controlling the pathogenesis of a viral disease. we found that bid-deficient mice are less susceptible to lethal encephalitis produced by a neurotropic reovirus strain following either po or ic inoculation. reovirus replicates with slower kinetics in the absence of bid, and virus-induced apoptosis and cns injury are diminished in biddeficient animals. although bid contributes significantly to reovirus pathogenesis, our data do not allow us to determine whether diminished reovirus virulence in bid-deficient animals is attributable to reduced capacity of reovirus to replicate in the cns, diminished capacity of reovirus to injure neurons by apoptosis, or both effects. it is also not clear whether the decreased capacity of reovirus to evoke apoptosis in the cns is a cause or effect of the lower viral titers at that site. because bid serves to amplify the death response, it is not universally required for apoptosis induction. in some cell types, known as type i cells, caspase- activation results in direct, bidindependent activation of the apoptosis effectors, caspase- and caspase- [ ] . in others, known as type ii cells, apoptosis requires amplification of death signals through stimulation of the mitochondrial pathway. in these cases, bid serves to link the extrinsic and intrinsic apoptotic pathways [ ] . since the requirement for bid in reovirus virulence is dependent on viral dose, we think that the role of bid as an apoptosis regulator contributes to viral replication and consequent neurovirulence. thus, we hypothesize that neurons function like type i cells when infected at a higher dose of virus and do not require the amplification of the mitochondrial apoptotic pathway via bid to undergo apoptotic cell death. however, at lower infectious doses, neurons function like type ii cells and require bid-driven activation of the intrinsic mitochondrial apoptotic cascade to elicit cell death. this model also may explain why primary neuronal cultures infected with reovirus at a high moi do not appear to require cytochrome c release and caspase- activation for apoptosis induction [ ] . it is not known how bid controls the efficiency of reovirus replication in the cns. one possibility is that bid-regulated apoptosis is required for efficient release of virus from neurons. therefore, cell-to-cell spread of reovirus within the cns may be inefficient in the absence of bid. as an extension of this idea, blockade of apoptosis by other means also should cause a delay in reovirus replication. however, although symptoms of encephalitis are alleviated in nf-kb p -deficient mice or in wild-type mice treated with a jnk inhibitor due to a reduction in virus-induced neuronal apoptosis, reovirus replication kinetics are not substantially diminished [ , ] . consistent with these findings, diminished virulence of apoptosis-defective reovirus mutants is not accompanied by significant decreases in reovirus replication efficiency in the cns [ , ] . apoptosis following reovirus infection can occur in absence of p , albeit at low efficiency [ ] . similarly, apoptosisdefective reovirus mutants retain some capacity to induce apoptotic cell death [ , ] . therefore, it is possible that in comparison to reovirus infection of bid-deficient mice, cns apoptosis was incompletely blocked in these other studies. such a difference in the efficiency of apoptosis inhibition could explain the observed discrepancy in the requirement for bid and other host or viral modulators of apoptosis for efficient replication of reovirus. a second possibility is that a bid function not related to its capacity to regulate apoptosis contributes to reovirus replication in the cns. analogous to its role in reovirus-induced cell death, bid is implicated in apoptosis caused by many viruses [ , , , , , , , , , , , ] . however, prior to our study, it was not known whether bid modulates the pathogenesis of viral disease. the function of bid in viral pathogenesis has been examined in a previous study, which found that the bh -only protein, puma, but not bid, contributes to apoptosis-mediated elimination of antigen-specific t cells following acute infection with herpes simplex virus- [ ] . here, we demonstrate a pathogenic function for bid in viral infection. should bid similarly modulate disease outcomes following infection by other virulent viruses, antiapoptotic compounds targeting bid [ , , ] may serve as useful antiviral therapeutics. murine l cells were maintained in joklik's minimal essential medium supplemented to contain % fetal bovine serum (fbs), mm l-glutamine, u/ml penicillin, mg/ml streptomycin, and ng/ml amphotericin b (invitrogen). wild-type and biddeficient mefs were maintained in dulbecco's minimal essential medium (dmem) supplemented to contain % fbs, mm lglutamine, u/ml penicillin, mg/ml streptomycin, and ng/ml amphotericin b. trail-r-deficient mefs, prepared from d embryos, were maintained in dmem supplemented to contain % fbs, mm l-glutamine, mem nonessential amino acids, . mm -mercaptoethanol, mm hepes, u/ ml penicillin, mg/ml streptomycin, and ng/ml amphotericin b. reovirus strain t d is a laboratory stock. t sa+ was generated by reassortment of reovirus strains t l and type clone -ma as described [ ] . purified reovirus virions were generated from second-or third-passage l-cell lysate stocks of twice-plaque-purified reovirus [ ] . viral particles were freon-extracted from infected cell lysates, layered onto . rabbit antisera raised against t l and t d have been described [ ] . rabbit antiserum specific for procaspase- was purchased from cell signaling. goat antiserum specific for bid was purchased from r & d systems, and goat antiserum specific for actin was purchased from santa cruz biotechnology. hrp-conjugated antirabbit and anti-goat secondary antibodies were purchased from amersham ge biosciences. alexa fluor-conjugated anti-mouse immunoglobulin (ig) g, anti-rabbit igg, and anti-goat igg secondary antibodies were purchased from invitrogen. plasmids prenilla-luc and pnf-kb-luc [ ] were obtained from dr. dean ballard (vanderbilt university). l cells or wild-type, bid-deficient, or trail-r-deficient mefs were either adsorbed with reovirus at an moi of pfu/cell or mock-infected in serum-free medium at uc for h, followed by incubation in serum-containing medium at uc for various intervals. whole cell lysates were prepared by washing cells in phosphate-buffered saline (pbs) followed by lysis using ripa buffer ( mm tris [ph . ], mm nacl, % tx- , % doc, . % sds, and mm edta) containing a protease inhibitor cocktail (roche). following centrifugation at , g to remove debris, the lysates were resolved by electrophoresis in polyacrylamide gels and transferred to nitrocellulose membranes. membranes were blocked for at least h in blocking buffer (pbs containing % milk or . % bsa) and incubated with antisera against bid ( : ), actin ( : ), or procaspase- ( : ) either at room temperature for h or uc overnight. membranes were washed three times for min each with washing buffer (pbs containing . % tween- ) and incubated with : dilution of horseradish peroxidase (hrp)-conjugated or alexa fluor-conjugated goat anti-rabbit ig (for procaspase- ) or donkey anti-goat ig (for bid and actin) in blocking buffer. following three washes, membranes were incubated for min with chemiluminescent peroxidase substrate (amersham biosciences) and either exposed to film (for hrp-conjugated secondary antibodies) or scanned using an odyssey infrared imager (licor). wild-type, bid-deficient, or trail-r-deficient mefs ( ) were seeded into black clear-bottom -well plates (costar) and adsorbed with reovirus in serum-free medium at room temperature for h. following incubation of cells at uc for h, caspase- / activity was quantified using the caspase-glo- / assay (promega). wild-type, bid-deficient, or trail-r-deficient mefs ( ) were grown in -well plates (costar) and adsorbed with reovirus at room temperature for h. the percentage of apoptotic cells after h incubation was determined using ao staining as described [ ] . for each experiment, . cells were counted, and the percentage of cells exhibiting condensed chromatin was determined by epi-illumination fluorescence microscopy using a fluorescein filter set (zeiss photomicroscope iii; thornwood, ny). wild-type or bid-deficient cells ( ) were grown in -well plates and adsorbed with reovirus at room temperature for h. following removal of the inoculum, cells were washed with pbs and incubated in complete medium at uc for h. monolayers were fixed with methanol, washed twice with pbs, blocked with . % ig-free bovine serum albumin (sigma-aldrich) in pbs, and incubated successively for h with polyclonal rabbit anti-reovirus serum at a : dilution and for h with alexa fluor labeled anti-rabbit igg at a : dilution. monolayers were washed with pbs, and infected cells were visualized by indirect immunofluorescence using a zeiss axiovert fluorescence microscope. reovirus antigen-positive cells were quantified by counting fluorescent cells in at least two random fields of view in triplicate wells at a magnification of . wild-type, bid-deficient, or trail-r-deficient mefs ( ) in -well plates were adsorbed with reovirus at room temperature for h in serum-free medium, washed once with pbs, and incubated in serum-containing medium for various intervals. cells were frozen and thawed twice prior to determination of viral titer by plaque assay using l cells [ ] . wild-type and bid-deficient cells in -well plates were transfected with . mg/well of an nf-kb reporter plasmid, which expresses firefly luciferase under nf-kb control (pnf-kb-luc), and . mg/well of control plasmid prenilla-luc, which expresses renilla luciferase constitutively, using fugene (roche). after incubation for h, transfected cells were adsorbed with reovirus in serum-free medium at room temperature for h and incubated at uc in serum-containing medium for h. luciferase activity in the cultures was quantified using the dual-luciferase assay kit (promega) according to the manufacturer's instructions. wild-type c bl/ j mice were obtained from jackson laboratory. bid-deficient mice backcrossed on to a c bl/ j background for at least generations have been previously described [ ] . two-day-old mice were inoculated either perorally or intracranially with purified virus diluted in pbs. po inoculations were delivered in a volume of ml by passage of a polyethylene catheter . mm in diameter (bd) through the esophagus and into the stomach [ ] . the inoculum contained . % (vol/vol) green food coloring to allow the accuracy of delivery to be judged. ic inoculations were delivered to the left cerebral hemisphere in a volume of ml using a hamilton syringe and a -gauge needle (bd biosciences) [ ] . for analysis of viral virulence, mice were monitored for weight loss and symptoms of disease for days. for survival experiments, mice were euthanized when found to be moribund (defined by rapid or shallow breathing, lethargy, or paralysis). for determination of viral titer and immunohistochemical staining, mice were euthanized at various intervals following inoculation and organs were resected. for analysis of virus growth, organs were collected into ml of pbs and homogenized by freezing, thawing, and sonication. viral titers in organ homogenates were determined by plaque assay using l cells [ ] . for immunohistochemical staining, organs were fixed overnight in % formalin, followed by incubation in % ethanol. fixed organs were embedded in paraffin, and -mm histological sections were prepared. consecutive sections were stained with h&e for evaluation of histopathologic changes or processed for immunohistochemical detection of reovirus antigens or activated caspase- [ ] . animal husbandry and experimental procedures were performed in accordance with public health service policy and the recommendations of the association for assessment and accreditation of laboratory animal care and approved by the vanderbilt university school of medicine institutional animal care and use committee. apoptosis in the mouse central nervous system in response to infection with mouse-neurovirulent dengue viruses alphavirus-induced apoptosis in mouse brains correlates with neurovirulence apoptosis plays an important role in experimental rabies virus infection reovirus apoptosis and virulence are regulated by host cell membrane penetration efficiency linkage between reovirus-induced apoptosis and inhibition of cellular dna synthesis: role of the s and m genes differences in the capacity of reovirus strains to induce apoptosis are determined by the viral attachment protein s independent regulation of reovirus membrane penetration and apoptosis by the mu phi domain reovirus-induced apoptosis requires activation of transcription factor nf-kappab reovirus infection activates jnk and the jnk-dependent transcription factor c-jun jnk regulates the release of proapoptotic mitochondrial factors in reovirus-infected cells organ-specific roles for transcription factor nf-kb in reovirus-induced apoptosis and disease a novel strategy for the treatment of viral cns infection utilizing a cell-permeable inhibitor of c-jun n-terminal kinase reovirus outer capsid protein m induces apoptosis and associates with lipid droplets, endoplasmic reticulum, and mitochondria jam-a-independent, antibody-mediated uptake of reovirus into cells leads to apoptosis virion disassembly is required for apoptosis induced by reovirus reovirus-induced apoptosis requires both death receptor-and mitochondrial-mediated caspase-dependent pathways of cell death reovirus-induced apoptosis is mediated by trail essential requirement for caspase- / flice in the initiation of the fas-induced apoptotic cascade ) tbid, a membrane-targeted death ligand, oligomerizes bak to release cytochrome c cytochrome c and datp-dependent formation of apaf- /caspase- complex initiates an apoptotic protease cascade identification of diablo, a mammalian protein that promotes apoptosis by binding to and antagonizing iap proteins smac, a mitochondrial protein that promotes cytochrome c-dependent caspase activation by eliminating iap inhibition essential role of the mitochondrial apoptosis-inducing factor in programmed cell death reovirus-induced apoptosis of mdck cells is not linked to viral yield and is blocked by bcl- bcl- is an innner mitochondrial membrane protein that blocks programmed cell death cleavage of bid by caspase mediates the mitochondrial damage in the fas pathway of apoptosis posttranslational nmyristoylation of bid as a molecular switch for targeting mitochondria and apoptosis ikb kinase subunits a and c are required for activation of nf-kb and induction of apoptosis by mammalian reovirus two cd (apo- /fas) signaling pathways bid-deficient mice are resistant to fas-induced hepatocellular apoptosis calpain-mediated bid cleavage and calpain-independent bak modulation: two separate pathways in cisplatin-induced apoptosis bid is cleaved by calpain to an active fragment in vitro and during myocardial ischemia/ reperfusion trail causes cleavage of bid by caspase- and loss of mitochondrial membrane potential resulting in apoptosis in bjab cells bid, a bcl interacting protein, mediates cytochrome c release from mitochondria in response to activation of cell surface death receptors caspase cleaved bid targets mitochondria and is required for cytochrome c release, while bcl-xl prevents this release but not tumor necrosis factor-r /fas death lysosomal protease pathways to apoptosis. cleavage of bid, not pro-caspases, is the most likely route cathepsin d links tnf-induced acid sphingomyelinase to bid-mediated caspase- and - activation cathepsin d mediates cytochrome c release and caspase activation in human fibroblast apoptosis induced by staurosporine selective disruption of lysosomes in hela cells triggers apoptosis mediated by cleavage of bid by multiple papain-like lysosomal cathepsins reovirusinduced apoptosis is preceded by increased cellular calpain activity and is blocked by calpain inhibitors addition of exogenous protease facilitates reovirus infection in many restrictive cells reovirus-induced neuronal apoptosis is mediated by caspase and is associated with the activation of death receptors fas-mediated apoptotic signaling in the mouse brain following reovirus infection mek kinase gene disruption alters cell migration and c-jun nh -terminal kinase regulation but does not cause a measurable defect in nf-kappa b activation trail-r as a negative regulator of innate immune cell responses dr knockout mice are compromised in radiation-induced apoptosis trail-r deficiency in mice promotes susceptibility to chronic inflammation and tumorigenesis regional differences in viral growth and central nervous system injury correlate with apoptosis minocycline delays disease onset and mortality in reovirus encephalitis utilization of sialic acid as a coreceptor is required for reovirus-induced biliary disease junctional adhesion molecule-a is required for hematogenous dissemination of reovirus early activation of the mitochondrial apoptotic pathway in vesicular stomatitis virus-infected cells influenza virus pb -f protein induces cell death through mitochondrial ant and vdac potential dengue virustriggered apoptotic pathway in human neuroblastoma cells: arachidonic acid, superoxide anion, and nf-kappab are sequentially involved mechanisms of apoptosis induction by the hiv- envelope nonstructural protein of infectious bursal disease virus inhibits apoptosis at the early stage of virus infection thiol agents and bcl- identify an alphavirus-induced apoptotic pathway that requires activation of the transcription factor nf-kappa b linkage between reovirus-induced apoptosis and inhibition of cellular dna synthesis: role of the s and m genes reovirus-induced alteration in expression of apoptosis and dna repair genes with potential roles in viral pathogenesis reovirus induction of and sensitivity to beta interferon in cardiac myocyte cultures correlate with induction of myocarditis and are determined by viral core proteins identification of an nf-kb-dependent gene network in cells infected by mammalian reovirus reovirus induces and benefits from an integrated cellular stress response nf-kappab signaling pathway governs trail gene expression and human t-cell leukemia virus-i tax-induced t-cell death regulation of fasl by nf-kappab and ap- in fas-dependent thymineless death of human colon carcinoma cells dna damaging agents induce expression of fas ligand and subsequent apoptosis in t lymphocytes via the activation of nf-kappa b and ap- regulation of fas-ligand expression during activation-induced cell death in t lymphocytes via nuclear factor kappab nf-kappab-mediated upregulation of bcl-x(s) and bax contributes to cytochrome c release in cyanideinduced apoptosis the major apoptotic pathway activated and suppressed by poliovirus murine coronavirus-induced apoptosis in cl- cells involves a mitochondria-mediated pathway and its downstream caspase- activation and bid cleavage hcv e may induce apoptosis of huh- cells via a mitochondrial-related caspase pathway porcine reproductive and respiratory syndrome virus induces apoptosis through a mitochondria-mediated pathway bovine ephemeral fever virus-induced apoptosis requires virus gene expression and activation of fas and mitochondrial signaling pathway role of the mitochondrial signaling pathway in murine coronavirus-induced oligodendrocyte apoptosis severe acute respiratory syndrome coronavirus a protein activates the mitochondrial death pathway through p map kinase activation the equine arteritis virus induces apoptosis via caspase- and mitochondria-dependent caspase- activation activation of the extrinsic caspase pathway in cultured cortical neurons requires p -mediated down-regulation of the x-linked inhibitor of apoptosis protein to induce apoptosis screening of pro-apoptotic genes upregulated in an experimental street rabies virus-infected neonatal mouse brain apoptosis induced by semliki forest virus is rna replication dependent and mediated via bak vesicular stomatitis virus induces apoptosis primarily through bak rather than bax by inactivating mcl- and bcl-xl bh -only protein puma contributes to death of antigen-specific t cells during shutdown of an immune response to acute viral infection targeting apoptosis via chemical design: inhibition of bid-induced cell death by small organic molecules structureactivity relationships by interligand noe-based design and synthesis of antiapoptotic compounds targeting bid bid-induced release of aif from mitochondria causes immediate neuronal cell death utilization of sialic acid as a coreceptor enhances reovirus attachment by multistep adhesion strengthening sigma protein of mammalian reoviruses extends from the surfaces of viral particles polypeptide components of virions, top component and cores of reovirus type efficiency of viral entry determines the capacity of murine erythroleukemia cells to support persistent infections by mammalian reoviruses persistent activation of nf-kb by the tax transforming protein involves chronic phosphorylation of ikb kinase subunits ikkb and ikkc antibody protects against lethal infection with the neurally spreading reovirus type (dearing) molecular basis of viral neurotropism: experimental reovirus infection molecular basis of reovirus virulence: role of the m gene we thank karl boehme, christina melki, and denise wetzel for helpful suggestions and review of the manuscript. we thank dean ballard (vanderbilt university) for providing the nf-kb reporter plasmids and wafik el-deiry (university of pennsylvania) for providing trail-rdeficient mice. conceived and designed the experiments: pd ssz tsd. performed the experiments: pd ajp akb ghh. analyzed the data: pd ajp akb ghh ssz tsd. wrote the paper: pd tsd. key: cord- -tkxpjlyn authors: dermody, terence s.; kirchner, eva; guglielmi, kristen m.; stehle, thilo title: immunoglobulin superfamily virus receptors and the evolution of adaptive immunity date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: tkxpjlyn nan obligate intracellular pathogens depend on cell-surface molecules to attach and enter into host cells. pathogen receptors may be highly specialized proteins, such as complement receptors or neurotransmitter receptors, or more ubiquitous components of cell membranes, such as integrins or sialic acid-containing oligosaccharides. the immunoglobulin superfamily (igsf) of molecules contains several members that are expressed at the cell surface, bind diverse ligands, and contribute to a variety of cellular activities, including adhesion and immune responses. many viruses have usurped the adhesive properties of igsf proteins to mediate attachment (table ) . strategies used by viruses to engage igsf receptors provide clues to general mechanisms by which igsf proteins bind different types of ligands, including antigens. members of the igsf have diverged in sequence and function. however, all contain domains with the characteristic immunoglobulin fold, which is defined by two opposing antiparallel b-sheets connected in a unique manner [ , ] . the core of the immunoglobulin fold is formed by four b-strands (b, c, e, and f) augmented with three to five additional b-strands (a, c , c , d, and g) to yield several distinct subtypes [ , ] . most common are the vset and c-set immunoglobulin domains, which are named according to their occurrence in the variable and constant regions of immunoglobulins, respectively. a third type, the i-set, is an intermediate structure between the v-and c-sets found frequently in cell-surface receptors. immunoglobulin domains rarely occur in isolation but typically form concatenated chains, often with a v-set or i-set domain at the n-terminus. biochemical and structural analyses of interactions between viruses and their cognate igsf receptors reveal several striking similarities. first, in cases in which structural information about virus-receptor complexes is available, the viral attachment proteins exclusively bind to the most membrane-distal, n-terminal domain (d ) of the igsf receptors [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . while structural information about com-plex formation is lacking for the igsf receptors carcinoembryonic antigen-related cell adhesion molecule, nectin- , nectin- , and signaling lymphocyte-activation molecule (slam), biochemical studies also implicate their respective d domains in virus binding [ ] [ ] [ ] [ ] . second, viruscontacting residues lie towards the upper ''tip'' of the igsf d domain. third, the viral receptor-binding region engages the cc fg b-sheet of the igsf receptor d domain. fourth and finally, almost all of the receptor domains interacting with viruses belong to the v-type igsf fold. the single exception, the d domain of icam- , belongs to the i-set type, which is structurally similar to the v-set domain. although the database of viral proteins in complex with igsf receptors is still quite small, interactions of viruses with their receptors parallel the recognition mode of immunoglobulins, which also recognize their cognate antigens via residues at the tip of their n-terminal, v-set domains. the case of the receptor-binding head domain of reovirus attachment protein s in complex with the d domain of its receptor, junctional adhesion molecule-a (jam-a) [ ] , serves to illustrate this point ( figure a ). the jam-a homodimer strikingly resembles the dimer formed by the v-set domains of the light and heavy chains of immunoglobulins. in both structures, the two v-set domains face each other with similar orientations. moreover, residues in the receptor required for virus attachment reside in bstrands and intervening loops that juxta-pose the complementarity determining regions (cdrs) of antibody molecules. thus, residues known to interact with ligands map to corresponding regions near the tip and one side of the v-set domains. these similarities extend beyond reovirus receptor jam-a. other igsf virus receptors, such as the coxsackievirus and adenovirus receptor (car) [ ] and hiv receptor cd [ ] , also recognize their viral ligands via residues that partially overlap with the cdr region of immunoglobulins ( figure b -f). car forms a homodimer via its d domain that is very similar to the jam-a homodimer [ ] . cd also forms homodimers, albeit via its d domain [ ] . the immunoglobulin fold predates the evolution of vertebrates. genomes of invertebrate organisms encode numerous molecules that belong to two families with homologs in vertebrates: the jam/cortical thymocyte marker of xenopus (ctx) family and the nectin family [ ] . vertebrate counterparts of these genes are found in discrete blocks, and many are now diversified to encode molecules that function in adaptive immunity, including cd and slam [ ] . invertebrates do not encode recombination-activating genes (rags) and generally display only limited antigen-specific immunity. therefore, the core structural element of adaptive immunity, the immunoglobulin fold, evolved prior to a mechanism to generate a highly diversified antigen-specific repertoire. similarities in mechanisms of ligand engagement by igsf pathogen receptors and immunoglobulins, coupled with the evolution of the immunoglobulin fold prior to the existence of the vertebrate adaptive immune system, suggest the possibility that primitive members of the jam/ctx and nectin families evolved to become soluble adaptive immune mediators in modern vertebrates. one attractive hypothesis is that soluble forms of pathogen receptors served as precursors to molecules of the adaptive immune system. soluble receptors would neutralize viral [ ] . expression of a soluble pathogen receptor followed by duplication within the primitive genome and acquisition of mutations that permitted recognition of additional pathogens could confer a strong selective advantage. upon introduction of rags into the vertebrate genome, such a gene family would have been primed to express molecules akin to present-day immunoglobulins. alternatively, membrane-anchored forms of igsf molecules that arose in primitive invertebrates may have been maintained in the genome due to their cell-adhesion functions, followed by the serendipitous introduction of mechanisms for the secretion and generation of diversity. in this scenario, pathogens may have contributed to the evolution of the modern adaptive immune system at much later evolutionary times. is there evidence that favors either of these potential evolutionary mechanisms? in addition to similarities in their ligandbinding strategies, many of the closest structural homologs of jam-a are immunoglobulins, which raises the possibility that immunoglobulins are more closely related to jam-a than to other igsf molecules. a search for structural homologs of the jam-a d domain using the dali algorithm [ ] provides support for this hypothesis. the closest structural homologs of the jam-a d domain are immunoglobulin domains, with the highest dali z-score of . for an igak variable domain (pdb code fbj) ( table ) . other igsf proteins with similarity to jam-a d have significantly lower z-scores. the z-scores correlate well with root mean square deviations for superpositions of jam-a d with immunoglobulins, which also are lower (i.e., more similar) than the corresponding values for superpositions of jam-a d with other igsf proteins. this homology search can be extended to car, neural cell adhesion molecule, and nectin-like molecule , which result in z-scores that are generally higher for the superposition of their d domains with immunoglobulins than with other cell adhesion molecules. in urochordates (ciona) and cephalochordates (branchiostoma), evolutionarily close relatives of the vertebrates, there are homologs of jam/ctx and nectin igsf molecules with features of membrane receptors. ciona encodes only a single jam/ctx-like molecule and two nectin-like molecules [ ] . in humans, these molecules are all part of a single linkage group involved in immune function [ , ] . taken together, these results suggest that relatively few jam/ctx and nectin family igsf molecules were maintained in invertebrates, and the expansion and duplication resulting in the evolution of immunoglobulins may have occurred after the introduction of these molecules into the vertebrate genome. there also is evidence of expansion of igsf molecules in invertebrates. for example, like many immunoglobulins, chitin-binding protein (cbp) of branchiostoma is a close structural homolog of jam-a (table ). variable region-containing (v) cbps contain a v-type immunoglobulin domain with extensive sequence diversity in the n-terminal region [ , ] . this diversity is thought to result from high haplotype variation, including variable copy number, polymorphisms, and potential for alternative splicing [ ] . another of the closest structural homologs of jam-a is down syndrome cell adhesion molecule (dscam), an igsf member of the more evolutionarily distant invertebrate drosophila (table ) . dscam is an immune mediator found in clusters of variable exons flanked by constant exons [ , ] . thousands of different dscam molecules can be generated via alternative splicing, a mechanism that is highly conserved across insect orders [ ] . secreted isoforms of dscam circulating in insect hemolymph contribute to phagocytic uptake of bacteria. while the structural similarities between jam-a and vcbp or dscam may not indicate a direct evolutionary relationship, it is clear that diversification and secretion of soluble forms of igsf molecules can occur in invertebrates and raise the possibility that pathogens have had selective influence on the diversification and secretion of these molecules. thus, igsf proteins that served as precursors to soluble adaptive immune effectors may have diversified both prior to and following their introduction into the vertebrate genome. a more thorough examination of igsf members in invertebrates may clarify mechanisms that led to the evolution of modern adaptive immune mediators and the role of jam/ctx family molecules in this evolutionary process. the evolution of jam family members prior to the biochemical means to efficiently and extensively diversify antigen receptor genes, along with the structural similarities in the binding surfaces of virus receptors and immunoglobulins, provides strong support for the contention that viruses and perhaps other pathogens that engage igsf receptors contributed to the selection of humoral mediators of adaptive immunity. these observations provide a new framework for understanding how pathogen-host interplay during a prolonged period of evolutionary struggle may have led to the development of antigen-specific immune responses in vertebrates. we thank jim chappell and the plos pathogens reviewers for insightful suggestions and critique of the manuscript. the immunoglobulin fold: structural classification, sequence patterns and common core many of the immunoglobulin superfamily domains in cell adhesion molecules and surface receptors belong to a new structural set which is close to that containing variable domains the structure of the two amino-terminal domains of human icam- suggests how it functions as a rhinovirus receptor and as an lfa- integrin ligand structure of an hiv gp envelope glycoprotein in complex with the cd receptor and a neutralizing antibody structural analysis of the mechanism of adenovirus binding to its human cellular receptor three-dimensional structure of poliovirus receptor bound to poliovirus interaction of coxsackievirus b with the full length coxsackievirusadenovirus receptor interaction of coxsackievirus a with its cellular receptor, icam- structure of reovirus s in complex with its receptor junctional adhesion molecule-a crystal structure of cd and electron microscopic studies of its complexes with polioviruses mouse hepatitis virus strain a and blocking antireceptor monoclonal antibody bind to the n-terminal domain of cellular receptor the first immunoglobulin-like domain of hvec is sufficient to bind herpes simplex virus gd with full affinity, while the third domain is involved in oligomerization of hvec v domain of human slam (cdw ) is essential for its function as a measles virus receptor soluble v domain of nectin- /hvec enables entry of herpes simplex virus type (hsv- ) into hsv-resistant cells by binding to viral glycoprotein d structural similarities in the cellular receptors used by adenovirus and reovirus dimeric association and segmental variability in the structure of human cd immunoglobulin superfamily receptors in protochordates: before rag time human rhinovirus selectively modulates membranous and soluble forms of its intercellular adhesion molecule- (icam- ) receptor to promote epithelial cell infectivity searching protein structure databases with dalilite v speculations on the origin of the vertebrate immune system identification of diversified genes that contain immunoglobulin-like variable regions in a protochordate ancient evolutionary origin of diversified variable regions demonstrated by crystal structures of an immunetype receptor in amphioxus genomic complexity of the variable region-containing chitin-binding proteins in amphioxus structural basis of dscam isoform specificity drosophila dscam is an axon guidance receptor exhibiting extraordinary molecular diversity extensive diversity of ig-superfamily proteins in the immune system of insects refined structure of an intact igg a monoclonal antibody crystal structure of human junctional adhesion molecule : implications for reovirus binding kinetic and structural analysis of mutant cd receptors that are defective in hiv gp binding dimeric structure of the coxsackievirus and adenovirus receptor d domain at . Å resolution isolation of a common receptor for coxsackie b viruses and adenoviruses and hcar and mcar: the human and mouse cellular receptors for subgroup c adenoviruses and group b coxsackieviruses receptor for mouse hepatitis virus is a member of the carcinoembryonic antigen family of glycoproteins mouse hepatitis virus utilizes two carcinoembryonic antigens as alternative receptors several members of the mouse carcinoembryonic antigen-related glycoprotein family are functional receptors for the coronavirus mouse hepatitis virus-a entry of alphaherpesviruses mediated by poliovirus receptorrelated protein and poliovirus receptor a cell surface protein with herpesvirus entry activity (hveb) confers susceptibility to infection by mutants of herpes simplex virus type , herpes simplex virus type , and pseudorabies virus the cd (t ) antigen is an essential component of the receptor for the aids retrovirus the t gene encodes the aids virus receptor and is expressed in the immune system and the brain slam (cdw ) is a cellular receptor for measles virus cellular receptor for poliovirus: molecular cloning, nucleotide sequence, and expression of a new member of the immunoglobulin superfamily the neural cell adhesion molecule is a receptor for rabies virus junction adhesion molecule is a receptor for reovirus the major human rhinovirus receptor is icam- a cell adhesion molecule, icam- , is the major surface receptor for rhinoviruses cdna cloning reveals that the major group rhinovirus receptor on hela cells is intercellular adhesion molecule key: cord- -d hetoq authors: meng, xiangzhi; zhang, fushun; yan, bo; si, chuanping; honda, hiroaki; nagamachi, akiko; sun, lu-zhe; xiang, yan title: a paralogous pair of mammalian host restriction factors form a critical host barrier against poxvirus infection date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: d hetoq host restriction factors constitute a formidable barrier for viral replication to which many viruses have evolved counter-measures. human samd , a tumor suppressor and a restriction factor for poxviruses in cell lines, is antagonized by two classes of poxvirus proteins, represented by vaccinia virus (vacv) k and c . a paralog of samd , samd l, is also encoded by some mammals, while only one of two paralogs is retained by others. here, we show that samd l functions similarly to samd as a restriction factor and that the two paralogs form a critical host barrier that poxviruses must overcome to establish infection. in mice, which naturally lack samd , overcoming samd l restriction with viral inhibitors is essential for poxvirus replication and pathogenesis. while a vacv deleted of both k and c (vk l(-)c l(-)) was restricted by mouse cells and highly attenuated in mice, its replication and virulence were completely restored in samd l(-/-) mice. in humans, both samd and samd l are poxvirus restriction factors, although the latter requires interferon induction in many cell types. while knockout of samd with crispr-cas was sufficient for abolishing the restriction for vk l(-)c l(-) in many human cells, knockout of both paralogs was required for abolishing the restriction in interferon-treated cells. both paralogs are antagonized by vacv k , c and c homologs from diverse mammalian poxviruses, but mouse samd l is resistant to the c homolog encoded by a group of poxviruses with a narrow host range in ruminants, indicating that host species-specific difference in samd /samd l genes serves as a barrier for cross-species poxvirus transmission. emerging and reemerging infectious diseases have continued to pose a major threat to public health. in particular, zoonotic viral infections have caused such lethal human diseases as sars, avian influenza, human monkeypox, and ebola [ ] . for many viruses, including coronaviruses and influenza viruses, host species-specific difference in viral entry receptors presents a major hurdle for cross-species transmission [ ] . poxviruses, however, can enter nearly any animal cell [ ] . why many poxviruses show strict host species specificity and what it would take for them to jump to new hosts are less clear [ ] . poxviruses include many lethal animal and human pathogens [ ] , the most infamous of which is the smallpox-causing variola virus. smallpox was successfully eradicated mainly through a global immunization program with vaccinia virus (vacv), and routine vacv vaccination had since discontinued. the human population is now vulnerable to zoonotic orthopoxvirus infection, as some extant poxviruses related to variola virus are capable of infecting a wide variety of wild and domestic animals. there are also many poxviruses with a more restricted host range [ ] . for example, capripoxviruses, consisting of sheeppox virus, goatpox virus, and lumpy skin disease virus, have a very narrow host-range in ruminants, causing economically significant diseases in sheep, goats, and cattle, respectively. host-restricted poxviruses have been exploited as safe vectors for vaccines, gene therapy or oncolytic viral therapies, although the basis for their host restriction is largely unknown [ ] . poxvirus host range at the cellular level is governed by a group of poxvirus genes referred to as the host range genes [ , ] . the first discovered and perhaps the most important host range genes are k l and c l of vacv [ , ] . vacv replication in most mammalian cell lines requires either k l or c l [ ] , and the deletion of both genes from vacv aborts the replication prior to viral late gene expression [ ] . k l is only present in vacv and a few related orthopoxviruses, but a c l homolog that functions nearly identically to vacv c l is present in most mammalian poxviruses [ ] . samd (sterile alpha motif domain-containing ) was found to be the restriction factor in human cell lines that blocked the replication of poxvirus mutants that lack k l and c l-like genes [ ] . k and c are structurally distinct [ , ] , but k and many c homologs independently bind and inhibit human samd [ , , ] . samd was initially identified as a tumor suppressor whose loss-of-function mutations in humans cause normophosphatemic familial tumoral calcinosis (nftc) [ , ] . humans and many other mammals also encode a chromosomally adjacent paralog of samd named samd -like (samd l). human samd and samd l are ubiquitously expressed in many tissues [ ] , and their expression can be further induced by interferons [ , ] . evolutionary analysis suggested that the two paralogs derived from the duplication of an ancestral gene early in mammalian evolution and that some mammalian species suffered a loss of either samd or samd l [ ] . notably, mice lack samd , while many ruminants lack samd l. mouse samd l is also a tumor suppressor, and haploinsufficiency of mouse samd l resulted in myeloid malignancies [ ] . the molecular functions of samd family of proteins remain largely elusive, but recent sequence analysis predicted a complex domain architecture suggestive of a regulative function of a putative signal transduction network [ ] . although samd has been established as a critical poxvirus restriction factor in human cell lines, whether this restriction is important at the organismal level and in other mammalian species is unknown. moreover, whether samd l plays a role in host defense is unknown. we studied the function of samd l from a host that lacks samd (mice) as well as a host that maintains both paralogs (humans). we found that samd l functions nearly identically to samd as a host restriction factor and that overcoming samd /samd l (samd /l) restriction is essential for poxvirus replication and pathogenesis. we also discovered some host species-specific difference in samd /l and some poxvirus species-specific difference in antagonizing samd /l, suggesting that these differences contribute to the barrier for crossspecies poxvirus infection. the importance of human samd as a poxvirus restriction factor became evident only when viral inhibitors of samd were removed from poxviruses, so we used a vacv mutant deleted of both k l and c l (vk l -c l -) as the model poxvirus to study poxvirus restriction factors in different host species. mouse is one of the mammalian species that have lost samd gene, and a previous study suggested that mouse samd l was not a functional paralog of human samd in terms of the tumor suppressor function [ ] . however, mouse cells, such as nih t cells and mouse embryonic fibroblasts (mefs), restricted the replication of vk l -c l - [ ] . to test whether mouse samd l (msamd l) might be the restriction factor for vk l -c l -, we used the crispr-cas technology to knock out msamd l from t cells. to control for potential off-target effect, we performed two independent knockouts with different guide sequences. to confirm the gene knockout, cell clones were isolated, and the msamd l genotype of representative clones from the two knockouts (named Δmsamd l# and Δmsamd l# ) was determined by sequencing. all msamd l alleles of the cell clones were found to contain indels, resulting predominantly in frameshift (s a fig) . while vk l -c lwas unable to replicate in the parental t cells, it replicated well in Δmsamd l# ( fig a) and Δmsamd l# (s b fig) cells, resulting in nearly -fold increase of viral titer after hours of infection. moreover, while vk l -c lwas sensitive to interferons (ifns) in permissive human cells [ , ] , its growth in Δmsamd l cells was not inhibited by pretreating the cells with ifn-β ( fig a) . as an alternative to the crispr-cas knockout, we prepared mefs from samd l +/+ and samd l -/mice. while vk l -c lfailed to grow in samd l +/+ mefs, its replication was completely restored in samd l -/-mefs (fig b) , confirming that msamd l is the restriction factor for vk l -c lin mouse cells. vk l -c lis highly attenuated in mice [ ] . to determine whether msamd l functions as a restriction factor at the organismal level, we tested whether vk l -c lwould regain virulence in samd l -/mice. intranasal infection of samd l +/+ and samd l +/mice with plaque-forming unit (pfu) of vk l -c ldid not cause any disease symptom or sustained body weight loss, similar to the mock infection (fig a) . the infected mice developed an antibody response to vacv (s a fig), indicating that they were properly infected. in contrast to their samd l +/+ and samd l +/littermates, all samd l -/mice lost close to % of body weight by day post infection and had to be euthanized. a similar lethal effect on samd l -/mice was observed when the infectious dosage was reduced to or pfu (fig b) , indicating that vk l -c lwas highly virulent in samd l -/mice. in mice, samd l haploinsufficiency caused myeloid malignancies [ ] . to test whether samd l haploinsufficiency would increase susceptibility to vk l -c linfection, we infected samd l +/+ and samd l +/mice with the highest dosage of vk l -c lwe could reasonably obtain ( pfu in μl). again, neither group of mice developed any disease symptoms or had sustained body weight loss (fig c) , and no virus was detected in lungs when the mice were euthanized at five days post infection (s b fig), indicating that one copy of msamd l gene is sufficient for restricting vk l -c l -. altogether, the results from samd l knockout cells and mice demonstrate that msamd l constitutes an essential host barrier for vk l -c lreplication and pathogenesis. we previously used vk l -c las the parental virus in constructing a panel of recombinant viruses that expressed different c homologs from representative mammalian poxviruses [ , ] . this panel of viruses were used for comparing the function of different c homologs in the same viral background, and they served as a surrogate model for different mammalian poxviruses in terms of their ability to overcome samd restriction. studies of these viruses showed that the c homologs from many mammalian poxviruses, including myxoma virus (myxv; infect rabbits) myxv-m , yaba-like diseases virus (yldv; infect monkeys) yldv- , swinepox virus (swpv) swpv- , and sheeppox virus (sppv) sppv- , could bind human samd and overcome its restriction [ ] . all these c homologs, except for sppv- , could also overcome the restriction of vk l -c lby mouse cell lines [ ] . to determine vk l -c lvirulence was restored in samd l -/mice. littermates from samd l +/mice were grouped according to their samd l genotypes and infected intranasally with vk l -c lor mock infected (pbs). the body weight change following the infection (average and standard deviation) is shown for each group. " à " indicates statistically significant difference (p < . by two way anova analysis). n denotes group size. wt vacv wr strain was used as a control in c. (d,e,f). the c homolog from sheeppox virus (sppv- ) has a host-species specific defect in mice. same as a except the viruses used were vsppv- (d), vswpv- (e), or vvacv-c (f), which are vk l -c l --derived viruses that expressed sppv- , swpv- or vacv-c , respectively. whether the defect of sppv- in mouse cell lines reflects a host species-specific defect, we studied the mouse virulence of vsppv- , the vk l -c l --derived recombinant virus that expressed sppv- . swinepox virus swpv- is the closest homolog of sppv- , so vswpv- , the vk l -c l --derived recombinant virus that expressed swpv- , was used as the control. similar to vk l -c l -, vsppv- did not result in significant body weight loss in samd l +/or samd l +/+ mice but caused lethal intranasal infection in samd l -/mice ( fig d) . in contrast, the same dose of vswpv- was lethal to samd l +/+ and samd l +/mice as well as to samd l -/mice (fig e) . in a separate experiment, vvacv-c , the vk l -c l -derived recombinant virus that expressed vacv-c , was also lethal to samd l +/+ and samd l +/mice ( fig f) . as sppv- can overcome samd restriction in human cells, these data show that sppv- has a host species-specific defect in mice. vacv k , c and c homologs from diverse poxviruses could bind msamd l, but the sheeppox virus c homolog has a specific defect due to the variation of two amino acids a biochemical basis for samd antagonism by the poxvirus proteins is their binding of samd [ ] , so we next studied the binding of msamd l by the viral proteins. due to the lack of a specific antibody to msamd l, a plasmid encoding a flag-epitope tagged msamd l was used in transfection of cells, which were subsequently infected with recombinant viruses expressing different v -tagged viral proteins. pulldown of vacv-k , vacv-c , myxv-m , swpv- or yldv- also precipitated msamd l ( fig a) . however, sppv- failed to co-precipitate msamd l. as expected, myxv-m and myxv-m , which are two additional c homologs from myxv previously known not to bind human samd [ ] , also failed to co-precipitate msamd l. residue and of sppv- were previously shown to be largely responsible for the defect of sppv- in mouse cells, and substituting them with the corresponding ones found in swpv- and vacv-c was sufficient to restore the host-range function in mouse cells without compromising the function in human cells [ ] . the same substitution was found to result in the binding of sppv- with msamd l ( fig b) . as controls, individual substitutions of two neighboring residues (residue or ) did not result in msamd l binding, correlating with their lack of effect on the host-range function in mouse cells [ ] . interestingly, residues and are only adjacent to the three conserved loops of the c -like proteins ( fig b) that are critical for the binding with samd [ ] . the finding that mouse samd l functions similarly to human samd as a poxvirus restriction factor raised the question about the function of human samd l (hsamd l). since knockdown of human samd (hsamd ) was sufficient for abolishing the host restriction for vk l -c lin hela and a cells [ , ] , hsamd l was not previously suspected to play a role in host restriction. to find out whether hsamd l expression might be impaired in cell lines, we performed western blot on hela cells and normal human fibroblasts derived from foreskin, and found that both hsamd and hsamd l were constitutively expressed and their expression was further induced by ifn-β ( fig a) . to study hsamd l function independent of hsamd , we knocked out hsamd from hela cells with crispr-cas . in a hela cell clone with hsamd knockout (Δhsamd ), no hsamd was detected by western blot even after the cells were treated with ifn-β ( fig a) . Δhsamd cells were fully permissive for the replication of vk l -c l -, as reported previously https://doi.org/ . /journal.ppat. .g [ ] . however, pretreating the Δhsamd cells with ifn-β restored the host restriction for vk l -c l -, reducing the viral yield by more than -fold compared to that in untreated cells (fig b) . to test whether hsamd l was responsible for the restriction, we knocked out hsamd l from Δhsamd and the parental hela cells. both the hsamd l single-knockout cells (Δhsamd l) or hsamd and hsamd l double knockout cells (Δhsamd &l) had no detectable level of hsamd l protein in untreated cells and only a trace amount of the protein after ifn-β stimulation (fig a) . in contrast to Δhsamd cells, Δhsamd &l cells remained permissive for vk l -c lafter ifn-β treatment (fig b) , and the growth of vk l -c lwas similar to that of the wild type (wt) vacv wr strain (s fig), indicating that ifn-β-induced hsamd l restricted the replication of vk l -c lin the absence of hsamd . Δhsamd l cells were similar to the parental hela cells in restriction of vk l -c l - (fig b) . to ensure that the hela cell results are representative of the phenotype in human cells, we performed similar knockout studies in a variety of human cells that we identified to be restrictive of the replication of vk l -c l -. these include normal human foreskin fibroblasts (hffs) and cancer cells derived from skin (a ), breast (hs t and mda-mb- ), cervix (ht- ), prostate (pc- ) and ovary (skov ). we transduced these cells with a lentivirus expressing a grna targeting either hsamd or hsamd l and pooled the stably transduced cells. the samd and samd l protein level in the pooled cells was reduced but not eliminated as in the clonally selected hela knockout cells (s a & s a figs) , presumably because the targeted gene was repaired with in-frame indels in a fraction of the cells. thus, the cells were conservatively speaking gene knockdown instead of knockout cells. nevertheless, the results were similar to that in knockout hela cells. the hffs were less stringent than the hela cells in restricting vk l -c l -, allowing the viral titer increase by~ -fold after thus, in all human cells that we have tested, the basal level of hsamd as well as ifninduced hsamd l are both capable of restricting vk l -c lreplication. vacv k , c and c homologs from diverse poxviruses could overcome hsamd l restriction, but the sheeppox virus c homolog has reduced potency due to the variation of two residues to find out whether mammalian poxviruses could overcome the restriction of hsamd l, we induced hsamd l expression in Δhsamd hela cells with u/ml of ifn-β and then infected the cells with our panel of vk l -c l --derived vacvs. viruses that expressed vacv-k , vacv-c , myxv-m , sppv- , swpv- and yldv- (but not myxv-m and myxv-m ) grew in ifn-treated Δhsamd cells (fig a) , indicating that all known samd antagonists could also antagonize hsamd l. we then studied these viral proteins for binding of hsamd l. while myxv-m (s fig) and myxv-m did not co-precipitate hsamd l, vacv-k , vacv-c , myxv-m , yldv- , sppv- and swpv- co-precipitated hsamd l (fig b) . among them, sppv- precipitated a lower amount of hsamd l, indicating a reduced affinity. this defect was again due to residue and of sppv- , as substitution of these two residues increased the precipitation of hsamd l without affecting the precipitation of hsamd (s fig). to determine whether the weaker hsamd l binding affinity by sppv- resulted in reduced inhibitory potency, we induced an increasingly higher level of hsamd l from Δhsamd hela cells with ifn-β and then infected the cells with either vsppv- or vswpv- . the two viruses grew equally well in untreated Δhsamd cells, reaching similar titers after hours of infection (fig c) . the viral yields were gradually reduced by the increasing concentrations of ifn-β, and the magnitudes of the reduction were significantly larger for vsppv- than for vswpv- when the interferon concentration was greater than u/ml, indicating that sppv- is less effective than swpv- at antagonizing hsamd l. discussion samd was recently identified as a restriction factor for poxviruses in human cell lines [ , ] . however, whether samd is important for host defense against poxvirus pathogenesis and whether similar antiviral defense exists in other mammals, especially those that lack a samd ortholog, were not established. in this study, we showed that the samd paralog, samd l, from mammalian species as diverse as mice and humans, functions similarly to human samd as a poxvirus restriction factor. since at least one of the two paralogs is present in all mammals with a completely sequenced genome [ ] , our finding indicates that samd and/or samd l-mediated antiviral defense is conserved in mammals. this conservation allowed us to use the mouse model to reveal the critical role of samd /l in host defense against poxvirus pathogenesis. studies of samd /l from two different mammalian species and the samd /l inhibitors from diverse mammalian poxviruses revealed some host speciesspecific difference in samd /l, which could serve as a barrier for cross-species poxvirus infection. this knowledge could be useful in assessing the potential of a given poxvirus in switching or expanding its host range. mouse is one of the mammalian species that have lost samd but maintained samd l. mouse samd l is % and % identical to human samd and samd l at amino acid level, respectively. through gene knockout with both the conventional technique as well as the crispr-cas technique, we found that mouse samd l was essential for restricting the replication of a model poxvirus, a vaccinia virus mutant deleted of both k l and c l (vk l -c l -). t cells with crispr knockout of samd l as well as samd l -/-mefs were permissive for the replication of vk l -c l -. furthermore, vk l -c lcaused lethal intranasal infection only in samd l -/mice but was completely avirulent in samd l +/+ and samd l +/mice, demonstrating the potency of samd l as a restriction factor at the organismal level. human is one of the mammalian species that have both samd and samd l, located head-to-tail in adjacent positions of the same chromosome. previous phylogenetic studies suggested that samd and samd l originated from a common ancestor through an ancient gene duplication event [ ] . gene duplications have a major role in evolution of new biological functions [ ] . with about % amino acid sequence identity, human samd and samd l could have diverged to take on different functions. this idea would be consistent with the previous findings that loss-of-function mutations in only samd cause nftc in humans [ ] and that knockdown of samd was sufficient for abolishing the restriction for vk l -c lin several human cell lines [ , ] . we were thus initially surprised to find that human samd l functioned similarly to samd as a restriction factor for poxviruses. the advent of crispr-cas genome editing technique made it possible to readily knock out one or both of the human paralogs from various human cells. the knockouts in tumor cell lines from various tissues and the normal human foreskin fibroblasts showed that both samd and samd l, when sufficiently expressed, could inhibit vk l -c lreplication. in many human cells, the basal level of samd is sufficient for restricting vk l -c l -, but samd l has to be induced by ifn to have the same effect. this probably only reflects a difference in gene regulation of the two paralogs in human cells but not any real difference in their potency or mechanism of action. mouse samd l is also an interferon-stimulated gene [ ] , but the basal level of samd l in mouse cells is sufficient for restricting vk l -c l -, again suggesting that the role of ifn in samd l function is merely to induce samd l to a sufficient level in some cell types. similarly, the only contribution made by k and c in vaccinia virus antagonism of ifn or irf [ , ] is likely the inhibition of ifn-or irf -induced samd /l, as vk l -c lwas not sensitive to ifn in cells that had samd /l knockout. both human samd and samd l have strong anti-proliferative function, and gain-offunction (gof) mutations of samd or samd l cause multisystem developmental disorder. gof samd mutations cause mirage (myelodysplasia, infection, restriction of growth, adrenal hypoplasia, genital phenotypes, and enteropathy) disorder [ ] , while gof samd l mutations cause a similar disorder characterized with cytopenia, immunodeficiency, and neurological symptoms [ , ] . in both cases, the gof mutations predispose to myelodysplastic syndrome by facilitating the selection for a loss of the chromosome that contain the mutations [ , ] . so why are both samd and samd l kept in humans and many other mammals? we can only postulate that the duplication of samd gene and the divergence of sequence and expression pattern represent a strategy for retaining two drastically different "alleles" of samd /l in the same host without the deleterious effect of both genes being constitutively active. having two different alleles of samd /l would have given the hosts more flexibility to rapidly evolve samd /l to evade the viral inhibitors, perhaps during a time of mammalian evolution when the challenge from poxviruses was particularly rampant. phylogenetic analysis has showed that both samd and samd l genes have been subjected to sustained positive selection [ ] . corresponding to the conservation of samd /l in mammals, the distribution of samd / samd l inhibitors in mammalian poxviruses are also very broad. nearly all mammalian poxviruses encode a c homolog that provides a host-range function similar to vacv c [ , , ] , and a few poxviruses also encode k (fig ) . all the functional c homologs and k were previously shown to bind and inhibit human samd [ , , ] . the breadth of their antagonistic activities is now expanded to include human and mouse samd l. among the c homologs from a wide variety of different mammalian poxvirus, the one from sheeppox virus, sppv- , stands out as the only one that displays a specificity for samd . sppv- could bind and inhibit human samd , but it has reduced potency against human samd l and failed to inhibit mouse samd l. remarkably, its binding to mouse samd l could be restored simply by substituting two residues, suggesting that subtle difference between samd and samd l might be responsible for their different ability in resisting sppv- . interesting, these two residues are only adjacent to the "three-fingered molecular claw" that is critical for samd binding [ ] , indicating that they may modulate the conformation of "the claw" to influence its binding specificity. sheeppox virus, goatpox virus, and lumpy skin disease virus (lsdv) are members of the capripoxvirus genus with a narrow host-range in ruminants. all capripoxviruses encode a nearly identical c homolog (> % identical) as sppv- , with the same residues at position and , suggesting that they all preferentially antagonize samd . this correlates with the loss of samd l in ruminants. thus, one evolutionary scenario is that the ancestor of sppv- could tolerate genetic drift that only disrupted samd l binding, after the ancestor of extant capripoxviruses established a niche host in ruminants and was no longer restricted by samd l. while the resulted failure of capripoxviruses in antagonizing mouse samd l could account for the host restriction of capripoxviruses in mice, the resulted reduction in potency against interferon-induced human samd l could only partly explain the host restriction of capripoxviruses in humans. we suspect that similar genetic drift might have occurred to other capripoxvirus host-range genes, resulting in their specialization to the ruminant hosts and altogether contributing to the host restriction of capripoxviruses in non-ruminant hosts. this idea is supported by a previous report that the sheeppox virus homolog of vacv host range gene e failed to provide the host range function in hela cells [ ] . that mouse samd l is completely resistant to sppv- while human samd l is only partially resistant could have been the result of a stronger positive selection in rodents, perhaps by some poxviruses that have similar inhibitory profile as the capripoxviruses. overall, our studies suggest that host species-specific difference in samd /l gene repertoire contributes to the barrier for cross-species poxvirus transmission. the animal studies reported in this paper were approved by the institutional animal care and use committee at the university of texas health science center at san antonio (protocol no. - ) , and pc- (atcc crl- ) were originally from atcc. hek ft was from thermo fisher scientific (cat. no. r ). mouse embryonic fibroblasts (mefs) were generated from embryos of samd l −/− and samd l +/+ mice according to standard protocols. human foreskin fibroblasts (hffs), described in [ ] , were kindly provided by dr. zhilong yang. wt vacv wr strain, k l and c l deletion vacv (vk l -c l -) and a panel of vk l -c l -derived recombinant viruses expressing vacv-k (vvacv-k l) or a c homolog from different poxviruses (vvacv-c l, vyldv- r, vmyxv-m r, vmyxv-m r, vmyxv-m r, vsppv- and vswpv- ) were described before [ , , ] . vsppv- with mutations at residue , , & of sppv- were also described before [ ] . all viruses were propagated and titrated on vero cells. the plasmids used for the gene knockout were constructed from lenticrisprv (a gift from feng zhang, addgene plasmid # ) according to the published protocol [ ] . in brief, len-ticrisprv was digested with bsmbi and ligated with a pair of oligonucleotides with the specific guide sequence. for each target gene, two guide sequences were chosen from the human or mouse genome-wide sgrna library [ ] . they were as follows: '-tatccgggaaccac ggttcg- ' (msamd l # ), '-acaccaacaatttccccgtg- ' (msamd l # ), '-ttgactgaacaagacgtgaa- ' (hsamd # ), '-gagaatttgttcttcgatac- ' (hsamd # ), '-cctgaccagttagacgacgc- ' (hsamd l # ), and '-ggctagctc tagggatatcc- ' (hsamd l # ). the lenticrisprv -derived plasmid was either transfected directly to the target cells or used in making lentiviruses for transduction of the target cells. in the latter case, the lentiviral plasmid and the packaging plasmids pmd .g and pspax (gifts from didier trono, addgene plasmid # & ) were transfected into hek ft cells with lipofectamin (thermo fisher scientific). hr post transfection, culture supernatants were collected, clarified by centrifugation, passed through a . μm filter, and used for transduction. for lentiviral transduction, the lentiviruses and the target cells in medium containing μg/ml polybrene were centrifuged at , rpm for hr. hr after either transfection or transduction, the cells were subjected to puromycin selection for (transfection method) or (transduction method) days. the puromycin concentrations are μg/ml for hela, μg/ml for t , and μg/ml for all other cells. for each gene knockout in t and hela cells, two separate knockouts with different guides were performed. clones of the knockout cells were isolated and validated by western blotting for hsamd or hsamd l or genotyping for msamd l. for genotyping, the genomic dna of the clones was extracted using the quickextract dna extraction kit (epicentre).~ bp of dna flanking the target site was pcr-amplified with the primer pair ( '-ggccactcaatctcattgacccaat- ' and '-tgcccaggatattcttagagctag c- ') and cloned to pgem vector (promega). the sequence of - pgem clones was determined by sanger sequencing. for knockout of hsamd or hsamd l in additional human cell lines and hffs, only guide # described above was used for the knockout. the stably transduced target cells were pooled without clonal selection, and the knockout was validated by western blot. mice with different samd l genotypes used in the infection experiment were generated from breeding pairs of samd l +/mice [ ] . at around to weeks old, the mice were infected intranasally with viruses in μl pbs as described previously [ ] . the viruses used were purified through a sucrose cushion sedimentation according to the standard protocol [ ] . individual mice were weighed every day and euthanized when % of the body weight was lost. statistical comparisons of body weight changes among groups were analyzed by two way anova using graphpad prism . . values of p < . were considered statistically significant. the anti-vacv serum antibody titers of some infected mice were determined by elisa with purified vacv virions as described previously [ ] . all mouse protocols were approved by uthscsa iacuc. msamd l expression plasmid msamd l orf was pcr-amplified with the primer pair ( '-agtggacaagtaactcaac caaaattg- ' and '-gatcacttttatgccatatgcc- ') from cdna synthesized from t cellular mrnas. a xflag tag sequence and a ha tag sequence were then appended to the ' and ' end of the msamd l orf by recombinant pcr, and the final pcr product was inserted between kpni and sacii sites of the pcdna . /v -his-topo vector (thermo fisher scientific). the msamd l orf was completely sequenced and found to have one amino acid difference (val instead of ile at ) compared to the msamd l reference sequence in gen-bank (np_ . ). the difference was not corrected, as samd l from most rodents also has val at this position. cells in -well plates were incubated with pfu per cell of different viruses for h at room temperature. following adsorption, the cells were washed twice with phosphate-buffered saline (pbs). one set of the cells was harvested immediately as the hr post infection sample, while the other set were moved to ˚c incubator to initiate viral entry and harvested at hr post infection. the viral titers in the cell lysates were determined by plaque assays on vero cells. for testing the effect of ifn, human or murine cells were treated with u/ml of human or murine ifn-β (pbl biomedical laboratories) for hr, before the cells were infected with vacv as described above. for assessing viral late protein expression, western blot with a mab against the vacv late protein wr (clone he ) [ ] was performed. for immunoprecipitation of hsamd or hsamd l, hela cells or ifn-treated Δhsamd hela cells were infected with different vacv viruses. for immunoprecipitation of msamd l, ft cells were transfected with the msamd l expression plasmid and then infected with different vacvs at hr post transfection. after hr of infection, the cells were lysed on ice with a lysis buffer ( . % (w/v) np- , mm tris, ph . , mm nacl) supplemented with protease inhibitor cocktail tablets (roche molecular biochemicals). the cleared cell lysates were mixed with μl of % (vol/vol) v -agarose beads (sigma-aldrich) for min at ˚c. after washing with lysis buffer, the beads were resuspended in sds sample buffer, the eluted proteins were resolved by sds-page and detected with western blot as described previously [ ] . the detection antibodies were mouse monoclonal antibodies (mab) against v (sigma-aldrich; clone v - ), flag tag (sigma-aldrich) or hsp (santa cruz), and rabbit polyclonal antibodies against hsamd (sigma-aldrich, hpa- ) and hsamd l (proteintech, - -ap). supporting information s fig. related to fig a. (a) . genotyping Δmsamd l t cells. the msamd l knockout cell lines (Δmsamd l) were constructed by transient transfection of t cells with a plasmid encoding cas and a grna targeting msamd l (guide# or guide# ). the targeted samd l sequence is underlined with the pam sequence in bold italics and the encoded amino acid sequence shown above the dna sequence. shown below the target are the genomic sequences from representative cell clones (Δmsamd l# and Δmsamd l# ). gray and crossed-out sequence indicates deletion.^indicates insertion. the number after the + and − denotes the number of indels, and the number before the "x" denotes the number of times the sequence was detected from a total of - cloned pcr products. (b). the restriction of k l and c l deletion vaccinia virus (vk l -c l -) in t cells was abolished by knocking out msamd l with crispr-cas . a validated msamd l knockout cell clone (Δmsamd l # ) and the parental t cells were infected with vk l -c lat an moi of pfu/cell. viral growth was determined by measuring viral titers at and hour-post-infection (hpi). (pdf) s fig. (a) . vk l -c linfection induced anti-vacv antibody response in mice. mice described in fig a were emerging viral diseases: confronting threats with new technologies cross-species virus transmission and the emergence of new epidemic diseases poxvirus cell entry: how many proteins does it take? viruses poxviridae: the viruses and their replication poxviruses and the evolution of host range and virulence vaccinia virus host range genes localization and sequence of a vaccinia virus gene required for multiplication in human cells the cowpox virus host range gene, cp , affects phosphorylation of eif alpha and vaccinia viral translation in apoptotic hela cells identification from diverse mammalian poxviruses of host-range regulatory genes functioning equivalently to vaccinia virus c l m is a host range factor essential for myxoma virus pathogenesis and functions as an antagonist of host samd in human cells structural basis for antagonizing a host restriction factor by c family of poxvirus host-range proteins structure function studies of vaccinia virus host range protein k reveal a novel functional surface for ankyrin repeat proteins identification of restriction factors by human genome-wide rna interference screening of viral host range mutants exemplified by discovery of samd and wdr as inhibitors of the vaccinia virus k l-c l-mutant a deleterious mutation in samd causes normophosphatemic familial tumoral calcinosis normophosphatemic familial tumoral calcinosis is caused by deleterious mutations in samd , encoding a tnf-alpha responsive protein human sterile alpha motif domain , a novel gene identified as down-regulated in aggressive fibromatosis, is absent in the mouse sterile alpha motif containing domain is involved in death signaling of malignant glioma treated with inactivated sendai virus particle (hvj-e) or type i interferon gain-of-function samd l mutations cause a syndrome of cytopenia, immunodeficiency, mds, and neurological symptoms evolution and divergence of the mammalian samd /samd l gene family haploinsufficiency of samd l, an endosome fusion facilitator, causes myeloid malignancies in mice mimicking human diseases with monosomy the complex domain architecture of samd family proteins, predicted stand-like ntpases, suggests new links to inflammation and apoptosis mouse samd l is not a functional paralogue of the human samd , the gene mutated in normophosphataemic familial tumoral calcinosis c l family of poxvirus host range genes inhibits antiviral activities induced by type i interferons and interferon regulatory factor vaccinia virus k l and c l inhibit antiviral activities induced by type i interferons samd is an innate antiviral host factor with stress response properties that can be antagonized by poxviruses gene duplication as a mechanism of genomic adaptation to a changing environment interferome v . : an updated database of annotated interferon-regulated genes samd mutations cause a novel multisystem disorder, mirage syndrome, and are associated with loss of chromosome ataxia-pancytopenia syndrome is caused by missense mutations in samd l the poxvirus c l host range factor superfamily comparative analysis of poxvirus orthologues of the vaccinia virus e protein: modulation of protein kinase r activity, cytokine responses, and virus pathogenicity the '-poly(a) leader of poxvirus mrna confers a translational advantage that can be achieved in cells with impaired cap-dependent translation vaccinia virus k l protein supports viral replication in human and rabbit cells through a cell-type-specific set of its ankyrin repeat residues that are distinct from its binding site for acap improved vectors and genome-wide libraries for crispr screening genome-scale crispr-cas knockout screening in human cells preparation of cell cultures and vaccinia virus stocks. current protocols in protein science / editorial board enhancement of immune response to an antigen delivered by vaccinia virus by displaying the antigen on the surface of intracellular mature virion. vaccine generation and characterization of a large panel of murine monoclonal antibodies against vaccinia virus we thank jouni uitto for shipping the samd +/mice. we thank zhilong yang and nicholas wallace for providing human foreskin fibroblasts. conceptualization: yan xiang. key: cord- -wk u a w authors: lamb, daniel; schüttelkopf, alexander w.; van aalten, daan m. f.; brighty, david w. title: charge-surrounded pockets and electrostatic interactions with small ions modulate the activity of retroviral fusion proteins date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: wk u a w refolding of viral class- membrane fusion proteins from a native state to a trimer-of-hairpins structure promotes entry of viruses into cells. here we present the structure of the bovine leukaemia virus transmembrane glycoprotein (tm) and identify a group of asparagine residues at the membrane-distal end of the trimer-of-hairpins that is strikingly conserved among divergent viruses. these asparagines are not essential for surface display of pre-fusogenic envelope. instead, substitution of these residues dramatically disrupts membrane fusion. our data indicate that, through electrostatic interactions with a chloride ion, the asparagine residues promote assembly and profoundly stabilize the fusion-active structures that are required for viral envelope-mediated membrane fusion. moreover, the blv tm structure also reveals a charge-surrounded hydrophobic pocket on the central coiled coil and interactions with basic residues that cluster around this pocket are critical to membrane fusion and form a target for peptide inhibitors of envelope function. charge-surrounded pockets and electrostatic interactions with small ions are common among class- fusion proteins, suggesting that small molecules that specifically target such motifs should prevent assembly of the trimer-of-hairpins and be of value as therapeutic inhibitors of viral entry. bovine leukemia virus (blv) and human t-cell leukemia virus type- (htlv- ) are related deltaretroviruses that cause aggressive lymphoproliferative disorders in a small percentage of infected hosts [ , , , , , ] . like other enveloped viruses, retroviruses must catalyse fusion of the viral and target cell membranes to promote entry of the viral capsid into the target cell. the retroviral class i fusion protein consists of the transmembrane glycoprotein (tm) component of the envelope glycoprotein complex [ ] . envelope is displayed on the surface of the virus or infected cell as a trimer, with three surface glycoprotein (su) subunits linked by disulphide bonds to a spike of three tm subunits [ ] . experimentally validated models suggest that su-mediated receptor engagement induces isomerisation of the inter-subunit disulphide bonds and initiates a cascade of conformational changes that activate the fusogenic properties of tm [ , ] . membrane fusion is achieved by re-folding of the tm from a native non-fusogenic structure through a rod-like pre-hairpin intermediate, in which the c-and n-terminal segments are embedded in the viral and target cell membranes respectively [ , ] . the pre-hairpin intermediate then resolves to a trimer-of-hairpins structure, which pulls the membranes together and facilitates lipid mixing and membrane fusion [ , , , ] . for several viruses membrane fusion is sensitive to inhibition by peptides that mimic a c-terminal region of the trimer-of-hairpins [ , , , , , , ] . the c-terminal fragment of the htlv- trimer-of-hairpins exhibits a short a-helical motif embedded in an extended non-helical peptide structure referred to as the leash and a-helical region (lhr) [ , ] . the lhr-based mimetics are structurally distinct from the prototypic extensively a-helical peptide inhibitors of human immunodeficiency virus but are reminiscent of the leash regions observed in influenza haemagglutinin [ , , , ] . importantly, amino acid residues that are required for potent inhibitory activity of the htlv- and blv peptides are not fully resolved in the available htlv- tm structure, yet this information is critical to the development of therapeutically relevant peptide or low-molecular-weight inhibitors of htlv- entry [ , ] . moreover, other class i fusion proteins have extended non-helical elements in the c-terminal region of the trimer-of-hairpins and understanding how these elements contribute to the leash in a groove mechanism of fusion protein function will have broad relevance to anti-viral therapies [ , ] . we therefore sought to examine the structure and function of the blv tm and to compare this information with data derived from diverse class i fusion proteins and the related htlv- tm structure. here we show the structure of the post-fusion conformation of the blv tm ectodomain and demonstrate that coordinated ions and a network of hydrogen-bonded water molecules make critical contributions to the assembly and stability of the trimer-of-hairpins form of the blv tm and are essential for tm-mediated fusion. additionally, we resolve a region of the lhr that is critical to the activity of peptide inhibitors of htlv- and blv entry. we provide evidence that basic residues in a membrane proximal helical element of the lhr interact with charged residues that surround an extended hydrophobic pocket on the coiled coil. this charge-surrounded pocket represents an attractive target for antiviral drugs. our data indicate that coordinated ions and charge-surrounded hydrophobic pockets are functionally significant leitmotifs of class i fusion proteins. the structure of the blv tm ectodomain to obtain crystals for structural studies, the n-and c-terminal limits of the coiled coil region of blv tm were identified using learncoil vmf [ ] . the tm ectodomain, including the predicted coiled coil and lhr from ala to trp , were fused to the c-terminal end of maltose binding protein via a threealanine spacer following the methodology of center et al [ ] . the soluble purified protein was crystallised and the structure solved to a resolution of . Å (fig. ). as anticipated, the overall fold of the blv tm ectodomain is that of a trimer-of-hairpins, with the nterminal a-helices twisting around each other to produce the central triple-stranded coiled coil that is characteristic of class i fusion proteins. buried leucine and isoleucine residues within the coiled coil predominantly mediate the interactions between monomers, but strikingly there are two polar layers within the coiled coil that establish interactions with ordered water molecules and a chloride ion respectively (see below). at the base of the coiled coil the peptide backbone undergoes a u loop forming the chain reversal region (fig. a, b) , within which is a short helical segment containing the first cysteine of the conserved cx cc motif [ , ] . the predicted disulphide between cys and cys is reduced in the resolved structure but this does not appear to affect the overall protein fold. in support of this view, preliminary lower resolution structures obtained for crystals formed in the absence of tcep-hcl reveal an intact disulphide (data not shown). these data indicate that the disulphide is not essential for constraining the chain reversal region in the folded protein, even though it might play a role in the folding process. therefore, the known defects in membrane fusion associated with substitution of these cysteines are likely due to direct perturbation of the inter-subunit disulphide isomerisation step of the envelopemediated membrane fusion process [ , ] . after cys , the lhr begins with an extended non-helical leash followed by an eightresidue a-helix. this helix is followed by a three-residue linker incorporating a single proline, allowing a sharp kink in the lhr prior to the start of a second a-helix that adopts an orientation almost u to the first helical segment of the lhr (fig. a , b, c). a panel of mutants in blv envelope are processed and expressed identically in vivo guided by the crystal structure, a panel of mutants in blv envelope were designed to perturb key features of the trimer-ofhairpins structure. a common difficulty with mutagenesis of viral envelopes, particularly the tm, is that at particular positions even conservative substitutions can dramatically impair the proteolytic processing and cell surface expression of envelope [ , ] . therefore, we compared each of the mutants with the parental ''wild-type'' envelope and confirmed appropriate expression, processing and cell surface presentation of the tm mutants. no significant differences in either the expression level of gp or the cleavage of the precursor protein, gp , were observed ( fig. a) . moreover, flow cytometry analysis confirmed that in each case the mutant envelope was displayed on the cell surface at levels equivalent to wild type (fig. b) . subsequently, the envelopeexpressing cells were used as effector cells in syncytium formation assays, and the efficiency of each mutant envelope relative to wildtype in catalysing membrane fusion was calculated as the relative fusogenic index. the effect of each mutation on fusogenicity (fig. c) , and the mutant phenotypes are interpreted with reference to the crystal structure below. solvent molecules are critical for stable assembly of the core coiled-coil the trimerisation of the n-helices appears to be facilitated by presentation of aliphatic residues to the interacting faces of the tm monomers, thereby forming a hydrophobic core down the axis of the central coiled coil. notably, in the blv trimer-of-hairpins there are two positions along this interface that harbour polar residues, thr and asn (fig. a, b ). thr is located approximately half way up the n-helix, and it is oriented such that the methyl group of the side chain points toward the centre of the coiled coil and the hydroxyl group faces toward the neighbouring n-helix. in this position, thr participates in a complex network of hydrogen bonding via several buried ordered water molecules. in particular, thr makes a contact through one water molecule with the main-chain of glu and through a separate water molecule to the side-chain of his , one of the residues which forms the wall of the groove into which the lhr human t-cell leukaemia virus types- (htlv- ) and bovine leukaemia virus (blv) are divergent blood borne viruses that cause hematological malignancies in humans and cattle respectively. in common with other enveloped viruses, infection of cells by htlv- and blv is dependent on the membrane fusion properties of the viral envelope glycoproteins. here we have solved the crystal structure of the blv transmembrane glycoprotein, and, through a functional and comparative analysis with htlv- , we have identified features that are critical to fusion protein function. in particular, we demonstrate that electrostatic interactions with small ions dramatically stabilize the assembly and fusion-associated forms of the blv tm, but are not required for the cell surface display of native prefusogenic envelope. moreover, we show that charged residues that border a deep hydrophobic pocket contribute directly to appropriate folding of fusion-active envelope and are critical to membrane fusion. importantly, the charged residues that border the pocket are key features that determine the specificity and activity of peptide inhibitors of envelope function. our study demonstrates that charge-surrounded pockets and electrostatic interactions with small ions are significant leitmotifs of diverse class- fusion proteins and that these elements represent ideal targets for novel small-molecule inhibitors of viral entry. binds (fig. a ). in the context of envelope the t v substitution, which replaces threonine with a non-polar residue of equivalent size, completely abrogates envelope-mediated membrane fusion (fig. c) . moreover, the introduction of the t v substitution into the pmbp-blvhairpin vector yields a recombinant protein that completely fails to trimerise (fig. e) . interestingly, the data demonstrate that substitutions at thr do not affect expression, processing, or surface expression of native envelope but severely impair the fusogenic activity of envelope by preventing assembly of the trimer-of-hairpins. our data therefore provides further evidence that the trimeric coiled coil likely forms during the fusion process. a spherical density feature was observed on the central axis of the molecule; comparison of refined temperature factors with those of the surrounding protein atoms suggests an entity more electron-dense than water. based on the chemical environment, the shape of the density and b factor comparisons we have modeled this feature as a chloride ion. this chloride ion is situated towards the chain reversal end of the coiled coil between a group of three asparagines, one from each monomer of tm (asn ). these asparagine residues establish electrostatic interactions with the chloride ion by creating a slightly positively charged microenvironment between the n-helices. an alignment of envelope sequences from diverse viral groups (fig. c ) reveals that this asparagine is conserved. moreover, comparison of the crystal structures for the fusion protein ectodomains of htlv- [ ] , momlv [ ] , ebola [ , ] , and syncytin- [ ] , a protein derived from a human endogenous retrovirus that plays a critical role in the implantation of the trophoblast into the wall of the uterus [ ] , show that the ability to coordinate chloride is retained (fig. d) . to test the importance of the chloride interacting asparagines, we introduced amino acid substitutions of similar size and geometry. introducing a leucine residue at position of blv tm renders envelope entirely non-fusogenic. the n l substitution does not completely prevent trimerisation of the blv coiled coil in vitro but it significantly destabilises the trimer and a large broad peak corresponding to higher order oligomers and aggregated material is observed by gel filtration chromatography (fig. e ). interestingly, a substitution whereby asn is replaced by an aspartic acid residue produces a decrease in fusogenicity of almost % (fig. c ). the n d substitution replaces an amide with a carboxylate group in the centre of the coiled coil, which inverts the surface charge of the binding pocket, and therefore would not support interaction with a chloride. in contrast to n l, the n d mutation should maintain the hydrogen bonding in and around the chain-reversal region mediated by the carbonyl group that is common to both asparagine and aspartic acid, and therefore the disruption of fusion should be due primarily to the absence of the chloride ion, albeit we cannot exclude the possibility that the close proximity of negatively charged aspartate side chains impairs trimerisation. however, our biological assays demonstrate that the n d substitution, unlike n l, retains some membrane fusion activity. in vitro, rather than producing a range of higher order species, the introduction of the n d mutation into pmbp-blvhairpin produces a recombinant protein with a major peak at an elution volume consistent with the molecular weight of a tetramer rather than a trimer (fig. e ). tetramerisation is unlikely to be the reason for the compromised fusogenic activity of the n d-envelope; nonetheless, the results demonstrate the importance of the chloride-interacting asparagines to the stability of the trimeric coiled coil. along the length of the lhr multiple contacts are made with the coiled coil and such interactions are required for the activity of inhibitory lhr-mimetic peptides [ , , , ] . amino acid residues that are critical determinants of peptide potency map to the c-terminal region of the lhr [ , ] , but key residues and the manner in which they interact with the coiled coil are not resolved in the published htlv- tm structure [ ] . a detailed view of the blv lhr bound to the coiled coil and a sequence comparison of the htlv- and blv lhrs are shown in fig. c and d. towards the n-terminus of the blv lhr, conserved residues leu and ile dock into a hydrophobic pocket on the coiled coil, and an array of polar side chains at one side of the pocket interact with the lhr peptide backbone (fig. c) . we have previously demonstrated that interactions with the peptide backbone contribute to the activity of the blv and htlv- peptide inhibitors [ , ] . by contrast, the side chain of arg projects out into solvent suggesting that this residue is not essential for interaction of the lhr with the coiled coil or for trimer-ofhairpins formation (fig. c ). in keeping with this view, the r a substitution has no significant effect on envelope fusogenicity (fig. c) and therefore serves as a useful control for our analysis of envelope structure and function. as the n-helices twist around one another the groove between them accommodates the first conserved a-helical segment of the lhr. within this a-helix, blv residues ile and leu extend down and pack into the groove (fig. c) . significantly, substitution of leu with alanine results in an % loss of fusogenic activity relative to wild type (fig. c) , indicating that this interaction contributes substantially to the stability of the lhr/coiled coil interaction. the first a-helix of the blv lhr ends at asp , and between the end of this helix and the beginning of the next is a threeresidue linker, comprised of leu , gln and pro . although leu is positioned such that it faces the groove between n-helices, a ridge across this groove prevents leu from docking particularly deeply (fig. c ). this leucine is not conserved among leukaemia viruses. instead, there is an arginine residue at this position of the htlv- lhr that makes contact with the coiled coil. such differences may account, in part, for the specificity and significant differences in potency that are observed for the blv and htlv- peptide inhibitors [ ] . the conserved proline (blv pro ) of the extended non-helical linker induces a sharp kink in the lhr thereby allowing a second a-helix to bind almost directly across the groove, at right angles to the rest of the lhr. crucially, this second helix is not resolved in the published htlv- tm structure [ ] . at the point at which this change of direction occurs, a conserved leucine, leu , docks into the start of a deep hydrophobic pocket. adjacent to leu in this pocket and within the second lhr helix is val , which is also conserved in htlv- (fig. c, d) . substitution of either leu or val with alanine markedly impairs the fusogenic function of envelope (fig. c) . the substitution of l a is somewhat more detrimental than substitution of val , with an activity loss of % and % respectively. embedded within the second helical element of the lhr is an arginine (arg ) that participates in electrostatic interactions with the coiled coil (fig. c ). this residue is conserved in htlv- and is critical to the inhibitory activity of the htlv- lhr mimetic peptide p cr - [ ] . intriguingly, arg projects back along the axis of the coiled coil and binds between leu and leu , where it forms hydrogen bonds with gln from the same nhelix and asp from an adjacent n-helix and also donates a hydrogen bond, through the e-nitrogen, to the main chain carbonyl of glu of the lhr (fig. c) . the substitution of arg with alanine dramatically disrupts the fusogenic activity of envelope and reduces envelope function by more than % relative to wild type (fig. c) . moreover, arg cannot be functionally replaced by lysine. the r k mutant, while more fusogenic than the alanine substituted derivative, is still % less effective at catalysing membrane fusion than wild-type envelope (fig. c) . these data indicate that the electrostatic and hydrogen bonding interactions made by arg are essential to the envelope-mediated membrane fusion process. contrasting effects of two arginine residues on the stability of the trimer-of-hairpins based on homology modelling, we previously suggested that arg of the lhr projects out from one side of the a-helix and is repelled electrostatically by n-helix residue arg [ ] . this prediction is substantiated by the crystal structure presented here (fig. c) . moreover, the r a substitution yields an envelope that produces extensive syncytia and is significantly more fusogenic than wild-type envelope (fig. c and fig. c) . thus, the substitutions r a and r a have very different effects on the fusogenicity of blv envelope, increasing activity by over two-fold and almost completely abolishing activity respectively (fig. c and c ). using a modified thermal aggregation assay [ ] , we assessed the relative effects of these two substitutions on the thermostability of the blv trimer-of-hairpins. control experiments revealed that after a five minute incubation at uc, on average % of the wild-type mbp-blv-hairpin protein was present in high-order aggregates (fig. a, b) . however, when the same heat treatment was applied to the protein bearing the r a mutation, we found that only % of protein aggregated. the difference to wild-type hairpin was significant (p# . , t-test) (fig. a, b) . by contrast, the r a substitution resulted in aggregation of over % of total protein, again a significant difference to wild type (p# . , t-test) ( fig. a and b) . notably, even without heat treatment over % of the r a protein was aggregated (fig. a) . furthermore, these relative differences in the propensity of the recombinant proteins to aggregate were maintained following heat treatment at and uc (fig. b) . hence, the contrasting effects of the arginine substitutions on envelope fusogenicity are directly due to changes in stability of the post-fusion trimer-of-hairpins structure. a prediction based on the increase in thermal stability of the r a substituted trimer-of-hairpins is that such substitutions should confer reduced sensitivity to lhr-mimetic peptide inhibitors. to test this view we compared the activity of the p blv - antagonist of envelope fusion [ ] against wild type or r asubstituted envelope. this peptide mimics residues cys to gln of blv env. in syncytium interference assays, p blv - inhibited fusion catalysed by wild-type envelope with an ic of . . mm (fig. d ). however, p blv - only inhibited fusion catalysed by r a envelope with an ic estimated to be . mm, and even at peptide concentrations up to mm inhibition of syncytium formation was markedly damped indicating that both the potency and efficacy of the peptide was reduced against r a substituted envelope (fig. d) . moreover, though more active against native envelope, a reciprocally substituted peptide antagonist, p blv -r a, failed to exhibit full potency against the r envelope derivative; p blv -r a inhibited wild-type envelope catalysed membrane fusion with an ic of . . mm, but inhibited the r a envelope with an ic of . mm (fig. e) . in both experiments, c (a peptide mimetic of hiv- gp residues gly to leu ) was used as a negative control. thus, improving the thermal stability of the trimer-of-hairpins form of a class- fusion protein significantly reduces the sensitivity of envelope to peptide inhibitors targeted to the coiled coil. the structure of the c-terminal segment of the blv lhr and the accumulated data suggest a key role for the conserved arginine (arg ) in the mechanism of envelope-mediated membrane fusion. moreover, our recent data indicate that the equivalent arginine residue, arg , of the htlv- lhr-mimetic peptide is critical to inhibitory activity [ ] . notably, for the htlv- lhrbased inhibitor residues equivalent to leu , arg and leu are also of importance to the biological activity of the peptide [ , ] . we therefore sought to establish whether or not the conserved arginine in the htlv- lhr docks with the coiled coil in a similar manner to that of blv and if this residue is important to membrane fusion mediated by the htlv- tm. we generated a small panel of mutants in htlv- envelope, substituting arg of the lhr with alanine and lysine (r a and r k), and making similar substitutions for arg (r a and r k). using a monoclonal antibody recognising htlv- su, we confirmed by western blotting and flow cytometry that all four mutant envelopes were expressed and post-translationally processed in a manner identical to wild type (fig. a, b) . in syncytium formation assays, the individual alanine substitutions of arg and arg resulted in envelopes that are % and % less fusogenic than wild-type envelope respectively (fig. c) . significantly, the lysine substitution of arg produced an envelope that was not significantly less fusogenic than wild type (p. . , t-test) (fig. c ). however, arg could not be functionally replaced with lysine, the r k mutation resulted in an % reduction in fusogenic activity. taken together with the data from the blv r k mutant, this suggests that htlv- r adopts a near-identical conformation to blv r at the cterminal segment of the lhr. the likely conservation of position and orientation of htlv- r in the trimer-of-hairpins allowed us to construct a model for the binding of the c-terminal segment of the htlv- lhr to the coiled coil (fig. d) . the model suggests that r could bind to a negatively charged ridge that is orientated across the groove between n-helices and that lies between the binding pockets for leu and leu and that r docks adjacent to r . as such, four critical interactions between lhr side chains and the coiled coil are contained within a binding hotspot of , Å x , Å and disruption of these interactions profoundly impairs envelope-mediated membrane fusion. there is greater sequence divergence between blv and htlv- than, for example, between siv and hiv- [ , ] , nonetheless the crystal structures of the blv and htlv- trimerof-hairpins are similar. the incorporation of ordered water molecules and ions to facilitate trimerisation of the coiled coil and assembly of the trimer-of-hairpins is seen in several viral fusion proteins [ , , , ] . the crystal structure of siv gp implicates multiple water molecules in the folding of the trimerof-hairpins [ ] . however, the number of water molecules concentrated in one region of the blv coiled coil is unusual. the interaction of thr with an array of water molecules appears to play a critical role in assembly of the coiled coil, as substitution of t eliminates both in vitro trimerisation and in vivo fusogenicity. notably, for htlv- the threonine residue is replaced by an asparagine, which by virtue of its larger side chain directly hydrogen bonds with the adjacent n-helix. by contrast, thr of blv makes this contact through water-mediated hydrogen bonds. however, the contribution of polar interactions to coiled coil assembly is maintained at this location in htlv- by interaction of a chloride ion on the symmetry axis of the coiled coil. moreover, for both viruses three conserved asparagine fusogenicity of htlv- c-terminal lhr arginine mutants. the fusogenic index was calculated as described (mean sd from triplicate assays). (d) a ''hotspot'' for binding of htlv- fusion inhibitors based on the previously published htlv- structure and modelled incorporating the data from this study. four key residues (leu , arg , leu and arg , shown as yellow sticks) interact with the htlv- coiled coil (grey space-filling model, region interacting with lhr residues shown in red) within a compact binding site. the contact made by arg is critical to the binding and functional activity of htlv- peptide inhibitors. doi: . /journal.ppat. .g residues located towards the membrane distal end of the coiled coil interact with a chloride ion. the properties of the n l substitution and the failure of n d to rescue envelope function indicate that the observed chloride-mediated interactions are essential for stable assembly of the coiled coil and for tmmediated membrane fusion. significantly, the asparagine (asn ) and adjacent arginine (arg ) residues of blv are conserved between blv, htlv- and a number of disparate viral fusion proteins, and the ability of the asparagines to interact with a chloride ion is maintained. critically, the chloride ion is present within the post-fusion conformation of ebola gp ( ), but is conspicuously absent in a pre-fusion structure resolved by lee et al [ ] wherein the conserved asparagines adopt a more open arrangement and the coiled coil is only partially trimerised. we suggest that asn , as a direct consequence of conformational changes in su resultant from receptor binding, and in concert with the equivalent asparagines from neighbouring monomers, mediates electrostatic interactions with a chloride ion, which brings the n-helices together, thus driving the trimerisation of the coiled coil to yield the pre-hairpin intermediate in an event pivotal to successful fusion. in addition, our results indicate that trimerisation of the n-terminus of tm is not a requirement for env maturation and surface display of trimeric pre-fusogenic envelope, offering a novel insight into the conformation of retroviral env spikes prior to activation. taken together, the data suggest that while the ''knobs-into-holes'' interactions of aliphatic residues at the centre of the coiled-coil regions of class viral fusion proteins are important for trimerisation of the fusion-active coiled coil the polar layers among these hydrophobic interactions are critical. this information should be considered when designing trimeric immunogens based on the coiled coil of tm. moreover, the structure in and around the chloride-interacting region has been conserved in diverse viral fusion proteins and may be susceptible to coiled coil destabilising drugs. it is interesting to note that removal of a steric clash between r and the blv coiled coil yields an envelope that is more fusogenic than wild type. for htlv- an isoleucine, at a position equivalent to residue arg in the blv lhr, also appears to make a steric clash with the coiled coil and an alanine substitution of this residue in the context of an lhr-based peptide inhibitor yields a peptide with improved coiled coil-binding properties and increased inhibitory activity [ ] . it is therefore clear that viral envelope has not evolved to optimise lhr binding to the coiled coil or to maximise fusogenicity. one plausible explanation is that membrane fusion needs to be kept in check. an overly fusogenic envelope may induce rampant membrane fusion and syncytium formation leading to cell death by apoptosis and thereby provide additional stimuli for a robust anti-viral immune response. clearly, sub-optimal lhr/coiled-coil interactions have been maintained during viral evolution and suggest that the development of compounds that bind to the coiled coil with higher affinity than the lhr may be an achievable anti-viral strategy. the most notable difference between the htlv- and blv tm structures is the resolution of a second helical motif within the lhr of the trimer-of-hairpins. residues that form the start of the second a-helix are conserved in htlv- and are critical to envelope-catalysed membrane fusion and to the inhibitory activity of lhr-mimetic peptides [ , ] . just three residues from the end of our structure is the first tryptophan of a conserved tryptophanrich membrane-proximal region (mpr). the mpr is believed to interact with the opposing membranes during the fusion process and substitution of the aromatic residues severely compromises, but does not abolish, membrane fusion [ ] . the conformation of the mpr has received considerable attention because broadly neutralising anti-hiv- antibodies bind epitopes contained within the mpr [ , ] . it is likely that the hiv mpr adopts a helical structure when interacting with membranes and the coiled coil during fusion [ , ] . examination of the blv envelope sequence and tm structure suggests that the second helical segment of the lhr is likely to continue and that in the fusion-activated state the tryptophan-rich mpr is also helical and inserted into the membrane at an oblique angle. for the leukaemia viruses, the conserved arginine (arg in blv), assists in defining the orientation of the mpr, as the interactions of arg with the coiled coil and the lhr backbone propagate a sharp kink in the lhr towards the membrane-proximal end of the coiled coil. a lysine substitution of arg retains only , % of the fusogenic activity of wild-type envelope lending further support to this view. the blv crystal structure and our analysis of htlv- and blv envelope-mediated membrane fusion reveal an important role for electrostatic interactions in binding of the lhr to the grooves of the coiled coil. for both blv and htlv- a conserved basic residue in the second helical segment of the lhr interacts with the charged rim of a deep pocket lined by non-polar residues. for htlv- the contribution of charge is enhanced by the interaction of an additional basic residue (arg ) with the opposite rim of the pocket. thus, while the insertion of non-polar residues into hydrophobic pockets likely drives the docking of the lhr with the coiled coil, we suggest that it is the electrostatic attraction and hydrogen bonding of charged side chains around these hydrophobic interactions that serves to ''cement'' the lhr in place and thereby establish the precise geometry and affinity of this interaction. in keeping with this view, substitution of these basic residues ablates envelope-mediated membrane fusion and dramatically impairs the inhibitory and coiled-coil-binding properties of lhr-based peptides [ ] . moreover, only one of these important basic residues is conserved in blv compared to htlv- and it is notable that the synthetic blv lhr-mimetic is approximately -fold less potent than the corresponding inhibitor of htlv- [ ] . examination of the electrostatic surfaces of blv, htlv, hiv and momlv coiled coils reveals, in each case, the presence of a pocket surrounded by charged residues, though the polarity of the charge and positioning along the groove is variable (fig. ). the importance of the pocket and surrounding charge to the assembly of the trimer-of-hairpins and the potency of c-helixbased mimetic peptides has been demonstrated for hiv [ , ] , and taken with our findings for blv and htlv- suggest that such charged regions are critical to retroviral envelope-mediated membrane fusion in general. we suggest that structure-assisted design of small molecules to target these charge-surrounded pockets is a viable objective for anti-retroviral therapy. we expect that the data produced in this study will be pivotal to the development of more drug-like inhibitors of htlv- envelope catalysed membrane fusion, and thereby provide therapeutic antiviral agents for adult t-cell leukaemia and htlv-associated myelopathy/tropical spastic paraparesis, diseases for which there are currently no effective treatments [ , ] . the plasmids pcmv-blvenv-rre and prsv-rev have been described [ , ] . to construct pcmv-htlvenv-rre the blv env sequences in pcmv-blvenv-rre were replaced by a htlv- env coding region amplified by pcr from phte- [ ] . to construct the plasmid pmal-gp hairpin (mbp-blv-hairpin), a modified fragment of the mbp open reading frame from pmal-c (new england biolabs) with a bglii site and a pvuii site replacing a saci site was pcr amplified. a fragment of blv env encoding amino acid residues to was pcr amplified with a pvuii site and a psti site. the pcr fragments were ligated into a pmal-c backbone digested with bglii and psti. the quikchange mutagenesis kit (stratagene) was used to introduce point mutations into pcmv-blvenv-rre and pmal-gp hairpin following the manufacturer's instructions. expression of the mbp fusion protein was carried out as previously described [ , ] . briefly, escherichia coli bl (de ) cells transformed with the mbp-blv-hairpin vector were grown at uc with vigorous shaking in lb supplemented with mm glucose and mgml ampicillin until an optical density at nm of , . was reached. protein expression was induced with isopropylthio-b-d-galactoside (iptg) at a final concentration of . mm for h at uc. cells were harvested by centrifugation and the pellet was resuspended in column buffer ( mm tris-hcl [ph . ], mm nacl, mm edta) supplemented with protease inhibitors ( mm phenylmethylsulfonyl fluoride and mgml aprotinin) and frozen o/n at uc. the cell suspension was thawed and the cells lysed by sonication. cell debris was pelleted by centrifugation at , g for min. the crude lysate was diluted : in column buffer and loaded onto an amylose column pre-equilibrated with column buffer. the column was then washed with column volumes of column buffer, and the bound protein was eluted with column buffer supplemented with mm maltose. the trimeric recombinant mbp-blv-hairpin protein was purified by superdex gel filtration, and the required fractions pooled and concentrated to mg ml . tris [ -carboxyethyl]phosphine hydrochloride (tcep-hcl) was then added to a final concentration of mm. protein concentration was estimated by absorbance measured at nm. crystallization conditions were identified using the sitting drop vapour diffusion technique, with crystal screens from hampton research and emerald biostructures. optimal crystallization was achieved by streak seeding with a reservoir solution composed of . % (v/v) isopropanol, . % (v/v) peg- and . m sodium citrate ph . , and optimal diffraction was achieved using a cryoprotectant composed of mother liquor supplemented with % (v/v) glycerol. diffraction data was collected at beamline bm at the european synchrotron radiation facility (esrf) in grenoble. the structure was solved by molecular replacement using a truncated model of mbp-htlv hairpin (pdb id mg ). refinement proceeded through cycles of model building using coot and o [ , ] and refinement using refmac [ ] . data and refinement statistics are given in table peptides peptides were synthesised using standard solid-phase fmoc chemistry and unless stated otherwise have acetylated n-termini and amidated c-termini. the peptides were purified by reversephase high-pressure liquid chromatography and verified for purity by maldi-tof mass spectrometry. all peptides were dissolved in dimethyl sulfoxide (dmso), the concentration of peptide stock solutions was confirmed by absorbance at nm in m guanidine hydrochloride and peptides were used at the final concentrations indicated. hela cells were maintained in dulbecco's modified eagle medium supplemented with % foetal bovine serum (fbs). for syncytium formation assays, hela cells were transfected with equal quantities of pcmv-blvenv-rre (or empty pcdna . for mock samples) and prsv-rev using the genejuice transfection reagent (novagen). after h, transfected cells were added to . non-transfected hela target cells in -well dishes (nunc), and cocultured for hrs, in the presence of peptides where appropriate. the cells were washed with phosphatebuffered saline ph . (pbs), fixed using % paraformaldehyde in pbs, and stained with giemsa. assays were performed in triplicate. to calculate fusogenic indices, the fraction of nuclei contained within syncytia in a x light microscope field was expressed as a percentage of the total number of nuclei within the field. western blotting was carried out using standard methods; bmercaptoethanol was used in sample preparation. blv and htlv- envelope was detected using supernatant from murine hybridoma cell lines expressing monoclonal antibodies raised against recombinant antigen derived from blv or htlv- envelope, and anti-mouse horseradish peroxidase conjugated secondary antibody at a dilution of : , in pbs containing % (w/v) marvel and . % triton x- . to detect surface-expressed protein, cells transfected with the appropriate envelope expression vector were detached from culture flasks using pbs + mm edta, and washed twice with pbs. . cells were incubated with agitation for hr at room temperature in ml dmem + % fbs containing mgml immunoglobulin purified from the blv anti-env antibodyexpressing hybridoma supernatant, or ml supernatant from the htlv anti-env antibody expressing hybridoma. cells were washed twice with pbs and incubated in the dark at room temperature for mins in dmem containing anti-mouse fitc (sigma) at / , dilution. cells were washed once with pbs and once with pbs + . % sodium azide before fixing with pbs + . % paraformaldehyde. bound fluorescence was detected using a facscan flow cytometer (becton dickinson). the oligomerisation states of mbp-blv-hairpin and mutant derivatives were examined by gel filtration using a superdex column equilibrated with mbp elution buffer. fractions containing the trimeric species were retained. to assess the thermostability of the trimers, samples from these fractions were re-run over the column either without heat treatment or with heat treatment at the temperature specified for min, followed by cooling for min on ice. the areas under the peaks observed were calculated, and the area of the peak corresponding to the aggregate was expressed as a percentage of the total area under both this peak and the peak corresponding to the trimer. human t-cell leukaemia virus types i and ii. fields virology mechanisms of leukemogenesis induced by bovine leukemia virus: prospects for novel antiretroviral therapies in human detection and isolation of type c retrovirus particles from fresh and cultured lymphocytes of a patient with cutaneous t-cell lymphoma isolation and characterization of retrovirus from cell lines of human adult t-cell leukemia and its implication in the disease global epidemiology of htlv-i infection and associated diseases modelling the risk of adult t-cell leukemia/lymphoma in persons infected with human t-lymphotropic virus type i mechanisms of viral membrane fusion and its inhibition viral membrane fusion isomerization of the intersubunit disulphide-bond in env controls retrovirus fusion turning of the receptor-binding domains opens up the murine leukaemia virus env for membrane fusion evidence that the transition of hiv- gp into a six-helix bundle, not the bundle configuration, induces membrane fusion hiv- envelope proteins complete their folding into six-helix bundles immediately after fusion pore formation a synthetic peptide inhibitor of human immunodeficiency virus replication: correlation between solution structure and viral inhibition an antiviral peptide targets a coiled-coil domain of the human t-cell leukemia virus envelope glycoprotein peptides from conserved regions of paramyxovirus fusion (f) proteins are potent inhibitors of viral fusion a synthetic peptide corresponding to a conserved heptad repeat domain is a potent inhibitor of sendai virus-cell fusion: an emerging similarity with functional domains of other viruses highly specific inhibition of leukaemia virus membrane fusion by interaction of peptide antagonists with a conserved region of the coiled coil of envelope inhibition of feline immunodeficiency virus infection in vitro by envelope glycoprotein synthetic peptides inhibition of cell-free human t-cell leukemia virus type infection at a postbinding step by the synthetic peptide derived from an ectodomain of the gp transmembrane glycoprotein crystal structure of human t cell leukemia virus type gp ectodomain crystallized as a maltosebinding protein chimera reveals structural evolution of retroviral transmembrane proteins nonhelical leash and alpha-helical structures determine the potency of a peptide antagonist of human t-cell leukemia virus entry basic residues are critical to the activity of peptide inhibitors of human t cell leukemia virus type entry leash in the groove mechanism of membrane fusion crystal structure of the ebola virus membrane fusion subunit, gp , from the envelope glycoprotein ectodomain learncoil-vmf: computational evidence for coiled-coil-like motifs in many viral membrane-fusion proteins crystallization of a trimeric human t cell leukemia virus type gp ectodomain fragment as a chimera with maltose-binding protein human t-cell leukemia virus type i envelope protein maturation process: requirements for syncytium formation functional implications of the human t-lymphotropic virus type transmembrane glycoprotein helical hairpin structure retrovirus envelope domain at . angstrom resolution core structure of the envelope glycoprotein gp from ebola virus at . -a resolution crystal structure of a pivotal domain of human syncytin- , a million years old endogenous retrovirus fusogenic envelope gene captured by primates direct involvement of herv-w env glycoprotein in human trophoblast cell fusion and differentiation evidence that the transmembrane domain proximal region of the human t-cell leukemia virus type fusion glycoprotein gp has distinct roles in the prefusion and fusion-activated states blv and htlv-i: their unique genomic structures and evolutionary relationship sequence of simian immunodeficiency virus from macaque and its relationship to other human and simian retroviruses central ions and lateral asparagine/glutamine zippers stabilize the post-fusion hairpin conformation of the sars coronavirus spike glycoprotein the crystal structure of the siv gp ectodomain at . a resolution structure of the ebola virus glycoprotein bound to an antibody from a human survivor a conserved tryptophan-rich motif in the membrane-proximal region of the human immunodeficiency virus type gp ectodomain is important for env-mediated fusion and virus infectivity the membraneproximal external region of the human immunodeficiency virus type envelope: dominant site of antibody neutralization and target for vaccine design structure and mechanistic analysis of the anti-human immunodeficiency virus type antibody f in complex with its gp epitope the membrane-proximal tryptophan-rich region of the hiv glycoprotein, gp , forms a well-defined helix in dodecylphosphocholine micelles conserved salt bridge between the nand c-terminal heptad repeat regions of the human immunodeficiency virus type gp core structure is critical for virus entry and inhibition conserved residue lys in the cavity of hiv- gp coiled-coil domain is critical for six-helix bundle stability and virus entry natural history of adult t-cell leukemia/ lymphoma and approaches to therapy human t-lymphotropic virus : recent knowledge about an ancient infection mutational analysis of the human immunodeficiency virus type rev transactivator: essential residues near the amino terminus htlv-i p rex regulates gag and env protein expression resistance to neutralization by antibodies targeting the coiled coil of fusionactive envelope is a common feature of retroviruses coot: model-building tools for molecular graphics improved methods for building protein models in electron density maps and the location of errors in these models refinement of macromolecular structures by the maximum-likelihood method we thank the european synchrotron radiation facility for synchrotron beam time, and the staff at bm for their support. we thank dr luc willems for generously providing an infectious molecular clone of blv. key: cord- - d rn authors: suthar, mehul s.; ma, daphne y.; thomas, sunil; lund, jennifer m.; zhang, nu; daffis, stephane; rudensky, alexander y.; bevan, michael j.; clark, edward a.; kaja, murali-krishna; diamond, michael s.; gale, michael title: ips- is essential for the control of west nile virus infection and immunity date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: d rn the innate immune response is essential for controlling west nile virus (wnv) infection but how this response is propagated and regulates adaptive immunity in vivo are not defined. herein, we show that ips- , the central adaptor protein to rig-i-like receptor (rlr) signaling, is essential for triggering of innate immunity and for effective development and regulation of adaptive immunity against pathogenic wnv. ips- (−/−) mice exhibited increased susceptibility to wnv infection marked by enhanced viral replication and dissemination with early viral entry into the cns. infection of cultured bone-marrow (bm) derived dendritic cells (dcs), macrophages (macs), and primary cortical neurons showed that the ips- -dependent rlr signaling was essential for triggering ifn defenses and controlling virus replication in these key target cells of infection. intriguingly, infected ips- (−/−) mice displayed uncontrolled inflammation that included elevated systemic type i ifn, proinflammatory cytokine and chemokine responses, increased numbers of inflammatory dcs, enhanced humoral responses marked by complete loss of virus neutralization activity, and increased numbers of virus-specific cd + t cells and non-specific immune cell proliferation in the periphery and in the cns. this uncontrolled inflammatory response was associated with a lack of regulatory t cell expansion that normally occurs during acute wnv infection. thus, the enhanced inflammatory response in the absence of ips- was coupled with a failure to protect against wnv infection. our data define an innate/adaptive immune interface mediated through ips- -dependent rlr signaling that regulates the quantity, quality, and balance of the immune response to wnv infection. west nile virus (wnv) is a neurotropic flavivirus and is an emerging public health threat. infection with wnv now constitutes the leading cause of mosquito-borne and epidemic encephalitis in humans in the united states [ ] . wnv is enveloped and contains a single strand positive sense rna genome of approximately kb in length that encodes three structural (c, prm/m, and e) and seven non-structural proteins (ns , ns a, ns b, ns , ns a, ns b, and ns ). it cycles enzootically between birds and culex mosquitoes, with humans infected as dead-end hosts. wnv infection has been modeled in inbred mice wherein infection and pathogenesis recapitulate many of the features of human infection (reviewed in [ ] ). following subcutaneous inoculation, wnv replicates in dendritic cells (dcs) at the portal of entry and in the draining lymph node. a primary viremia develops and virus spreads to visceral organs including the spleen, where further amplification occurs, leading to central nervous system (cns) dissemination and encephalitis. in humans, wnv causes an acute febrile illness that can progress to severe and sometimes lethal neuroinvasive disease, especially in the elderly and immunocompromised [ ] . however, healthy young adults are also afflicted with severe neurological disease [ , , ] , indicating that virulence can occur independently of immune deficiencies or aging. intracellular innate immune defenses and the actions of type i interferon (ifn) provide a first-line of defense against virus infection and are essential for the control of wnv replication, dissemination, and neurovirulence [ ] . innate antiviral immune defenses are triggered through the recognition of conserved pathogen associated molecular pattern (pamp) motifs within viral products by intracellular pathogen recognition receptor (prr) proteins in infected cells. prr signaling directs downstream activation of latent transcription factors, including nf-kb, interferon regulatory factor (irf)- and irf- , in a cell type-specific manner to induce antiviral response programs that include expression of proinflammatory cytokines, chemokines, type i ifn, and interferon stimulated genes (isgs) [ , , , ] . the isg products induced through autocrine and paracrine actions of ifn confer antiviral activity by limiting virus replication and cell-to-cell virus spread. modulation of ifn signaling has been identified as a virulence feature of pathogenic strains of wnv [ , ] . the rlrs, retinoic acid inducible gene-i (rig-i) and melanoma differentiation antigen (mda ) [ , , , ] , are prrs that play critical roles in triggering immune defenses against rna virus infection, including wnv. rig-i and mda are cytosolic rna helicases that contain an amino terminal tandem caspase activation and recruitment domain (card). upon engaging rna substrates, the rlrs undergo a conformational change and bind to the mitochondrial associated protein, interferon promoter stimulator- (ips- ) through a card-card interaction, leading to ips- -dependent signaling of ifn production and expression of immune response genes [ , ] . rlr signaling and ips- function have an essential role in triggering ifn defenses during wnv infection of mouse embryo fibroblasts (mefs) and human cell lines in vitro. cells lacking either rig-i or mda were attenuated in their ability to generate an effective innate immune response to infection, whereas cells lacking both rig-i and mda or those deficient in ips- alone were unable to respond to infection with wnv and related flaviviruses [ , , , ] . recent studies examined the role of another class of pattern recognition receptors, toll like receptor (tlr) and tlr , and show that these receptors are also important prrs of wnv infection, as they play a role in signaling ifn production and an inflammatory response upon viral ligand recognition [ , , ] . tlr has been shown to contribute to both enhancement and protection of cns inflammation and neurovirulence of wnv in vivo [ , ] , while tlr -dependent signaling was shown to be essential for directing proper immune cell homing to sites of wnv infection during the adaptive immune response in vivo [ ] . type i ifn, a major product of prr signaling, has been shown to link innate and adaptive immune responses. however, the specific prr pathways that mediate this during acute wnv infection have not been delineated nor has the rlr pathway been evaluated in this context. the quantity and quality of the innate and adaptive immune responses after infection must be carefully regulated to avoid aberrant inflammation and immunopathogenesis. regulatory t (t reg ) cells and inflammatory dendritic cell (dc) subsets regulate inflammation during acute virus infection through t cell suppression and by modulating the trafficking and inflammatory cytokine production of immune cells into infected tissues [ , , ] . thus, the level of local and peripheral t reg cells, and the composition of local dc subsets that develop during wnv infection may determine immune control and wnv disease. here, we assessed the role of rlr signaling and ips- in wnv infection and immunity. our studies define ips- as an essential modulator of immunity in vivo and demonstrate that ips- -dependent signaling orchestrates an innate/adaptive immune interface that regulates immune responses to effectively control wnv infection. wnv infection of primary embryonic fibroblasts recovered from rig-i / mice revealed that rig-i was important in eliciting innate antiviral immune defenses early during infection, whereas mda was important for enhancing and sustaining this response [ ] . we further evaluated wnv infection of rig-i / or mda / mice and confirmed that rig-i serves a dominant role among the rlrs for the acute induction of innate immune defenses and protection against wnv infection in vivo (data not shown). since the rlrs signal innate defenses through the ips- adaptor protein [ ] , we also examined the role of ips- in protection against wnv infection upon a sub-lethal virus challenge of wild type and ips- / mice. ips- / mice were highly susceptible to wnv infection and exhibited % mortality with an average survival time (ast) of . days as compared to wild type mice ( . % mortality with an ast of . days; p, . ; fig a) . thus, rig-i and ips- -dependent signaling are essential for protection against wnv infection. to define the role of ips- in controlling wnv in vivo, wild type and ips- / mice were infected subcutaneously (s.c.) with pfu of wn-tx and viral burden within peripheral tissues and the cns was measured over time post-infection (pi). ips- / mice exhibited increased viremia compared to wild type mice ( . fold enhancement at day pi, p, . ) throughout the course of infection (fig b) . similarly, viral loads in the spleen were elevated in the infected ips- / mice (fig c) . wnv infection of ips- / mice displayed an expanded tissue tropism as infectious virus was found in the kidneys, a tissue that is not normally permissive to infection in wild type mice ( fig d) . wnv is typically detected in the cns of wild type mice after s.c. challenge between and days pi [ ] . consistent with this time course, infected wild type mice exhibited detectable viral loads (average viral titer of . pfu/gram of tissue) in the brain by day p.i., although virus was not detected in the spinal cord (fig e and f ). in contrast, wnv spread to the brain ( fig e) and spinal cord of ips- / mice (fig f) by day pi, with viral loads rising through day pi. together these results indicate that ips- , likely west nile virus (wnv) is a mosquito-transmitted rna virus that has emerged in the western hemisphere and is now the leading cause of arboviral encephalitis in the united states. however, the virus/host interface that controls wnv pathogenesis is not well understood. previous studies have established that the innate immune response and interferon (ifn) defenses are essential for controlling virus replication and dissemination. in this study, we assessed the importance of the rig-i like receptor (rlr) signaling pathway in wnv pathogenesis through analysis of mice lacking ips- , the central adaptor molecule of rlr signaling. our studies revealed that ips- is essential for protection against wnv infection and that it regulates processes that control virus replication and triggering of innate immune defenses. we found that ips- plays an important role in establishing adaptive immunity through an innate/adaptive interface that elicits effective antibody responses and controls the expansion of regulatory t cells. thus, rlrs are essential for pathogen recognition of wnv infection and their signaling programs help orchestrate immune response maturation, regulation of inflammation, and immune homeostasis that define the outcome of wnv infection. through rlr signaling of innate immune defenses, limits wnv replication, viremia, and peripheral spread, and is essential for the control of viral invasion of the cns. myeloid cells, including tissue and lymphoid dc and macrophages (mw), are among the first cells to encounter wnv during infection and thus function to restrict the spread of virus to distant tissues and the cns [ ] . to define the role of ips- in controlling virus replication and innate immunity in myeloid cells, we analyzed wnv infection and host responses in primary bone marrow-derived dc and mw recovered from wild type and ips- / mice. dc and mw were infected at an moi of . (relative to viral plaque assay quantification of bhk- cells; see methods) and evaluated for virus replication, ifn induction, and innate immune triggering of isg expression (fig ) . ips- / dcs sustained significantly higher wnv replication at and hours pi compared to wild type infected cells (fig a) . wnv infection of wild type dcs induced ifn-b secretion but this response was completely abolished in ips- / dcs (fig b) . the lack of ifn-b induction in ips- / dcs correlated with a lack of isg expression including rig-i, mda , and stat- ( fig c) . in addition, expression of isg and isg , which are direct irf- target genes [ , ] , were not induced during wnv infection of ips- / dcs (fig c) . moreover, isg , another irf- target gene [ ] , was induced late during infection and to lower levels as compared to isg and isg in wild type, infected dcs. wnv infection of ips- / mw resulted in significantly higher virus replication between and hours pi as compared to infected wild type cells ( fig d) . whereas wild type infected mw expressed ifn-b, this response was completely abolished in ips- / mw (fig f) . we also observed a differential expression of isgs and irf- target genes within wnv-infected mw. rig-i, mda , and stat- were not induced in ips- / mw, whereas, isg , isg , and pkr were expressed at reduced levels and with delayed kinetics. these data establish that ips- -dependent rlr signaling is the major innate immune signaling pathway that controls virus replication in conventional dcs and mw. the rlr signaling pathway triggers the innate immune response to wnv infection in primary cortical neurons neurons represent the target cell of wnv infection in the cns and their death after infection is a key factor in pathogenesis and neurological sequelae [ , ] . to define the role of rlr signaling in restricting virus replication in neurons, primary cortical neurons were generated from wild type and ips- / mice. cells were infected at an moi of . with wn-tx and virus yield, ifn-b induction, and isg expression were evaluated. in the absence of ips- , wnv replicated faster and to higher levels resulting in a . and . -fold (p, . ) increase in viral production at hrs and pi, respectively as compared to infected wild type neuronal cells ( fig a) . this relatively modest virologic effect in neurons compared to that observed in ips- / dc and mw was expected, as ifn-a or -b pre-treatment only inhibits wnv infection in cortical neurons to a maximum of to -fold [ ] , suggesting that the ifn response is comparably less potent in neurons. ifn-b expression was induced to lower levels in ips- / neurons compared to wild type infected neurons at ( -fold, p, . ) and hours pi ( -fold, p, . ) despite the higher levels of virus replication (fig a and b) . expression of isgs, (including rig-i and mda ) and irf- target genes (including isg and isg ) followed this pattern and were dependent on ips- for rapid and high level expression ( fig c) . the presence of ifn-b and isg transcripts in ips- / cells at hrs pi is consistent with the finding that tlr has an independent and subordinate role in triggering innate immune responses in cortical neurons at later time points after wnv infection [ ] . these results demonstrate that the rlr signaling pathway controls virus replication and induces innate immune responses against wnv infection in cortical neurons. neuronal destruction and cns inflammation are enhanced in wnv infected ips- / mice to determine the role of the rlr pathway in protection of neurons against wnv pathogenesis in vivo, we conducted histological analysis of brain tissue from wild type and ips- / mice infected with wn-tx ( fig a) . analysis of brain sections from infected wild type mice revealed little or no inflammation or neuronal damage, with sparse and focal cell infiltrates restricted to the hippocampus and cerebral cortex on day pi. by day pi (a timepoint in wild type mice in which peak virus replication in the cns occurs [ ] ) cellular infiltrates were present in the parenchyma and neuronal destruction was observed throughout the cortex and hippocampus. in contrast, brain sections from infected ips- / mice recovered on day pi displayed extensive injury to neurons in the cortex and granular neurons of the hippocampus. damaged neurons appeared pyknotic with vacuolation, degeneration and cell dropout. somewhat surprisingly, we observed extensive inflammation in the brains from infected ips- / mice within the cortex, hippocampus, and cerebellum (data not shown) displaying prominent perivascular and parenchymal immune cell infiltrates. to evaluate the composition and antigen-specificity of the inflammatory cells within the brains of wnv-infected mice, lymphocytes were isolated from infected brains on day pi and were characterized from pools (n = ) of wild type and ips- / infected mice. brains from ips- / infected mice showed an . fold increase in the total number of immune cells as compared to wild type infected mice (fig b) , and this was associated with an increase in absolute numbers of infiltrating cd + and cd + t cells ( fig c) . among the brain cd + t cells isolated from ips- / mice, there was a remarkable -fold increase in the number of antigen-specific cells that expressed ifn-c after treatment with an immundominant ns b peptide ( fig d) [ , ] . analysis of microglia/mw cells, based on relative surface expression of cd and cd b [ ] , revealed increased numbers of microglial cells (cd + lo /cd b+) and infiltrating macrophages (cd + hi / cd b+) within the brains of infected ips- / mice when compared to wild type mice (fig e) . similar findings were observed in the spinal cords from infected ips- / mice (data not shown). combined with the histological analysis, these results demonstrate that in the absence of ips- , wnv infection induces a strong inflammatory response in the cns. while this response is likely associated with increased viral loads, the failure of this increased inflammatory response to elicit protection or control cns pathology, in the absence of ips- , suggests a role for the rlr signaling pathway as a regulatory program that controls the quality of the inflammatory response to wnv infection. to further characterize how ips- modulates the inflammatory response to wnv infection, we measured levels of systemic type i ifn, proinflammatory cytokines, and chemokines present in the serum of wnv-infected mice at and days pi. paradoxically, a trend towards more rapid induction and increased levels of type i ifn were observed in the serum of ips- / mice compared to wild type mice (fig a) . we note that in this case type i ifn was detected and quantified through a mouse-specific type i ifn bioassay, which does not differentiate between the ifn-a or -b species. this result is consistent with our recent studies showing that serum type i ifn levels accumulate during wnv infection in an irf- -dependent but irf- -independent manner [ , ] . in this case ifn-a species are likely accumulating through a tlr dependent signaling pathway involving plasmacytoid dcs, which do not require the rlr pathway for ifn production [ ] . more recently, town et al. assessed the role of tlr and myd / during wnv infection and found that mice lacking myd produced elevated levels of systemic ifn during wnv infection [ ] . thus, during wnv infection systemic ifn is regulated through rlr-dependent and independent processes. correspondingly, when compared to wild type mice, ips- / infected animals, which show higher viremia (fig b) produced increased serum levels of proinflammatory cytokines and chemokines in response to wnv infection. elevated levels of systemic il- , tnfa, cxcl , and ifn-c were observed at and/or days pi in ips- / mice (fig b) . serum cytokine levels were also compared between uninfected wild type and ips- / mice and showed no differences in basal cytokine expression (data not shown). these results demonstrate that in the absence of ips- , greater proinflammatory cytokine and chemokine responses are induced during wnv infection. altered wnv-specific antibody profiles in ips- / mice wnv-specific antibody responses are essential for suppressing viremia and virus dissemination and limiting lethal wnv infection [ , ] . to determine if a deficiency in ips- modulated the quality and quantity of the humoral immune response, we characterized the antibody profile in sera during wnv infection. in wild type mice, neutralizing virus-specific igm antibodies are typically detectable by day pi with wnv and production of neutralizing virus-specific igg antibodies follow between days and pi [ ] . a time course analysis in wild type and ips- / infected mice showed that between and days pi, wnv-infected ips- / mice exhibited significantly higher levels of virus-specific igm, igg, and igg subclasses as compared to infected wild type mice ( table ) . wnv-specific igg antibodies were detected at low levels on day pi in sera from wild type and ips- / mice. additionally, we observed a , . -fold increase in wnv-specific igg a levels in infected ips- / as compared to wild type mice on day pi and , . -fold increase on day pi. assessment of the virus-specific antibody responses through a prnt assay revealed that neutralization titers in sera from wild type mice increased dramatically between and days pi. sera from ips- / infected mice exhibited a modest increase in neutralization titer to : , despite having much higher levels of virusspecific antibodies. this difference translated into a serum neutralization index that was , -fold lower on day pi in the infected ips- / mice compared to wild type mice. these results demonstrate that the humoral responses in wnv-infected ips- / mice are distinct from responses in wild type infected mice. thus, rlr signaling and ips- actions likely contribute to regulatory processes that govern the levels, igg class switching, and neutralizing capacity of antibodies generated in response to wnv infection. to characterize the immune parameters associating with the dysregulated inflammatory and humoral responses in wnv infected ips- / mice, we analyzed the immune cell composition in draining lymph node and spleen tissues. wild type and ips- / mice were challenged with diluent alone or with wn-tx, and draining popliteal lymph node (dln) and the spleen were harvested at and days pi, respectively. analysis of the popliteal dln provides insight into how ips- modulates the inflammatory response immediately after infection whereas assessment of the spleen elucidates characteristics of the adaptive immune response prior to the infection endpoint. immune cells were isolated from the popliteal dln and were characterized by flow cytometry (fig ) . analysis of cd a dc subsets, which are phenotypically the major antigen presenting cells within lymphoid tissues and are implicated in eliciting virus-specific cd t cell in response to acute wnv infection [ ] , showed that infected wild type and ips- / mice exhibited similar increases in the numbers of cd a + and cd a dcs compared to mock-infected mice (fig a, b) . however, a significant increase (, -fold; p, . ) of a proinflammatory dc subset, characterized as cd c + cd b hi ly c + , was observed within the popliteal dlns of ips- / infected mice (fig c) . this dc subset is monocytederived and typically recruited to sites of acute inflammation where they propagate the inflammatory response [ ] . we found that these dc subsets were not significantly expanded and showed no differences in their recruitment to the dln in either wild type or ips- / infected mice at hours pi (data now shown). thus, as early as hours pi, an elevated cellular inflammatory response is initiated in the ips- / mice. in contrast, similar increases in plasmacytoid dcs were observed within infected wild type and ips- / infected mice (fig d) , demonstrating that an absence of ips- did not directly affect expansion and/or recruitment of this dc subset. within the popliteal dlns, mock-infected ips- / mice compared to wild type mice generally showed elevated numbers of b cells, cd + t cells (p, . ), and cd + t cells (fig e, f, and g) . we further analyzed the lymphocyte composition of the spleen on day after wnv infection (fig ) . gross pathologic analysis revealed that wnv infection of ips- / mice results in massive splenomegaly whereas infection of wild type mice induces only a slight increase in spleen size (fig a) . cell analysis revealed increased numbers of total lymphocytes in the spleens of infected ips- / mice as compared to wild type mice (fig b) . regulatory t (t reg ) cells have recently been shown to contribute to the dampening of inflammation and adaptive immune responses during acute virus infections [ , , ] . moreover, a reduction in the number of circulating t reg in mice leads to enhanced lethality after wnv infection [ ] . a time course analysis of t regs in wild type mice revealed that wnv infection results in a significant increase in the numbers of foxp + t cells as compared to mock-infected mice beginning on day and peaking by day pi (fig c) , indicating the expansion of t regs during acute wnv infection. despite their marked increase in viral load, the infected ips- / mice did not display an increase in the numbers of foxp + t cells at any timepoint analyzed. thus, ips- signaling directly or indirectly impacts t reg proliferation and does so independently of viral load. we also observed that spleens from infected ips- / mice exhibited significantly increased numbers of cd + t cells in comparison to those from infected wild type mice, whereas the expansion of splenic cd + t cells in wild type and ips- / mice were not different (fig d) . the spleens from wnv-infected ips- / mice showed significantly higher numbers ( . -fold; p, . ) of wnv-specific cd + t cells producing ifnc. to further define the phenotype associated with wnv-induced splenomegaly in ips- / mice, we also assessed the numbers of nk cells and neutrophils. spleens from infected ips- / mice contained greater numbers of nk cells (cd cd nk . + cells), nkt cells (cd +/cd +/nk . + cells) and neutrophils (cd b + gr + cells) (fig e) . although wnvinfected wild type mice infected displayed slight increases in the absolute numbers of these specific cell types, a deficiency of ips- resulted in a more marked enhancement of these immune cell populations. in this study, we establish a major role for rlr signaling in protection from wnv pathogenesis, and demonstrate that ips- is critical for the control of wnv infection in vivo. our studies indicate that ips- -dependent rlr signaling functions to establish balanced, effective, and protective innate and adaptive immune responses, and that ips- links adaptive immune regulation with the innate immunity triggered by rlr signaling during wnv infection. in the absence of ips- in vitro, innate immune defense programs of myeloid dcs and macrophages were ablated or severely attenuated. moreover, in vivo analysis of infected ips- / mice showed altered igg and igm antibody responses with diminished virus neutralization activity. the inflammatory response to wnv infection is regulated by ips- -dependent processes such that a deficiency of ips- resulted in elevated proinflammatory cytokine and chemokines and increased numbers of inflammatory dcs, antigen-specific t cells, natural killer cells, and neutrophils in lymphoid organs, and activated macrophage/ microglial cells within the cns. the dysregulated inflammatory response to wnv infection in ips- / mice was associated with a reduction in the numbers of t reg cells and their failure to expand during acute infection. these observations demonstrate the critical role of ips- in mediating rlr signaling of innate antiviral immunity against wnv infection, and reveal novel features of ips- function in regulating immune homeostasis, inflammation, and adaptive immunity to infection. although infection of primary dcs, macrophages, and neuronal cells failed to induce type i ifn upon wnv infection, wnvinfected ips- / mice showed enhanced systemic type i ifn responses. this finding agrees with previous studies that indicate both ips- -dependent and -independent pathways contribute to the systemic type i ifn production in vivo [ , , , ] . most importantly, the enhanced tissue tropism and rapid viral entry into the cns observed in the ips- / mice is not affected by the elevated systemic ifn responses. this suggests that local tissuespecific and intracellular responses triggered by rlr-dependent signaling are more essential for reducing viral burden and dissemination. one possible explanation is that a distinct set of rlr-responsive genes function to control virus replication at the site of infection. this could explain, in part, the elevated levels of virus replication, enhanced tissue tropism and cell-to-cell spread in mice or cells deficient in irf or irf- , each of which are downstream transcription factors of rlr signaling [ , , ] . additionally, it is likely that pdcs, which are specialized dendritic cells for producing systemic type i ifn during a viral infection [ ] , are likely contributing to the ifn responses observed during wnv infection. studies by silva et al. have shown that wnv triggers ifn induction in pdcs through a replication-independent manner [ ] . interestingly, within the dln, we observed similar expansion of pdcs between wild type and ips- / infected mice, yet at the same timepoint ( hours pi), elevated systemic type i ifn responses were observed in ips- / mice. this suggests two possibilities: ) splenic pdcs or circulating pdcs are likely responding to the high levels virus in the serum from the ips- / infected mice to produce ifn at hours pi and/or ) various other cell types that express tlr and/or tlr are responding to wnv infection and contributing to systemic ifn responses. taken together, these studies indicate that rlr signaling and the actions of irf- / are important in triggering ifn production and isg expression to limit wnv replication and spread, and that tlr signaling may impart additional, rlrindependent defenses that regulate immunity against wnv infection. the production of and response to type-i ifn is a major linkage point between innate and adaptive immunity, as ifn-a and ifn-b sustain b cell activation and differentiation [ , , ] , expand antigen-specific cd + t cells [ , ] , cd + t cells [ ] , and activation of nk cells [ ] . one of the most intriguing aspects of this study was the global alteration of the immune response elicited in the ips- / mice, indicating that rlr signaling couples innate immunity with regulation of the adaptive immune response. infection of ips- / mice exhibited increased igm and igg wnv-specific antibodies, enhanced wnv-specific cd +t cell response, and increased expansion of neutrophils, nk cells and nk-t cells. one trivial explanation for these differences is that there is an increased antigen load in the absence of ips- and, as a result, enhanced virus-specific (e.g. cd + t cells, igg and igm antibodies) and nonspecific (e.g. neutrophils, nk cells) responses. however, there are several key findings from this study that argue against these responses simply being attributed to higher antigen load: ( ) in the absence of ips- , the cd and cd t cells, which are protective against wnv infection [ , , , , , , ] , were significantly enhanced in the peripheral and cns compartments but failed protect against infection. one explanation for this observation is that the expansion and migration of cd and cd t cells to different tissues was itself uncontrolled, resulting in t cell-mediated pathology rather than t cell-mediated protection. ( ) while the quantity of virus-specific igm and igg antibody responses were greatly enhanced in the absence of ips- , there was a marked reduction in antibody quality in terms of neutralization capacity. in contrast deficiencies in tlr or myd (data not shown) did not alter virus-specific antibody responses and neutralization capacities. collectively, these findings suggest that rlr-dependent signaling coordinates effective antibody responses during wnv infection through as yet undefined pathway. ( ) while systemic ifn responses provide a link between innate and adaptive immune responses, our studies suggest that the prr signaling pathways (rlr-dependent vsindependent) and levels of ifn production in combination with production other proinflammatory cytokines or chemokines regulate the quantity and quality of the immune response during virus infection. thus, in the absence of ips- signaling, infected conventional dcs or mw, two integral cell types in establishing adaptive immunity, likely do not produce ifn or the normal array and level of proinflammatory cytokines/ chemokines. instead, ifn and other mediators may be strictly produced by infected or bystander cells during wnv infection, occurring with altered kinetics and magnitude, through tlr-dependent pathways, such as tlr and/or tlr [ , ] . ( ) in the absence of ips- , the enhanced expansion of ly c+ ''inflammatory'' dcs failed to limit early wnv replication and dissemination. this inflammatory dc subset migrates to sites of infection, secretes pro-inflammatory cytokines, and promotes cd + t cell expansion during a secondary virus infection, suggesting it sustains the effector t cell response [ ] . our data indicate that ly c+ dc recruitment and/or expansion is governed by ips- -dependent events of rlr signaling. thus, the aberrant recruitment/expansion of these inflammatory dcs may contribute to immunopathogenesis and limit development of an effective immune response to control wnv virus infection. ( ) the lack of t reg expansion during wnv infection correlated with altered ifn levels, increased proinflammatory cytokines and chemokine levels, and an increased number and distribution of antigen-specific cd + t cells. these observations implicate an indirect or direct role for ips- in regulating t reg levels during wnv infection, and provide evidence that links a lack of t reg expansion to immune dysregulation. while their importance in autoimmunity is established [ ] , recent studies have implicated an integral role for t regs in controlling inflammation and adaptive immune responses during acute virus infections [ , , ] . during acute infection t regs function to locally contain and control the immune response with the dual outcome of suppressing viral dissemination while decreasing the likelihood of immune-mediated pathology. in support of this model, infection studies with herpes simplex virus (hsv) and mouse hepatitis virus (mhv), two well established models of viral encephalitis, have demonstrated the importance of t regs in limiting proinflammatory cytokine and chemokine responses to protect the cns and enhance survival [ , ] . recent work also implicates t regs in the control of wnv pathogenesis, wherein peripheral expansion of t regs was associated with asymptomatic infection among wnv-infected blood donors but reduced t reg levels associated with wnv disease [ ] . furthermore, these studies revealed that the conditional depletion of t reg cells in mice results in enhancement of wnv virulence and expansion of antigen-specific cd t cells. interestingly, from our studies, type i ifn does not appear to be the major contributor to t reg expansion during wnv infection, as t regs failed to expand in the ips- / infected mice despite their elevated levels of systemic type ifn. these observations suggest that rlr signaling through ips- provides essential signals that directly or indirectly impart the expansion of t regs during wnv infection. we propose that ips- coordinates an innate/adaptive immune interface wherein ips- -signaling after rlr engagement regulates the quantity, quality, and balance of the subsequent immune response. the integrity of the innate/adaptive immune interface is central to the eliminating virus but also restricting immunopathogenesis and inflammation during infection. rlr signaling is essential for triggering the innate immune response to rna viruses that cause human disease, including the influenza viruses, respiratory syncytial virus and other paramyxoviruses, picornaviruses, reoviruses, flaviviruses, and hepatitis c virus [ , , ] . thus, in addition to wnv, ips- -dependent rlr signaling will likely have a broad impact for the control of inflammation, immune response quality, and viral disease. bhk and l cells were maintained in dulbecco's modified eagle medium (dmem) supplemented with % fetal bovine serum (fbs), mm l-glutamine, mm sodium pyruvate, antibiotic-antimycotic solution, and nonessential amino acids (complete dmem). wnv strain tx -hc (wn-tx) was isolated by as previously described [ ] . working stocks of wn-tx were generated by a single round of amplification on vero-e (ccl- ; atcc) cells, and supernatants were collected, aliquoted, and stored at uc. virus stocks were titered by a standard plaque assay on bhk cells as previously described [ ] . ips- / (c bl/ sv/ev) and their wild type littermate control mice have been published [ , ] and were obtained as a generous gift from dr. s. akira (osaka university, osaka, japan). mice were genotyped and bred under pathogen-free conditions in the animal facility at the university of washington. experiments were performed with approval from the university of washington institutional animal care and use committee. the methods for mice use and care were performed in accordance with the university of washington institutional animal care and use committee guidelines. age-matched six to twelve week old mice were inoculated subcutaneously (s.c.) in the left rear footpad with pfu of wn-tx in a ml inoculum diluted in hanks balanced salt solution (hbss) supplemented with % heatinactivated fbs. mice were monitored daily for morbidity and mortality. for in vivo virus replication studies, infected mice were euthanized, bled, and perfused with ml of phosphate-buffered saline (pbs). whole brain, spinal cord, kidney, and spleen were removed, weighed, homogenized in ul of pbs, and titered by plaque assay. bone-marrow derived dc and mw were generated as described previously [ ] . briefly, bone marrow cells from wild type and congenic deficient mice were isolated and cultured for days in either rpmi- supplemented with granulocyte-macrophagecolony stimulating factor, and interleukin- (peprotech) to generate myeloid dc or in dmem supplemented with macrophage colony stimulating factor (peprotech) to generate mw. on day , dc or mw were infected with wn-tx at an moi of . and at , , , and hours post-infection (hpi), supernatants were collected for titration of viral burden by plaque assay on bhk cells and levels of ifn-b (described below). cells were collected in parallel for western blot analysis. cortical neurons were isolated from -day-old embryonic mice and cultured as described previously [ ] . on day of culture, neurons were infected with wn-tx at an moi of . and at , , , and hpi, supernatants were collected for virus titration by plaque assay on bhk cells and cells were collected for rna analysis by rt-qpcr (described below). cells were lysed in modified ripa buffer ( mm tris [ph . ], mm nacl, . % sodium deoxycholate, and % triton x- ) supplemented with protease inhibitor cocktail (sigma) and phosphatase inhibitor cocktail ii (calbiochem). protein extracts ( mg) were analyzed by immunoblotting as described previously [ ] . the following primary antibodies were used to probe blots: mouse anti-wnv from the center for disease control; rabbit anti-isg , rabbit anti-isg , rabbit anti-isg , kindly provided by dr. g. sen; mouse anti-pkr from santa cruz; rabbit anti-rig-i and rabbit anti-mda from ibl; mouse anti-tubulin from sigma; and rabbit anti-stat- from cell signaling. secondary antibodies included peroxidase-conjugated goat anti-rabbit, goat anti-mouse, donkey anti-rabbit, and donkey anti-mouse were from jackson immunoresearch. for analysis of viremia, serum was separated (bd microtainer tube sst) and rna was extracted as previously described [ ] . wnv rna copy number was measured by rt-quantitative pcr (rt-qpcr) as previously described [ ] . for cultured cells, total rna was extracted using the rneasy kit (qiagen), dnase treated (ambion) and evaluated for isg , isg , ifn-b, rig-i, and mda rna expression by one-step sybr green rt-qpcr. specific primer sets for isg- , isg- , rig-i, and ifn-b have been described previously [ , ] . primer sets for mda are: -gtggtcgagccagagctgat and -tgtctcatg-ttcgataactcctgaa. ifn-a and -b were measured in sera using a biological assay as previously described [ ] . briefly, l cells were seeded at cells/well in a well plate one day prior to the addition of interferon standards or experimental samples. mouse sera (diluted : in l media) were treated with uv light for minutes to eliminate residual virus. duplicate sera samples were then added to the -well plates in two-fold dilutions along with a murine ifnb standard. the following day, emcv challenge virus was added to the cells in ml/well at an moi of . . twenty-four hours later, cytopathic effect was measured by a blinded scorer and ifn levels in the sera was calculated based on the ifn standard. ifn-b in cell culture supernatants was analyzed using mouse-specific elisa kits from pbl biomedical laboratories according to the manufacturer's protocol. wnv-specific igm, total igg, igg , and igg a levels were determined by an elisa using purified recombinant e protein as previously described [ ] . the neutralization titer of serum antibody was determined by using a previously described plaque reduction neutralization assay [ ] . briefly, sera samples from mock or wn-tx infected mice were diluted in dmem followed by incubation at uc for minutes to inactivate virus and complement factors. sera were further diluted in two-fold increments and incubated with pfu of wn-tx at uc for hour. standard plaque assays were performed on bhk cells and the dilution at which % of plaques were neutralized was determined by comparing the number of plaques formed from wnv-infected sera samples to mock infected sera samples. cytokine/chemokine analysis wnv infected sera were analyzed for the presence and levels of tnf-a, ifn-c, cxcl (ip- ), and il- by a mouse-specific cytokine/chemokine milliplex elisa (millipore). mock-infected or wnv-infected mice were exsanguinated and perfused with pbs, % paraformaldehyde, ph . . brains were embedded in paraffin and -mm sections were prepared and stained with hematoxylin and eosin (h&e) by the uw histology pathology laboratory. sections were analyzed using a nikon eclipse e microscope (uw keck microscope facility). draining lymph nodes from mice were isolated and digested with collagenase (roche) and type i dnase in serum-free rpmi media at uc for minutes with mechanical disruption. cells were then incubated with rpmi media containing % fbs with edta and hepes for minutes at room temperature, pelleted, and resuspended in pbs containing % fbs and . % sodium azide (facs staining buffer). splenocytes were isolated, washed, and re-suspended in rpmi containing % fbs before in vitro stimulation. cells were washed twice before facs staining. for isolation of cns immune cells, mice were euthanized and perfused extensively with pbs to remove residual intravascular leukocytes. brains and spinal cords from mice per experimental group were isolated and pooled. tissues were minced in rpmi media, triturated, and digested with liberase (roche) and type i dnase in serum-free rpmi media at uc for min. immune cells were isolated after gradient centrifugation from a / % percoll interface and washed twice with facs staining buffer. immune cells were stained with antibodies specific to cd c, cd b, b , cd , cd , cd , cd , nk . , gr- , siglec h, and cd (all reagents from ebiosciences). intracellular foxp staining was performed as previously described [ ] . intracellular ifn-c staining was performed on splenocytes and cns immune cells as previous described [ , ] . briefly, lymphocytes were stimulated with mg/ml of the wnv ns b peptide (ssvwnat-tai) for h at uc. cells were washed and stained for cell surface markers followed by permeabilization-fixation using the cytofix-cytoperm kit (bd-pharmingen) and stained with a pacific-blue conjugated ifn-c antibody (ebiosciences) at uc for min, washed and analyzed by flow cytometry. flow cytometry was performed on a bd lsrii machine using bd facsdiva software. cell analysis was performed on flowjo (v. . . ) software. for in vitro studies and immune cell analysis an unpaired student t-test was used to determine statistical differences. for in vivo viral burden analysis, mann-whitney analysis was used to determine statistical differences. kaplan-meier survival curves were analyzed by the log-rank test. a p-value # . was considered significant. all data were analyzed using prism software (graphpad prism ). west nile virus activity-united states pathogensis of west nile virus infection: a balance between virulence, innate and adaptive immunity, and viral evasion virology, pathology, and clinical manifestations of west nile virus disease west nile virus meningoencephalitis in an immunocompetent adolescent severe west nile virus disease in healthy adults emerging viruses in transplantation: there is more to infection after transplant than cmv and ebv evasion and disruption of innate immune signalling by hepatitis c and west nile viruses cell-specific irf- responses protect against west nile virus infection by interferondependent and -independent mechanisms interferon regulatory factor irf- induces the antiviral alpha interferon response and protects against lethal west nile virus infection the host response to west nile virus infection limits viral spread through the activation of the interferon regulatory factor pathway resistance to alpha/beta interferon is a determinant of west nile virus replication fitness and virulence alpha/beta interferon protects against lethal west nile virus infection by restricting cellular tropism and enhancing neuronal survival mda- : an interferon-inducible putative rna helicase with double-stranded rnadependent atpase activity and melanoma growth-suppressive properties regulating intracellular anti-viral defense and permissiveness to hepatitis c virus rna replication through a cellular rna helicase, rig-i the rna helicase rig-i has an essential function in double-stranded rnainduced innate antiviral responses shared and unique functions of the dexd/h-box helicases rig-i, mda , and lgp in antiviral innate immunity lengthdependent recognition of double-stranded ribonucleic acids by retinoic acidinducible gene-i and melanoma differentiation-associated gene regulation of innate antiviral defenses through a shared repressor domain in rig-i and lgp distinct rig-i and mda signaling by rna viruses in innate immunity west nile virus evades activation of interferon regulatory factor through rig-i-dependent and -independent pathways without antagonizing host defense signaling establishment and maintenance of the innate antiviral response to west nile virus involves both rig-i and mda signaling through ips- differential roles of mda and rig-i helicases in the recognition of rna viruses toll-like receptor has a protective role against west nile virus infection toll-like receptor mediates west nile virus entry into the brain causing lethal encephalitis toll-like receptor mitigates lethal west nile encephalitis via interleukin -dependent immune cell infiltration and homing coordination of early protective immunity to viral infection by regulatory t cells natural regulatory t cells in infectious disease monocyte-derived dendritic cells in innate and adaptive immunity card games between virus and host get a new player novel characteristics of the function and induction of murine p family proteins transcriptional profiling of interferon regulatory factor target genes: direct involvement in the regulation of interferon-stimulated genes infection and injury of neurons by west nile encephalitis virus west nile virus infection in the golden hamster (mesocricetus auratus): a model for west nile encephalitis role of cd + t cells in control of west nile virus infection protective capacity and epitope specificity of cd (+) t cells responding to lethal west nile virus infection antigenspecific cytotoxic t lymphocytes protect against lethal west nile virus encephalitis replicon particles of venezuelan equine encephalitis virus as a reductionist murine model for encephalitis cell type-specific involvement of rig-i in antiviral response a critical role for induced igm in the protection against west nile virus infection b cells and antibody play critical roles in the immediate defense of disseminated infection by west nile encephalitis virus batf deficiency reveals a critical role for cd alpha+ dendritic cells in cytotoxic t cell immunity in vivo induction of immune responses to pathogens by conventional dendritic cells role of regulatory t cells in coronavirus-induced acute encephalitis regulatory t cells promote early influx of cd + t cells in the lungs of respiratory syncytial virus-infected mice and diminish immunodominance disparities tregs control the development of symptomatic west nile virus infection in humans and mice production of type i interferons: plasmacytoid dendritic cells and beyond differential activation of human monocyte-derived and plasmacytoid dendritic cells by west nile virus generated in different host cells early b-cell activation after west nile virus infection requires alpha/beta interferon but not antigen receptor signaling type i ifn receptor signals directly stimulate local b cells early following influenza virus infection early type i interferon-mediated signals on b cells specifically enhance antiviral humoral responses type i interferons act directly on cd t cells to allow clonal expansion and memory formation in response to viral infection innate inflammatory signals induced by various pathogens differentially dictate the ifn-i dependence of cd t cells for clonal expansion and memory formation cutting edge: the direct action of type i ifn on cd t cells is critical for sustaining clonal expansion in response to a viral but not a bacterial infection the reciprocal interaction of nk cells with plasmacytoid or myeloid dendritic cells profoundly affects innate resistance functions cd + t cells require perforin to clear west nile virus from infected neurons cd + t cells mediate recovery and immunopathology in west nile virus encephalitis west nile virus-specific cd t cells exhibit direct antiviral cytokine secretion and cytotoxicity and are sufficient for antiviral protection cd + t-cell responses are required for clearance of west nile virus from the central nervous system dendritic cell-induced memory t cell activation in nonlymphoid tissues tregs are regulated by cytokines: implications for autoimmunity essential role of ips- in innate immune responses against rna viruses pkr and rnase l contribute to protection against lethal west nile virus infection by controlling early viral spread in the periphery and replication in neurons detection of west nile virus lineages and by real-time pcr innate immunity induced by composition-dependent rig-i recognition of hepatitis c virus rna differential induction of type i interferon responses in myeloid dendritic cells by mosquito and mammalian-cell-derived alphaviruses we thank kristy szetter and jessica briley for technical help and robert immormino for generation of purified wnv e protein for elisa. key: cord- -tph n ak authors: kim, yunjeong; liu, hongwei; galasiti kankanamalage, anushka c.; weerasekara, sahani; hua, duy h.; groutas, william c.; chang, kyeong-ok; pedersen, niels c. title: reversal of the progression of fatal coronavirus infection in cats by a broad-spectrum coronavirus protease inhibitor date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: tph n ak coronaviruses infect animals and humans causing a wide range of diseases. the diversity of coronaviruses in many mammalian species is contributed by relatively high mutation and recombination rates during replication. this dynamic nature of coronaviruses may facilitate cross-species transmission and shifts in tissue or cell tropism in a host, resulting in substantial change in virulence. feline enteric coronavirus (fecv) causes inapparent or mild enteritis in cats, but a highly fatal disease, called feline infectious peritonitis (fip), can arise through mutation of fecv to fip virus (fipv). the pathogenesis of fip is intimately associated with immune responses and involves depletion of t cells, features shared by some other coronaviruses like severe acute respiratory syndrome coronavirus. the increasing risks of highly virulent coronavirus infections in humans or animals call for effective antiviral drugs, but no such measures are yet available. previously, we have reported the inhibitors that target c-like protease ( clpro) with broad-spectrum activity against important human and animal coronaviruses. here, we evaluated the therapeutic efficacy of our clpro inhibitor in laboratory cats with fip. experimental fip is % fatal once certain clinical and laboratory signs become apparent. we found that antiviral treatment led to full recovery of cats when treatment was started at a stage of disease that would be otherwise fatal if left untreated. antiviral treatment was associated with a rapid improvement in fever, ascites, lymphopenia and gross signs of illness and cats returned to normal health within days or less of treatment. significant reduction in viral titers was also observed in cats. these results indicate that continuous virus replication is required for progression of immune-mediated inflammatory disease of fip. these findings may provide important insights into devising therapeutic strategies and selection of antiviral compounds for further development for important coronaviruses in animals and humans. coronaviruses comprise a large family of rna viruses that infect a wide variety of mammalian and avian hosts causing a broad spectrum of diseases. coronaviruses have a single-stranded, positive-sense rna genome and are classified into four genera of alpha-, beta-, gamma-, and deltacoronaviruses [ ] . coronaviruses are prone to mutation and recombination during replication and this propensity has contributed to the existing diversity of coronaviruses [ , ] . sudden emergence of new coronaviruses transmitted from animal hosts, severe acute respiratory syndrome coronavirus (sars-cov) and, more recently, middle east respiratory syndrome coronavirus (mers-cov), has raised awareness about the potential risks of highly virulent coronavirus infections in humans with increasing close contact between humans and animals harboring coronaviruses. however, effective therapeutic measures for coronavirus infections have been elusive so far despite the extensive efforts in the development of anti-coronavirus agents [ ] [ ] [ ] [ ] [ ] . shifts in tissue or cell tropism and resulting changes in virulence have also been reported for coronaviruses; porcine respiratory coronavirus causes mild respiratory infection in pigs and presumably arose from transmissible gastroenteritis virus (tgev), the etiologic agent of gastroenteritis in young pigs with a high fatality, by spontaneous mutations and/or deletions in its genome [ ] . seemingly innocuous coronavirus infection can also be turned deadly by changing its tropism, exemplified by mutation of feline enteric coronavirus (fecv) to feline infectious peritonitis virus (fipv) [ , ] . feline infectious peritonitis (fip) has intrigued researchers for half a century since its first description in the s [ ] . infection with fecv which causes inapparent or mild enteritis is widespread among cats, especially in high-density environments, and has little clinical consequence. however, a small portion of cats develop fip during the course of fecv infection and succumb to the disease. published studies support that fip arises in individual cats through mutation of the virus to gain tropism for macrophages [ ] [ ] [ ] [ ] [ ] and that the immune system of the infected cats plays an important role in the pathogenesis of fip [ ] . fip occurs in two major forms, effusive (wet) form or non-effusive (dry) form. the wet form is more common ( - % of fip cases) and characterized by accumulation of fluids in the abdominal and/or, to a lesser degree, chest cavities [ ] . granulomatous vasculitis is frequently found in the omentum, mesenteric lymph nodes, and serosal surface of the large intestine, resulting in the characteristic exudates rich in protein and inflammatory cells in the body cavities in wet fip [ ] . the majority of exudate cells are virusinfected macrophages and high virus load is detected in these cells [ ] . multiple granulomatous lesions composed of macrophages laden with viruses and other inflammatory cells typically form in various tissues and organs, such as the omentum, mesenteric lymph nodes, spleen and liver, in both forms of fip [ ] . clinical symptoms of fip reflect the organs involved and include fever, jaundice, bodily effusions and weight loss and may also affect the central nervous system and the eyes [ ] . virus-induced immunopathogenesis and lymphopenia in cats with fip are features also frequently associated with other coronavirus infections, such as sars and mers in humans. the causes for lymphopenia observed in these coronavirus infections are not fully elucidated but the published reports support that lymphopenia is related to the indirect effects of virus infection [ ] [ ] [ ] . lymphopenia associated with massive apoptosis of uninfected t-cells is a prominent feature of both experimental and natural fip [ , , , ] and implicated with cytokines secreted by the virus-infected macrophages and other immune cells [ , ] . lymphopenia precedes the onset of clinical signs and is associated with disease progression and death in experimental fip, which indicates that impaired cellular immune responses associated with lymphocyte depletion is important in fip pathogenesis [ , ] . once cats develop classic clinical signs, fatality to fip is virtually % [ , [ ] [ ] [ ] and the median survival time from the time of diagnosis to death or euthanasia is about - days [ , ] . fip is a leading cause of death among young cats under years of age and estimated to kill in to cats worldwide [ , ] . fip also affects endangered exotic cats in zoos, such as jaguars and cheetahs [ ] . however, vaccines have proven ineffective and treatment is only palliative [ ] . studies of anti-coronavirus drugs have mainly focused on the discovery of anti-sars-cov agents. effective treatment intervention for coronavirus infections with an immunopathological component, such as sars, mers and fip, is speculated to involve the judicious use of immunomodulatory agents to enhance protective host immunity and decrease pathological immune responses and antiviral drugs to directly inhibit viral replication. we have previously reported several series of small synthetic peptidyl compounds that target a virally-encoded protease, c-like protease ( clpro) [ ] [ ] [ ] . coronavirus clpro and papain-like protease (plp) process viral polyproteins into functional individual proteins and their structures are highly conserved among coronaviruses. since viral proteases are indispensable for virus replication, many synthetic small molecules or natural compounds targeting clpro or plp of coronaviruses have been investigated using the in vitro systems [ ] [ ] [ ] [ ] [ ] . however, only few studies tested the in vivo efficacy of protease inhibitors in experimental animals [ , ] . deng et at [ ] reported that a plp inhibitor failed to reduce virus titers in the lung or increase the survival of mice infected with a mouse-adapted sars-cov, presumably due to low bioavailability or stability of the inhibitor. recently, we demonstrated that our clpro inhibitors significantly decreased the virus titers and pathology in the liver of mice infected with murine hepatitis virus (mhv), a murine coronavirus [ ] . in those studies, treatment was started shortly before or after virus infection in asymptomatic mice. here we extended our previous work on coronavirus clpro inhibitors and investigated the pharmacokinetics (pk), safety and efficacy of a clpro inhibitor in cats. gc is a clpro inhibitor which is previously reported to be active against the clpro of multiple coronaviruses, including sars-cov [ ] , but with highest potency against fipv in cell culture. in this study, we determined that gc exhibited favorable bioavailability and safety in cats. in the in vivo efficacy study using gc in cats experimentally infected with fipv, antiviral treatment was started after the cats reached a clinical stage that would ultimately lead to death, if untreated. antiviral treatment caused a rapid reversal of clinical signs and lymphopenia and reduction in viral titers in the macrophages from the ascites. active infection was no longer apparent after - days of antiviral treatment and the treated cats have remained normal under observation for as long as eight months. these results provide important first evidence that a clpro inhibitor is effective at reversing disease progression when administered to cats in an advanced and invariably fatal stage of experimentally induced fip. pharmacokinetics study of gc in cats gc (fig ) is a representative compound of the dipeptidyl transition state clpro inhibitors [ ] [ ] [ ] ] whose synthesis was described previously [ ] . npi shares homologous structural elements with gc , except that npi has an additional residue of -naththylalanine compared to gc in a position that corresponds to the p position [ ] , using the nomenclature of schechter and berger [ ] (fig ) . the comparable antiviral activity of gc and npi against the replication of feline coronavirus in a cell culture system was previously reported (fig ) [ , ] . however, their pk properties have not been reported. in this study, we investigated the drug plasma concentration changes in healthy specific pathogen free (spf) [ , ] and the % cytotoxic concentration (cc ) values of gc or npi determined in various cell lines were previously reported [ , ] and summarized in a table. cats of - month age (n = for each compound) following single subcutaneous (s.c.) dose of mg/kg gc or mg/kg npi . serial blood samples were then collected and the plasma drug concentrations were measured. previously, we reported that gc is converted into an aldehyde form by the removal of the bisulfite group, and the aldehyde form forms a reversible covalent bond with the nucleophilic cysteine residue of clpro in the x-ray crystallography studies [ ] . we also observed the conversion of npi into its aldehyde form in the blood. therefore the aldehyde forms of gc or npi were measured in the plasma samples. fig a shows the plasma drug concentrations over time following single-dose administration of gc (red triangles) or npi (black circles). the pk study results indicate that gc is rapidly absorbed after s.c. administration and the peak plasma level was reached within hr after injection. the mean plasma drug concentrations remained above the % effective concentration (ec ) value of the aldehyde form of gc ( ng/ml) for hrs post injection (fig a, red triangles) . the plasma drug concentrations following injection of mg/kg npi stayed above the ec value of the aldehyde form of npi ( ng/ml) for hrs post injection (fig a, black circles) . the maximum detected plasma drug concentration following npi administration was substantially lower than that of gc by . -fold. this result indicate that gc was more easily absorbed than npi via the tested route, even when the lower dose of npi ( mg/kg), compared to gc ( mg/kg), was taken into account. after the dosage regimen of gc was determined in the pk study, safety of gc was evaluated in four healthy spf cats of - months of age. the cats were administered with mg/kg gc by s.c. injection twice daily at am and pm for weeks. for the duration of the study, they were observed daily for adverse effects. blood samples were taken weekly and the complete blood counts and blood chemistry panels were conducted. during the study period, in the single-dose pharmacokinetics study, two healthy specific pathogen free (spf) cats were subcutaneously injected with gc at mg/kg/dose or npi at mg/kg for the determination of serial plasma drug concentrations. gc and npi are readily converted into aldehyde forms in the blood [ ] . the red triangles and black circles indicate the plasma concentrations of the aldehyde forms of gc and npi , respectively (means and standard error of the means are shown). (b) in the safety study, four healthy spf cats were subcutaneously given gc at mg/kg/dose daily at am and pm for weeks. during that time, plasma drug concentrations were measured at and hr post-injection for the first three days and weekly thereafter (red and black triangles, respectively, means are shown). the dotted red line indicates the ec value of gc . the % cytotoxic concentration (cc ) value of gc (> μm) is greater than the dotted blue line. there were no clinically significant changes in vital signs and clinical lab parameters (s a-s d fig) , indicating that the dosage and the route of administration of gc was well-tolerated in cats for the duration of the safety study. during the safety study, additional blood samples were taken at and hr post drug administration for the first three days and then weekly for weeks. the plasma drug concentrations at hr post administration were determined from the blood collected immediately before next drug administration and thus represent the minimum drug levels in the plasma. the results show that the lowest plasma drug concentrations remained above the ec value (fig b black triangles) and that the highest determined drug concentrations were well below the cc value which is greater than μm in cell culture [ ] (fig b red triangles) . based on the results from the safety and the pk studies, the dose and administration route of gc was determined to be suitable for the in vivo efficacy study. the experimental infection of cats with serotype i fipv that induces wet fip has been reported previously [ , , ] . fipv is classified into serotypes i and ii based on virus neutralization tests. serotype i fipv is responsible for the majority ( - %) of naturally-occurring fip [ , [ ] [ ] [ ] [ ] . in this experimental infection, an absolute lymphopenia, fever, weight loss, jaundice and inapparent to mild ascites appear within - weeks after infection. increasing jaundice and ascites occur during the next - weeks. all the cats that develop lymphopenia and clinical signs following experimental infection do not spontaneously recover but succumb to the disease [ , , ] . to investigate the efficacy of gc , we conducted two independent studies. in these studies, antiviral treatment was started after the infected cats developed the typical laboratory finding of absolute lymphopenia and clinical symptoms to determine whether treatment with gc is effective in reducing the severity of symptoms or fatality. in both studies, the infected cats were monitored daily for fever, body weight, and outward disease signs and weekly for lymphocyte counts. in the first efficacy study, four spf cats of - months of age (p , p , p and p ) were intraperitoneally administrated with a cat-passaged serotype i fipv (fipv-m c- ) [ , , ] . following infection, they developed lymphopenia and clinical symptoms including inapparent or mild ascites within - days post infection (dpi) ( table ). in the second study, the ascites of four spf cats of - months of age inoculated with the same virus (p , p , p and p ) were allowed to progress to more profound, classical abdominal effusions, which closely resemble those of cats with naturally-occurring fip frequently presented at the clinics (table ) . however, in order to alleviate suffering, the latter four cats were given meloxicam, a non-steroidal anti-inflammatory drug, and subcutaneous fluids prior to antiviral treatment. this supportive treatment was discontinued before antiviral drug treatment commenced. the eight cats from both studies developed jaundice, inapparent to profound ascites, absolute lymphopenia ( ~ /μl, reference range , to , /μl) and high fever (up to . °c) (fig b and d , table ) before antiviral treatment was started. they also lost body weight up to . % of their pre-infection weight during this same period (fig c) . when they reached this stage, twice daily s.c. administration of gc at - mg/kg/dose was started. these cats were treated for - days, except for p and p that were euthanized after and days after starting antiviral treatment based on the severe nature of their clinical signs ( fig a) . all six remaining cats showed rapid improvement in attitude and resolution of fever ( fig b) . the profound absolute lymphopenia observed in all cats prior to antiviral treatment also returned to normal before the next blood testing one week later ( fig d) and weight losses were reversed and normal growth resumed ( fig c) . ascites and scrotal swelling indicative of peritonitis also gradually resolved after a week of antiviral treatment. all cats that received antiviral treatment for - days appeared normal by clinical observation and laboratory testing. the six recovered cats from both studies have remained healthy showing no signs of relapse during an observation period up to months. these experiments demonstrate that the protease inhibitor was able to reverse disease progression when treatment was initiated at advanced clinical stages of fip. since fipv is highly associated with tissues and is not reliably detected in blood at high levels in cats with fip [ ] , assessment of the efficacy of antiviral drugs in reducing the viral load poses a difficulty in live animals. although measuring virus titers of the exudate macrophages from the ascites allows to determine the effects of antiviral drug against the replication of fipv, ascites rapidly decreased with antiviral treatment and we were not able to collect ascites in the recovered cats. however, we determined the viral load in two cats from the second study (p and p ) prior to and during antiviral treatment. these cats were euthanized after and days of antiviral treatment. on necropsy, both cats had severe pancreatitis, a possible complication of meloxicam treatment, but no lesions (p ) or mild lesions (p ) typical of fip were found. virus titers in the macrophages from the ascites were determined by real-time quantitative rt-pcr and the ct values were analyzed by the comparative ct method using the β-actin as a reference gene [ ] . the results showed that viral rna level in the macrophages from the ascites decreased commensurately with the duration of antiviral treatment in these cats. the fold reduction of viral rna level determined using the delta delta ct method was , . in p that received day-antiviral treatment ( fig a) and , . in p that received dayantiviral treatment (fig b) , compared to the pre-treatment viral rna level in the macrophages of each cat. the viral rna levels ( -Δct ) in the macrophages from the ascites are summarized in fig d. the viral rna level in the omentum of p and p is also shown in fig c. based on these results, the reduction in virus titers in p and p seems to correlate with the necropsy findings of mild or no fip lesions in those cats. these results on viral titers show that fipv clpro is a valid target for fipv antiviral drugs and gc can effectively reduce the virus load in the macrophages from the ascites and the omentum of cats with fip. serial passages of fipv- in crandell rees feline kidney (crfk) cells in the presence of gc or npi (an aldehyde form of npi ) were conducted to compare the emergence of viral resistance under drug pressure. at passage number , the ec value of npi against fipv increased by -fold, compared to wild-type virus at the same passage number. however, a decrease in antiviral activity of gc against fipv was not observed at up to passages. the sequence analysis of the clpro gene of npi -resistant fipv viruses collected from passage revealed a single mutation of serine to cysteine at the position of , which is located between the β-strands cii and dii in the domain ii (s a fig). since these compounds share similar structure, we also investigated whether npi -resistant viruses retain susceptibility to [ ] and fipv (red) modelled based on tgev clpro (pdb id: f ) [ ] . the clpro of tgev and fipv are highly conserved with the amino acid sequence identity of > %. however, clpro of tgev, mers-cov and sars-cov have low amino acid sequence identity of about %. nonetheless, they share well conserved overall structure (s a fig). the activity of gc was previously reported against the clpro of sars-cov using a fret assay [ ] . however, its activity against the clpro of mers-cov and fipv is unknown. therefore, we cloned and expressed the full-length clpro of fipv and mers-cov following the procedures described previously [ ] . the results are summarized in fig . the data show that gc was most effective against fipv clpro by a fret assay but it also substantially inhibited the activity of mers-cov and sars-cov clpro. since fip disease progression is quite rapid and the pathogenesis of fip is primarily immunemediated, an important question has remained unanswered as to whether antiviral drug treatment can effectively reverse disease progression in symptomatic hosts. it was previously shown that anti-inflammatory agent or antiviral immunity enhancing agents increased survival of mice infected with mouse-adapted sars-cov and treated with a nf-kb inhibitor [ ] or various toll-like receptor agonists [ , [ ] [ ] [ ] , which was started shortly before or after virus infection. these reports indicate that controlling immune responses may prove an effective therapeutic strategy for coronavirus infections where inflammation plays an important role in pathogenesis. however, the available data on the efficacy of antiviral compounds failed to show sufficient effectiveness in mice infected with mouse-adapted sars-cov, even when treatment was started at the same time or shortly after virus infection [ , ] . the observed low effectiveness of antiviral treatment is largely thought to be due to the use of compounds with weak anticoronavirus activity and/or bioavailability. however, the lack of available potent antiviral compounds against coronaviruses has made it difficult to investigate the effects of antiviral treatment in animals with lethal coronavirus infection. our clpro inhibitors were previously reported to be potent against fipv in the in vitro assays [ , ] and effective at significantly reducing viral titers and tissue pathology in mice infected with mhv [ ] . however, these clpro inhibitors have not been tested in cats. in this study, a clpro inhibitor, gc , was determined to be safe with good bioavailability in cats. in the in vivo efficacy study using cats with fip, the antiviral treatment started for cats at clinically advanced stages led to rapid normalization of the numbers of lymphocytes, during which time, fever, jaundice and ascites also resolved. the granulomatous lesions typically found in various organs in the cats infected with fipv were not found or greatly reduced in the two cats that were euthanized after only and days of antiviral treatment. these results demonstrate that continuous virus replication is important in the progression of the immune-mediated pathogenesis of fip and that controlling virus replication by a directly-acting antiviral compound targeting coronavirus clpro is effective at reversing fip disease. our results provide the first evidence, to our best knowledge, that a direct-acting antiviral agent is effective at reversing the immune-mediated disease progression caused by coronavirus infection, even when antiviral treatment was started at clinically advanced stages. this finding may have important implication in devising effective therapeutic strategies for other coronavirus infections. the conserved active site of coronavirus clpro has been considered as a promising target for the design of broad-spectrum inhibitors for coronavirus infections [ , ] and our group [ ] and others [ , , ] have previously reported the synthesis of clpro inhibitors with antiviral activity against multiple coronaviruses. gc was previously shown to be active against fipv in cell culture [ , ] and sars-cov clpro in a fret assay [ ] . in this study, we compared the activity of gc against the clpro of fipv, mers-cov and sars--cov by a fret assay and determined that gc has most potent activity against the clpro of fipv. the ic values of gc against mers-cov and sars-cov were . and . -fold, respectively, higher than that against fipv in a fret assay. these results indicate that gc is active against the clpro of coronaviruses belonging to alphacoronavirus (fipv) or the multiple clades in betacoronavirus (mers-cov and sars-cov), despite the low sequence identity of clpro among fipv, sars-cov, and mers-cov. the varying degree of effectiveness of gc against clpro of different coronaviruses may reflect a subtle difference in spatial structure fit of the compound in the active site of coronavirus clpro (s b and s c fig) . however, the antiviral activity of gc against the replication of these human coronaviruses has not yet been determined in cell culture or in the animal models. a majority of reported protease inhibitors that are shown to have inhibitory effects against various coronaviruses in the enzyme assay are tripeptidyl or bulkier compounds [ , [ ] [ ] [ ] [ ] and their antiviral activities in cell culture or in vivo properties are often not available. in this study, we compared the subcutaneous absorption of dipeptidyl and tripeptidyl clpro inhibitors. gc , a dipeptidyl compound, consists of a warhead, a gln surrogate structure in a position that corresponds to the p position, leu in the p position and a cap structure [ ] (fig ) . npi , a tripeptidyl compound, has homologous structural elements with gc , except that npi has an additional residue of -naththylalanine (fig ) . these two compounds have comparable antiviral activity against fipv in cell culture (fig ) [ ] . however, the peak plasma concentration following a subcutaneous injection of npi was considerably lower than that of gc , which indicates that gc is absorbed better than npi via the subcutaneous route. our results on these closely related compounds indicate that relatively small structural change (addition of a residue) can have profound effects in absorption, and therefore the bioavailability of compounds needs to be taken into consideration early during drug selection process. gc was also found to be well-tolerated in cats during the -week duration of twice daily administration, with plasma drug concentrations remaining above the ec value but well below the cc value. emergence of viral resistance is a major concern in antiviral therapy. the only available literature on protease inhibitor-resistant coronavirus [ ] reported that a clpro inhibitor (grl- ) has a low genetic barrier to mhv. in that study, resistant viruses were selected in passage numbers in the presence of the inhibitor in cell culture, but the resistant viruses were highly attenuated in mice. to study the development of viral resistance against our clpro inhibitors, we serially passaged fipv in the presence of mock (vehicle), gc or the aldehyde form (npi ) of npi . at passage , the ec value of npi increased by -fold, compared to wild-type viruses passaged without npi , indicating the emergence of npi -resistant viruses. the clpro gene of npi -resistant virus has a single mutation of s c which located between the cii and dii strands in the domain ii (s a fig). the role of this mutation in the clpro in conferring resistance to npi is currently not clear. however, the fact that serine at this position is conserved among all feline coronaviruses whose sequences are available and that c , the active site residue that forms a covalent bond with the warhead of the inhibitor, is on the same loop as s between the cii and dii strands (s a fig) suggest that this mutation may influence the conformation of the loop and positioning of the activesite residue for proteolysis. interestingly, deng et al [ ] also reported that one of the mutations on clpro of mhv which is partially resistant to grl- is located away from the catalytic site but in a position that may influence the conformation of the catalytic site. in contrast to npi , resistant viruses against gc have not been selected at up to passages. these results indicate that similarly structured compounds may have different levels of resistance barrier against coronavirus clpro. interestingly, npi -resistant viruses did not lose susceptibility to gc in cell culture, indicating that this mutation did not confer cross-resistance. the mechanisms underlying differences in resistance development to these inhibitors need to be defined, but it may be speculated that the small size of gc , compared to npi , makes it difficult for the virus to evade drug binding while retaining substrate cleavage capability. we are currently investigating the relative viral fitness of the resistant viruses and the role of the mutation in conferring resistance to npi . in summary, a representative compound, gc , of our dipeptidyl clpro inhibitor series was shown to be safe by the dosage regimen used in cats and effective at reversing the progression of fip even when the treatment was started at advanced clinical stages. based on these results, this compound may have the potential to be developed into a safe and effective drug for fip. furthermore, broad activity of this compound against important human coronaviruses, including mers-cov and sars-cov, suggest that our inhibitor series may serve as a platform for further optimization for those important viruses. the results of this study also suggest that similar intervention approaches targeting virally-encoded clpro warrant investigation for other existing and emerging coronavirus infections. random bred cats free of most common feline pathogens, including feline enteric coronavirus, were obtained from the feline nutrition breeding colony, school of veterinary medicine, uc davis. all animal experiments were conducted in strict compliance with the animal welfare act, phs policy and other federal statutes and regulations relating to animals and approved by the institutional animal care and use committee at university of california, davis (protocol number: ). the syntheses of gc and npi were previously described by our group [ , ] . in the single-dose pk study, two healthy spf cats of - months of age (n = for each compound) were subcutaneously injected with mg/kg gc or mg/kg npi dissolved in % etoh and % peg . blood samples were collected from each cat at , , , , , , and hrs following injection and plasma samples were prepared. the plasma drug concentrations were measured using routine high pressure liquid chromatography by frontage laboratories, inc (exton, pa). in the safety (multiple-dose) study, four healthy spf cats of - months of age were injected s.c. with a daily dose of gc ( mg/kg/dose dissolved in % etoh, % peg and % pbs) at am and pm for weeks. plasma samples were prepared for determination of drug concentrations at or hrs (immediately before the next dose) after drug administration for the first three days and weekly thereafter for weeks. measurement of plasma drug concentrations from the safety study was also performed by frontage laboratories, inc. during the safety study, cats were monitored twice daily for adverse effects and body weight was measured daily. prior to the first dose of gc and thereafter weekly, blood samples were collected from each cat for complete blood count and blood chemistry tests. a total of eight female or male spf cats of - months of age in two independently conducted studies were originally part of another published experiment concerning the role of genetics in susceptibility/resistance to fipv infection [ ] . in that study, cats were inoculated with catpassaged serotype i field strain fipv-m c- . among the cats that developed clinical and laboratory signs consistent with the abdominal effusive (wet) form of fip and progressed to the point where they would be inevitably fatal, eight cats were transferred to the drug efficacy study. four cats (p , p , p and p ) in the first study did not receive any medication other than gc . five doses of oral or subcutaneous meloxicam at . mg/kg/dose (once a day) as well as fluids were given to four cats (p , p , p and p ) in the second study for alleviation of pain and dehydration and discontinued before antiviral treatment was started. gc dissolved in % etoh, % peg and % pbs was given s.c. at am and pm for - days. p and p were given gc at mg/kg/dose for or days, respectively, and the dose was increased to mg/kg/dose until the end of antiviral treatment. p and p were euthanized following antiviral treatment of and days, respectively. all other cats received gc at mg/kg/dose during antiviral treatment. animals were observed daily for clinical signs and body weight and blood was collected weekly for lymphocyte counts. ascites were collected at multiple times before and during antiviral treatment from p and p for virus titration by real-time quantitative rt-pcr (qrt-pcr). the omentum samples were collected from p and p on necropsy. virus titers in the macrophages from the ascites and the omentum were determined by realtime qrt-pcr. ascites ( ml) collected from p and p were diluted at : in pbs containing units/ml heparin. after centrifugation, the cell pellets were incubated with μl rnalater (life technologies, ny, usa) for overnight at °c. cell pellets were then collected by centrifugation and stored at - °c until analysis. prior to total rna extraction, μl of pbs was added to the cell pellets. omentum was cut into a size of less than . cm and placed in volumes of rnalater. following overnight incubation at °c, samples were centrifuged for min at , rpm to remove supernatant and tissues were stored at - °c until analysis. total rna was extracted from the macrophages from the ascites and the omentum using rneasy mini kit (life technologies) and real-time qrt-pcr was conducted. the primers and a probe targeting the '-utr region of fipv are '-ggaggtacaagcaaccctatt- ' (a forward primer), '-gatccagacgttagct cttcc- ' (a reverse primer) and fam-agatccgc tatgacgagccaacaa-iowa black (a probe). the relative levels of viral rna in the samples were calculated by the comparative ct method [ ] using beta actin as a reference gene. the fold changes in viral rna level in the macrophages in the ascites collected during antiviral treatment were calculated using the viral rna level in the macrophage samples collected prior to the antiviral treatment. serial passages of fipv to generate viruses resistant to gc and npi sequential in vitro passage experiments using wild-type fipv- in the presence of gc or npi were performed to select resistant viruses. briefly, crfk cells were infected with fipv at an moi of . - in the presence of gc or npi ranging from . ~ μm. at each passage, supernatants containing viruses were passed on to fresh cells in the presence of gc or npi . control mock virus was passaged in the absence of drug following the same procedure. virus titers at certain passage numbers were determined by the % tissue culture infective dose assay and the fold changes in ec values relative to the wild-type virus were determined. after passages in the presence or absence of npi , total viral rna was isolated using the rneasy mini kit (invitrogen) and the clpro gene was sequenced following amplification by rt-pcr and analyzed for the presence of mutations. the viruses grown without the drug (mock) or npi -resistant viruses at passage number were purified three times by limiting dilution [ ] . to investigate if npi -resistant viruses are susceptible to gc , serial dilutions of gc or npi were added to confluent monolayers of crfk cells in -well plates or cells were mock-treated, and the cells were immediately infected with npi -resistant virus at an moi of . - . following incubation at °c until an extensive cytopathic effect was observed in the mock-treated well (up to hrs), cells were freeze-thawed for virus titration. the ec values were determined using graphpad prism software version (graphpad software, san diego, ca) following the procedures described previously [ , ] . the codon-optimized cdna encoding the full length clpro of fipv-m c- was amplified by rt-pcr using the omentum tissue from the cats infected with fipv-m c- . those of sars--cov (genbank: gu . ) and mers-cov (genbank: km ) were synthesized by genscript (piscataway, nj). the expression and purification of each clpro were conducted following a standard method described previously by our group [ ] . primers for mers clpro are; forward primer (attctagaaaggagatataccatgcat catcatcat catcatagcggtctggttaaaatgagcc) and reverse primer (atctcgagtcactg catcacaacacccataatc). primers for fipv clpro are; forward primer (attcta gaaaggagatataccatgcatcatcatcatcatcattctg gattgc gaaaaatggc) and reverse primer (atctcgaggcggccgctcactgact). fret assay was performed using a fluorogenic substrate (dabcyl-ktsavlq/sgfrkmeedans) derived from the cleavage sites on viral polyproteins of sars-cov [ ] and was synthesized by genscript. methods for fret assay were described previously by our group [ , ] . briefly, in the fret assay, clpro of fipv, mers-cov, or sars-cov were incubated with gc for min and the edans/dabcyl fret substrate derived from the cleavage sites on sars-cov polyprotein was added to the mixture. following incubation for min, the florescence signals were measured and the ic was calculated for each clpro [ ] [ ] [ ] . three-dimensional structural model of clpro the structural model of fipv clpro was built based on tgev clpro (pdb id: f ) [ ] using the easymodeller program (version . ) [ ] and superimposed on the clpro structure of mers-cov (pdb id: wme, teal) [ ] . the surface representation of the active sites of tgev (pdb id: f ) and mers-cov (pdb id: wme) were created with pymol (delano scientific) [ ] . [ ] and fipv clpro (red) modeled using modeller [ ] based on tgev clpro as a template (pdb id: f ) [ ] . the clpro of tgev and fipv are highly conserved with the amino acid sequence identity of > %. coronavirus clpro forms a dimer for function but only the monomer form is shown here. the tan rectangle contains the active site located in the cleft between the domains i and ii. the active site residues of clpro of mers-cov and fipv, cys and his, are shown in orange and blue colors, respectively. the residue (s ) mutated in the clpro of fipv resistant to npi , an aldehyde form of npi , is shown in purple. (b and c) surface representation of the active sites of clpro of tgev (pdb id: f ) [ ] (c) and mers-cov (pdb id: wme) [ ] (d). (b) the crystal structure of tgev clpro bound with gc (gray) in the s and s pockets of the active site of clpro was previously published by our group [ ] . the residues in the s and s pockets that form hydrogen bonds with gc are shown in yellow. (c) the s and s pockets of mers-cov clpro are shown in pink. the residues that can potentially form hydrogen bonds with gc are indicated. all images were newly prepared using pymol. (tif) virus taxonomy: ninth report of the international committee on taxonomy of viruses ed the molecular biology of coronaviruses discovery of seven novel mammalian and avian coronaviruses in the genus deltacoronavirus supports bat coronaviruses as the gene source of alphacoronavirus and betacoronavirus and avian coronaviruses as the gene source of gammacoronavirus and deltacoronavirus antiviral drugs specific for coronaviruses in preclinical development anti-sars coronavirus agents: a patent review ( -present) potential antivirals and antiviral strategies against sars coronavirus infections from sars to mers: crystallographic studies on coronaviral proteases enable antiviral drug design design of wide-spectrum inhibitors targeting coronavirus main proteases porcine respiratory coronavirus differs from transmissible gastroenteritis virus by a few genomic deletions a review of feline infectious peritonitis virus infection: - an update on feline infectious peritonitis: virology and immunopathogenesis feline infectious peritonitis: role of the feline coronavirus c gene in intestinal tropism and pathogenicity based upon isolates from resident and adopted shelter cats significance of coronavirus mutants in feces and diseased tissues of cats suffering from feline infectious peritonitis spike protein fusion peptide and feline coronavirus virulence phylogenetic analysis of feline coronavirus strains in an epizootic outbreak of feline infectious peritonitis mutation in spike protein cleavage site and pathogenesis of feline coronavirus levels of feline infectious peritonitis virus in blood, effusions, and various tissues and the role of lymphopenia in disease outcome following experimental infection mechanisms of lymphocyte loss in sars coronavirus infection a "possible" involvement of tnfalpha in apoptosis induction in peripheral blood lymphocytes of cats with feline infectious peritonitis tnf-alpha, produced by feline infectious peritonitis virus (fipv)-infected macrophages, upregulates expression of type ii fipv receptor feline aminopeptidase n in feline macrophages in vivo cytokine response to experimental feline infectious peritonitis virus infection natural history of a recurrent feline coronavirus infection and the role of cellular immunity in survival and disease effect of feline interferon-omega on the survival time and quality of life of cats with feline infectious peritonitis randomized, placebo controlled study of the effect of propentofylline on survival time and quality of life of cats with feline infectious peritonitis an update on feline infectious peritonitis: diagnostics and therapeutics. veterinary journal epidemiology of feline infectious peritonitis among cats examined at veterinary medical teaching hospitals prevalence and implications of feline coronavirus infections of captive and free-ranging cheetahs (acinonyx jubatus) broad-spectrum antivirals against c or c-like proteases of picornaviruses, noroviruses, and coronaviruses potent inhibition of feline coronaviruses with peptidyl compounds targeting coronavirus c-like protease broad-spectrum inhibitors against c-like proteases of feline coronaviruses and feline caliciviruses a chimeric virusmouse model system for evaluating the function and inhibition of papain-like proteases of emerging coronaviruses characterization and inhibition of norovirus proteases of genogroups i and ii using a fluorescence resonance energy transfer assay on the size of the active site in proteases. i. papain the influence of age and genetics on natural resistance to experimentally induced feline infectious peritonitis an eight-year epidemiologic study based on baculovirus-expressed type-specific spike proteins for the differentiation of type i and ii feline coronavirus infections feline coronavirus serotypes and : seroprevalence and association with disease in switzerland prevalence of feline coronavirus types i and ii in cats with histopathologically verified feline infectious peritonitis the prevalence of types i and ii feline coronavirus infections in cats analyzing real-time pcr data by the comparative c(t) method structures of the middle east respiratory syndrome coronavirus c-like protease reveal insights into substrate specificity fused-ring structure of decahydroisoquinolin as a novel scaffold for sars cl protease inhibitors inhibition of nf-kappab-mediated inflammation in severe acute respiratory syndrome coronavirus-infected mice increases survival a new mouse-adapted strain of sars-cov as a lethal model for evaluating antiviral agents in vitro and in vivo intranasal treatment with poly(i*c) protects aged mice from lethal respiratory virus infections coronavirus main proteinase ( clpro) structure: basis for design of anti-sars drugs design, synthesis and antiviral efficacy of a series of potent chloropyridyl ester-derived sars-cov clpro inhibitors structural basis of inhibition specificities of c and c-like proteases by zinc-coordinating and peptidomimetic compounds the crystal structures of severe acute respiratory syndrome virus main protease and its complex with an inhibitor characterization and inhibition of sars-coronavirus main protease individual and common inhibitors of coronavirus and picornavirus main proteases synthesis, crystal structure, structureactivity relationships, and antiviral activity of a potent sars coronavirus cl protease inhibitor coronaviruses resistant to a c-like protease inhibitor are attenuated for replication and pathogenesis, revealing a low genetic barrier but high fitness cost of resistance design, synthesis, and bioevaluation of viral c and c-like protease inhibitors natural resistance to experimental feline infectious peritonitis virus infection is decreased rather than increased by positive genetic selection. veterinary immunology and immunopathology isolation of fidelity variants of rna viruses and characterization of virus mutation frequency easymodeller: a graphical interface to modeller the pymol molecular graphics system vs, llc we thank monica durden for care of cats and david george for technical assistance. key: cord- -xs df a authors: tapia, karla; kim, won-keun; sun, yan; mercado-lópez, xiomara; dunay, emily; wise, megan; adu, michael; lópez, carolina b. title: defective viral genomes arising in vivo provide critical danger signals for the triggering of lung antiviral immunity date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: xs df a the innate immune response to viruses is initiated when specialized cellular sensors recognize viral danger signals. here we show that truncated forms of viral genomes that accumulate in infected cells potently trigger the sustained activation of the transcription factors irf and nf-κb and the production type i ifns through a mechanism independent of ifn signaling. we demonstrate that these defective viral genomes (dvgs) are generated naturally during respiratory infections in vivo even in mice lacking the type i ifn receptor, and their appearance coincides with the production of cytokines during infections with sendai virus (sev) or influenza virus. remarkably, the hallmark antiviral cytokine ifnβ is only expressed in lung epithelial cells containing dvgs, while cells within the lung that contain standard viral genomes alone do not express this cytokine. together, our data indicate that dvgs generated during viral replication are a primary source of danger signals for the initiation of the host immune response to infection. the recognition of virus-specific pattern associated molecular patterns (pamps) is a pivotal event in the initiation of the host innate response to infection. in recent years, it has been established that most viral danger signals are derived from oligonucleotide structures exposed during the replication of the viral genomes [ , , , , , ] . however, most viruses produce proteins that antagonize and effectively delay signaling by the primary viral oligonucleotide sensor molecules retinoic acid inducible gene i (rig-i) and melanoma differentiation-associated gene (mda ), allowing the virus to replicate to high titers and produce large amounts of danger signals prior to host intervention [ , ] . it is currently unclear how the host immune response overcomes viral evasion to initiate a protective antiviral response. defective viral genomes (dvgs) arise when the viral polymerase loses processivity during virus replication at high titers, thereby generating truncated versions of the viral genome that contain deletions and/or complementary ends (the later known as copyback or snap-back genomes) [ , ] . dvgs with the ability to interfere with standard virus replication were first described by von magnus in the early s as the genomes of incomplete forms of influenza virus called defective interfering (di) viral particles [ ] . dvgs have been identified in multiple distinct viral families when the viruses are grown in the laboratory at high multiplicity of infection and span a broad range of hosts, from plants to mammals [ ] . importantly, dvgs are found in patients infected with hepatitis a [ ] , hepatitis b [ , ] , hepatitis c [ ] , hiv [ ] , dengue virus [ ] , and influenza virus [ ] . however, the biological role of dvgs in the context of natural infections is not well understood. we and others have shown that stocks of sendai virus (sev) with a high content of copy-back dvgs with interfering activity trigger enhanced production of cytokines in vitro and more potently induce antigen presentation by mouse and human dendritic cells than do virus stocks lacking this kind of dvgs [ , , , , , ] . our group has also demonstrated that in contrast to standard viral genomes, sev copy-back dvgs induce the expression of mda and of a number of other interferonstimulated genes in the absence of type i ifn positive feedback [ , , ] . remarkably, sev copy-back dvgs show this potent in vitro stimulatory activity even in the presence of functional viral encoded antagonists of the host response [ , ] . here, we demonstrate that dvgs that trigger a robust activation of the transcription factors irf and nf-kb accumulate at a high rate in infected cells becoming the main source of viral pamps. these dvgs arise naturally during acute respiratory viral infections in mice and provide essential stimuli for the initiation of the antiviral innate immune response in the lung. these data demonstrate the generation of dvgs in vivo during acute respiratory viral infections and suggest a critical role of these kinds of viral genomes in determining the quality of the host response to infection. sev copy-back dvgs trigger a robust and sustained activation of irf and nf-kb independent of type i ifn feedback to further investigate the cellular mechanisms responsible for the efficient activation of the antiviral response by sev dvgs, we evaluated the phosphorylation of transcription factors that are critical for the expression of type i ifns in cells infected with equivalent amounts of infectious particles of a sev strain cantell stock containing high levels of copy-back dvgs (sev cantell hd) or with sev cantell depleted of dvgs (sev cantell ld). virus stocks were prepared from the same parental virus and their content of dvgs was determined by calculating the ratio of infectious particles to total particles (ratios are specified in the material and methods section). in addition, copy-back dvgs of these stocks were identified by pcr. one predominant copy-back genome was present in cells infected with sev cantell hd (amplicon of bp), while no copy-back defective genome was detected in cells infected with sev cantell ld up to six hours after infection (figs. a and s ). cloning and sequencing of the nt long amplicon confirmed that it corresponded to a previously described sev cantell copy-back dvg of nt in length (dvg- ) [ ] . phosphorylation of irf and of the nf-kb repressor ikba in response to sev cantell hd occurred rapidly and was sustained even in type i ifn receptor ko cells (ifnar / ) ( fig. b and c), while no phosphorylation of irf or ikba was observed for up to ten hours post-infection with sev cantell ld despite equivalent or higher expression of the viral protein np (fig. d) . corresponding with the strong activation of transcription factors, ifnb mrna was expressed in ifnar / cells infected with sev cantell hd (fig. e ). in contrast, type i ifn signaling was required for the cellular response to newcastle disease virus (ndv), an avian virus that only partially inhibits the type i ifn pathway, triggering the expression of type i ifn and other cytokines in the absence of dvgs. to further validate the role of sev copy-back dvgs as triggers of type i ifn-independent antiviral responses, we cloned dvg- under the control of the t polymerase promoter and used this construct to prepare a sev stock containing a single recombinant dvg (rdvg). for this purpose we used sev strain that normally does not produce highly immunostimulatory copy-back dvgs [ ] . equivalent infectious units of sev and sev plus rdvgs had similar levels of total rna (fig. f ) but infection with virus containing rdvgs strongly induced the antiviral response while virus that lacked dvgs did not (fig. g) confirming the dvg immunostimulatory activity. in addition, presence of rdvgs significantly reduced the expression of sev np mrna, demonstrating their strong interfering capacity (fig. g) . notably, mouse embryo fibroblasts lacking the type i ifn receptor expressed ifnb mrna in response to sev containing rdvg (fig. h ) and virus containing rdvgs triggered irf phosphorylation independently of type i ifn feedback (fig. i) , mirroring the response to sev cantell hd. altogether, this evidence conclusively shows that sev copy-back dvgs confer potent immunostimulatory ability to sev stocks, independent of type i ifn feedback. notably, potent ifnb mrna expression in response to sev dvgs was independent of irf , irf , and irf while only partially dependent on irf (fig. s ). this response was maintained in a variety of cell types (fig. s ) . dvgs accumulate at a high rate in infected cells and are a primary source of pathogen associated molecular patterns that trigger rlr signaling to determine whether standard viral genomes and dvg rnas have distinct intrinsic properties that explain their differential immunostimulatory activities, we compared naked rna purified from a stock of sev cantell ld with in vitro transcribed dvg- . rnas were transfected into cells before or after treatment with phosphatase or with rnase a that cleaves of single stranded c and u residues, and/or rnase v that cleaves base paired nucleotides. both genomic rna and dvg rna were susceptible to treatment with phosphatase, as well as to treatment with rnases ( fig. a) , corresponding with the literature that demonstrates a crucial role for -triphosphate-rna in the induction of type i ifns. while transfected dvgs induced stronger expression of ifnb than an equivalent concentration of gsev ( fig. a) , transfection of equivalent molar amounts of genomic and dvg rna resulted in higher immunostimulatory activity of genomic rna compared to dvg rna (fig. b) , demonstrating that sev ld rna can strongly trigger the host response to infection when delivered naked into the cells. paradoxically, cells infected with sev cantell ld alone failed to induce strong type i ifn production even when used at a times higher infectious dose than sev cantell hd (fig. c) . although the amount of gsev rna was significantly higher in cells infected with an moi of of sev cantell ld compared with ten times less sev cantell hd at h post-infection (fig. c) , dvgs were only detected in cells infected with sev cantell hd, confirming a strong correlation between the presence of dvgs in the infected cells and the induction of the host response to infection. to determine whether the amount of total input viral rna affected the immunostimulatory activity of sev cantell ld and hd, we measured the rna content in equivalent infectious doses of these stocks. sev cantell hd had less than two fold higher the amount of total rna than sev cantell ld and total rna levels were equivalent between sev cantell ld and hd when ld was at twice the infectious dose (fig. d) . thus, differences in the net input amount of viral rna cannot explain the more than . fold difference in the expression of ifnb mrna between cells infected with equivalent infectious doses of sev cantell ld and hd. dvgs have an increased rate of replication compared to standard viral genomes due to their shorter size and promoter properties [ ] . to determine whether dvgs replicate faster than gsev, we calculated the rate of replication of gsev and dvgs in cells infected with sev cantell hd. although at an early time point more copies of gsev than dvgs were detected in the cells, dvgs dominated by h post-infection (fig. e ) accumulating at a times faster rate than gsev (fig. f) . these data demonstrate that dvgs rapidly surpass the number of gsev in infected cells, providing large quantities of pathogen associated molecular patterns. in infections with viruses well adapted to the host virusencoded proteins that delay the cellular response allow the virus to replicate to high titers prior to host intervention. the mechanisms overcoming viral evasion of the immune system and leading to the production of the primary antiviral cytokine ifnb are not well established. here, we demonstrate that truncated forms of viral genomes that are generated in situ during virus replication are a primary source of danger signals for the initiation of the host immune response to respiratory viral infections in vivo. defective viral genomes (dvgs) are able to function as triggers of the immune response even in the absence of type i ifn signaling and are strong triggers of the host response to infection while overcoming viral antagonism. supporting previous observations that dvgs from sev stimulate the cellular antiviral response through signaling by rig-i like receptors (rlrs) [ , , ] , the essential rlr adaptor protein mitochondrial antiviral signaling protein (mavs) was required for the activation of the transcription factors irf and nf-kb and for expression of numerous antiviral and pro-inflammatory molecules upon infection with sev cantell hd. in contrast, mavs was not required for the response to herpes simplex virus, which can trigger the host response independently of rlrs (fig. s ). in addition, only dvg rna, but not standard viral genomes, could be amplified from endogenous rig-i and mda complexes immunoprecipitated from infected cells (fig. s c ), supporting published evidence that dvgs bind to rig-i preferentially over the standard viral genomes in infected cells [ ] . as predicted, association of dvgs with rlrs correlated with type i ifn induction, but not with the level of virus replication (fig. s d ). importantly, in addition to the primary role of rig-i in the response to sev dvgs, mda participates in the induction of type i ifn in primary mouse lung fibroblasts infected with sev hd (fig. s e) , similar to what we have observed in dcs [ , ] . overall, these data demonstrate that dvgs are produced in the infected cells at a higher rate than genomic rna and that dvgs are the predominant ligands for both rig-i and mda during sev infection. based on the potent ability of sev stocks containing a high content of copy-back dvgs to induce the host response to infection in vitro [ , , , ] (fig. ) and on our prior reports of strong host responses to dvgs regardless of the presence of functional virus-encoded antagonists [ , ] , we hypothesized that dvgs that arise in situ during viral infections provide essential stimuli to initiate an antiviral immune response. to test this hypothesis, we first determined if sev strains that accumulate copy-back dvgs early in infection induced faster ifnb mrna expression in vitro than viruses with delayed dvg accumulation. for these experiments we used sev preparations that did not show immunostimulatory activity or evidence of copy-back dvg accumulation by h post-infection and all the viruses were used at a multiplicity of infection of . tcid /cell. while standard viral genomes of all the different sev strains used were detected at all tested time points, copy-back dvgs of different sizes were detected starting at h post-infection in cells infected with sev z and at later time points in cells infected with sev , enders, or cantell ld in both murine lung epithelial cells (tc- ) and bone marrow-derived dendritic cells (bmdcs) (fig. a and b and data not shown). sequences of the starred pcr products confirming the amplification of copy-back dvgs are shown in fig. s . remarkably, accumulation of dvgs was directly associated with phosphorylation of irf (fig. c ) and with the expression of ifnb mrna (fig. d ), demonstrating that standard viral genomes alone are not sufficient to initiate this response during infection in vitro and strongly supporting a unique ability of naturally arising dvgs to initiate the cellular antiviral response. to evaluate the impact of dvgs during sev infection in vivo, we infected mice with sev cantell hd or ld. mice infected with sev cantell hd showed diminished morbidity than mice infected [ , , , , , ] . reduced virulence of sev cantell hd was associated with a stronger stimulation of the host antiviral response as shown by the expression of ifnb mrna (fig. c ). to conclusively demonstrate the role of dvgs in diminishing virulence in vivo, we co-infected mice with sev cantell ld and purified viral particles containing dvgs (defective particles; dps). confirming their critical role, dvgs reduced the pathogenicity of sev cantell ld in mice, while uv-inactivated dp particles did not provide significant protection (figs. d-f). interestingly, infection in the presence of dps resulted in reduced expression of sev np protein in the lung at day post-infection, suggesting that in this system, dps reduce virulence by interfering with virus replication. to determine whether immunostimulatory dvgs were generated in situ in the lung during infection, we infected mice with sev cantell ld, and we followed the appearance of copy-back dvgs in the lung by pcr. sev copy-back dvgs were detected in whole lung homogenates at the time of high viral replication (fig. a) . notably, upon infection with sev cantell ld, a copy-back dvg of high molecular weight was detected at day post-infection in the lung, while a dvg of low molecular weight (amplicon of bp) that predominates in the parent stock of sev cantell hd (fig. a) was only detectable at day post-infection. copy-back dvgs also appeared in the lung of mice infected with sev (fig. s ) , showing that dvgs naturally arise during infection in vivo independent on the virus strain. interestingly, accumulation of copy-back dvgs during infection with sev cantell ld was associated with the expression of ifnb and il- mrna in the lung (fig. b) . to determine whether dvgs were necessary for the expression of antiviral cytokines in vivo, we took advantage of ifnb-yfp reporter mice. to demonstrate that yfp expression serves as readout for dvg activity, we first infected bmdcs prepared from ifnb-yfp reporter mice with sev cantell ld alone, or together with increasing doses of purified dps. as shown in fig. c , at h post-infection, yfp was expressed only in the presence of dps and in a dose-dependent manner, and the yfp expression was lost when uv-treated dps were used. dps alone were also able to induce yfp in a dose-dependent manner, albeit at much lower levels than during co-infection with sev. these data agree with our previous reports that demonstrate that the immunostimulatory activity of dps is greatly amplified during dvg replication by the cognate polymerase provided by co-infecting sev [ ] and validate the ifnb-yfp reporter system as a readout for dvg activity. we then infected ifnb-yfp reporter mice with sev cantell ld and analyzed viral genomes in yfp + cells. we focused our analysis on the cd (non-hematopoietic) cellular fraction of the lung as sev replicates predominantly in the lung epithelium [ ] . although full-length viral genomes were detected in both yfp + and yfp cd populations sorted three days after infection, dvgs were only found in yfp + cells (fig. d) , suggesting that the presence of dvgs promotes ifnb production in response to virus infection in vivo. together, these findings show that dvgs are normally generated in situ in the lung during respiratory infection with sev, and that their accumulation is associated with the expression of ifnb in the lung. sev copy-back dvgs are generated in the lung independently of type i ifn signaling to determine whether type i ifns produced early upon infection promoted the generation of dvgs in the lung, we infected wild type or type i ifn receptor deficient mice (ifnar / ) with sev cantell ld and analyzed the lungs at different times post-infection. as shown in fig. e , dvgs accumulated in the lung at a higher rate in mice unable to respond to type i ifns compared with wild type mice, corresponding with the predicted enhanced rate of virus replication in the lack of type i ifn signaling and demonstrating that type i ifns are not required for the generation of sev copy-back dvgs in vivo. to investigate whether the content of dvgs in iav stocks affects virulence similar to sev, we obtained iav strain pr stocks with a high content of dvgs (hd) or lacking dvgs (ld). the stock of iav pr hd produced two predominant dvgs derived from the pa and pb genomic segments in infected cells, while no dvgs were detected in cells infected with iav pr ld (fig. a ) (strategy for iav detection and sequences for the iav dvgs present in infected cells can be found in fig. s ). mice infected with iav pr hd showed reduced morbidity compared to mice infected with iav pr ld (fig. b ) despite similar levels of virus replication (fig. c ). similar to sev cantell hd, reduced morbidity was associated with enhanced ifnb mrna expression in the lung (fig. d) . to determine whether iav dvgs were generated in situ in the infected lung, we tracked their appearance in mice infected with iav pr ld. accumulation of dvgs was clearly observed at day post-infection (fig. e) . representative sequences of starred iav dvgs products are shown in fig. s . similar to sev infection, accumulation of dvgs corresponded with enhanced expression of mrna for ifnb and il- ( fig. f ) despite evidence of reduced genomic viruses at that time point (figs. e and f) . these data demonstrate that dvgs are generated de novo in the lung during infections with iav, and suggest an important role of these types of genomes in promoting the host response to iav in vivo. we have shown that dvgs are naturally generated in the lung during infection with sev and iav and provide primary danger signals for the triggering of the host response to infection. the generation of dvgs during virus growth in tissue culture is a highly conserved phenomenon among viruses of different species and is tempting to speculate that dvgs provide an evolutionary advantage to the virus by contributing to the preservation of both the virus and the host. interestingly, immunostimulatory dvgs result from drastic truncations in the genome of the virus that render it a dead end product unable to persist in the absence of helper virus. it will be relevant to determine how dvgs relate to viral ''quasispecies'' that result from mutations as a consequence of having a viral polymerase with a lower fidelity and processivity [ ] . viral quasispecies have been shown to be essential for viral fitness and virulence [ ] . whether dvgs a tradeoff of this viral polymerase characteristic that enables more rapid virus evolution but makes the virus more vulnerable to innate immune detection remains to be established. our data demonstrate that dvg recognition is not necessary for the response to ndv, while is required for the response to sev. we speculate that the differential dvg requirement may be explained by the poor adaptation of the avian ndv to grow in mice while the murine sev is fully adapted to grow in this species. a critical factor of this adaptation is the activity of the virally encoded v and c proteins that effectively block the induction of type i ifns, as well as the type i ifn-mediated amplification of the type i ifn pathway [ , , , ] . as ndv is adapted to grow in birds, its antagonistic proteins are not fully functional in mammalian cells [ ] allowing unrestricted production and amplification of type i ifns. in contrast, the sev c and v proteins very effectively block the cellular response to sev [ , , , ] and no cellular response is observed unless dvgs are present. interestingly, in the absence of type i ifn feedback (or of the ifn-inducible transcription factor irf ) sev dvgs induce a more potent cellular response compared to ndv, suggesting that dvgs have a unique ability to bypass both virus antagonism and the requirement for irf for strong type i ifn production. we have reported that sev cantell hd, but not ndv, has the ability to induce the expression of the viral sensor mda independently of type i ifn feedback [ ] , and that mda is involved in the recognition of sev dvgs ( [ ] and data in fig. s ). although it is unclear why this newly synthetized mda is less susceptible to inhibition by the sev v protein, preferential binding of dvgs to both rig-i and mda compared to standard sev genomes, together with the availability of high levels of mda , may explain the strong activation of transcription factors and type i ifn expression in response to dvgs, regardless of type i ifn feedback. remarkably, dvgs arise in vivo independently of type i ifn feedback, demonstrating that dvgs do not appear in response to host pressure via type i ifns. notably, viruses containing dvgs have significantly reduced virulence. in a previous study, we reported that a sev strain with a lower propensity to produce dvgs (sev ) persisted longer in the lung than a sev strain able to produce high levels of dvgs (cantell) [ ] . consistently, we observed higher levels of viral np protein at day post-infection in the lung of mice infected with sev cantell ld, compared to mice infected with sev cantell ld plus dps (fig. g) . in additional studies, we have not observed significant differences in the rate of sev-specific t cells in the lung of mice infected with sev ld alone or in the presence of dps (data not shown and [ ] ). although interpretation of this observation is complicated by the reduced amount of sev antigen (np protein) present in infection with sev ld plus dps, we favor the hypothesis that dps diminish virulence by competing for the viral polymerase, thus interfering with the replication of the standard virus, a well-defined characteristic of dvgs. additionally, enhanced production of type i ifns in response to dvgs likely contributes to dampened viral replication. highly immunostimulatory sev dvgs are of the copy-back type. intriguingly, during sev infections in vivo, accumulation of dvgs of high molecular weight preceded the appearance of low molecular weight dvgs, suggesting that the smaller ones may be secondary to longer defective genomic products. copy-back dvgs are not transcribed due to their promoter properties [ ] , thus their stimulatory activity likely derives solely from their genomic composition. iav dvgs are truncated versions of one of the genomic segments that have natural complementarity among their and ends providing the theoretical capacity to form structures similar to copy-back dvgs. notably, it is apparent that both sev genomic and dvg rnas have the potential to induce a host response when delivered naked into the cells. based on our data, we predict that in the context of infection, dvg rna is more available for detection due to their enhanced rate of replication compared to standard viral genomes (fig. ) . interestingly, we have shown that dvgs have the ability to bypass viral-encoded antagonists of the immune response even upon overexpression of viral antagonistic proteins [ ] . it remains to be investigated what is the molecular mechanism behind this dvg property. notably, dvgs of different forms and compositions have been described in the sera of patients chronically infected with a number of different viruses [ , , , , ] and dvgs of various viruses have been shown to promote persistent infections in tissue cultures [ , , , , , , , , ] supporting a role for dvgs in the maintenance of chronic viruses. the role of naturally arising dvgs in promoting virus persistence in vivo remains to be investigated. in summary, we have demonstrated that dvgs arise naturally during an acute respiratory virus infection and that they play a critical role in regulating the virus-host cycle in vivo. importantly, the recognition of dvgs as stimuli for the onset of immunity has multiple practical implications, most directly: (i) dvgs represent novel determinants of virus pathogenesis that could be targeted for therapy, and (ii) dvgs are novel candidate biomarkers to predict the outcome of infections and the rate of virus spread in the population. this study was carried out in strict accordance with the recommendations in the guide for the care and use of laboratory animals of the national institutes of health. the protocol ( ) was approved by the institutional animal care and use committee, university of pennsylvania animal welfare assurance number a - . sev strains cantell, , enders, and z, and influenza a/pr / virus were grown in days hen embryonated eggs (spafas; charles river laboratories). sev cantell was passaged to retain its original high di particle content (hd) or to deplete it of di particles (ld) as we previously described [ ] . in brief, sev , enders, z, and cantell hd were grown in embryonated hen eggs inoculated with , medium tissue culture infectious dose (tcid ) for h. sev cantell hd tcid was calculated by end point dilution in llcmk cells in the presence of trypsin, as described below [ ] . sev cantell hd total particles were calculated by end point dilution of hemagglutination of chicken red blood cells. sev cantell hd stocks had consistently an infectious:total particle ratio of , - , . sev had an infectious:total particle ratio of , . sev enders had an infectious:total particle ratio of , . sev z had an infectious:total particle ratio of , . sev cantell ld was prepared by inoculating embryonated hen eggs with tcid for h. under these conditions only % of the eggs grew virus. allantoic fluid from those eggs was pooled and diluted / for subsequent inoculation into embryonated hen eggs in a total volume of ml. allantoic fluid containing virus ( % of the inoculated eggs) was pooled and tittered as described below. sev cantell ld stocks had consistently an infectious:total particle ratio of , - , . iav strain pr (ld) was grown by inoculating hen embryonated eggs with , tcid obtained directly from infected lung homogenates. allantoic fluid containing the virus was collected h later. egg's allantoic fluid was snap frozen in an ethanol/dryice bath and stored at uc. iav pr containing high dose of di particles (hd) was kindly provided by dr. laurence c. eisenlohr, v.m.d., ph.d (thomas jefferson university). iav hd was grown by inoculating hen embryonated eggs with , pfu of egg-passed virus. eggs were incubated at uc and allantoic fluid containing the virus was collected h later. iav pr hd and ld stocks were originated from the same parent stock but were extensively passaged in the different conditions described. permissive cells were infected with serial : dilutions of lung homogenates or virus stocks in the presence of mg/ml of trypsin to determine the medium tissue culture infectious dose (tcid ). llcmk cells were used for sev titration, while mdck cells were used for iav titration. after h of incubation at uc, ml of supernatant from each well was tested by hemagglutination of chicken red blood cells (rbcs) for the presence of virus particles at the end point dilution. to do this, : dilutions of the cell supernatant were incubated in . % chicken rbcs at uc for min. hemagglutination of rbcs indicated the presence of sev or influenza virus particles. bmdcs were generated as previously described [ ] . detailed procedure can be found in the supplemental information material and methods. bmdcs were infected after days in culture with viruses at an multiplicity of . as we have previously described [ ] . for sev infections, mice were anesthetized with tribromoethanol (avertinh; acros organics) and inoculated in the nostrils with ml of pbs containing or tcid of sev. for iav infections animals were infected intranasally with tcid / mouse in a ml volume. lungs were extracted at different times post-infection, homogenized in . % w/v gelatin-pbs and snap frozen in dry-ice/ethanol for preservation. total rna was extracted from cell lines or lungs with trizol (invitrogen) according to the manufacturer's specifications and total rna was reversed transcribed using the high capacity rna to cdna kit from applied biosystem. for sorted cells, ng of rna were reversed transcribed, for all other experiments - mg of rna were reversed transcribed. cdna was diluted to a concentration of mg/ml and amplified with specific primers in the presence of sybr green (applied biosystem). for the detection of dvgs, isolated total rna was reverse transcribed using superscript iii without rnase h activity, to avoid selfpriming by the dvgs complementary ends and recombinant rnase h (invitrogen) was added later to the samples. for the detection of the standard virus genome, the negative strand of the full-length genome was reverse transcribed with transcriptor first strand cdna synthesis kit (roche). pcr detection for iav was performed using as it has been previously described [ ] . primers and detailed pcr conditions can be found in the supplemental information material and methods. detailed primers and pcr conditions can be seen in the supplemental information material and methods. whole cellular extracts were prepared by lysing of cells in a np- -based lysis buffer containing phosphatase inhibitors, proteinase inhibitors (roche and thermo scientific), and . m edta. the concentration of protein was measured by bradford assay (themo scientific). samples ( mg) were boiled for min and resolved on % bis-tris pre-cast gels (bio-rad). resolved proteins were transferred to a polyvinylidene fluoride (pvdf) membrane (millipore). the membrane was blocked with % nonfat milk and immunoblotted with the indicated antibodies. antirabbit irf , anti-rabbit phospho-irf (ser ), anti-mouse ikba, anti-mouse phospho-ikba (ser / ), and anti-rabbit igg (hrp-conjugated) were purchased from cell signaling. antimouse gapdh was purchased from sigma. anti-mouse igg and anti-mouse igg (hrp-conjugated) were purchased from jackson immunologicals. lumi-light western blotting substrate was used for hrp detection (roche). dp purification was performed as previously described [ ] . in short, allantoic fluid from infected hen eggs was pooled and concentrated by high-speed centrifugation. pellets were suspended in . ml of pbs/ mm edta and incubated overnight at uc in a - % sucrose (fisher) gradient that was prepared using a gradient maker (biocomp). gradients were centrifuged at uc for . h at , rpm and fractions containing low-density viral particles were collected, pelleted, suspended and re-purified using the same procedure. collected low-density fractions were concentrated by centrifugation at uc for h at , rpm. pellets were suspended in pbs, snap frozen, and stored at uc. the content of di particles was determined by calculating the ratio of infectious over non-infectious particles as described above. a nt long product containing the sequence of the t promoter followed by the -nucleotide long copy back dvg from sev cantell, and flanked by the restriction enzymes spei and sapi at the an ends was synthetically synthetized (dna . ) and clone into the psl vector (amersham pharmacia biotech) containing the sequences for the hepatitis delta virus ribozyme and the t polymerase terminator. in order to optimize the transcription of the dvg, g residues were introduced downstream of the t promoter by site-directed mutagenesis (stratagene, ca) using the oligonucleotides ccactagttaa-tacgactcactatagggaccagacaagagtttaagag- and ctcttaaactcttgtctggtccctatagtgag tcgtattaactagtgg- . bsr-t cells were infected with a moi of approximately of partially inactivated sev strain . virus inactivation was performed by exposing diluted virus to uv light ( nm model mrl- , uvp upland, ca) for sec at a distance of inches from the light source. virus inactivation diminished the virus replication rate, while allowing the expression of viral proteins necessary for the replication of dvgs. cells were incubated at uc for h before transfection of mg of vector encoding dvg. transfection was performed with xtremegene transfection reagent (roche) according to manufacturer instructions. cells were cultured in dulbecco's modified eagle medium (dmem) supplemented with % bovine serum albumin, % naco , . mg/ml trypsin (worthington) and . % penicillinstreptomycin (invitrogen) and incubated in % co at uc. cells and supernatant containing sev and rdps were harvested after h and ml of the suspension were inoculated in the allantoic cavity of -day embryonated hen eggs (b & e eggs, silver springs, pa). after h allantoic fluid was harvest and ml of undiluted fluid were inoculate in -day embryonated eggs for virus growth and egg inoculation was repeated for three consecutive passages. allantoic fluid from the third passage was quick-frozen in dried ice/ethanol and used for infections. presence of recombinant dvg was confirmed by pcr. no other dvgs were detected. dvg rna was in vitro transcribed (ambion) from the t -dvg plasmid. standard genomic rna was extracted from sev cantell ld stocks. to remove -triphosphates, mg of rna was incubated with u of calf intestinal phosphatase (new england biolabs) for min at uc. to cleave single stranded rna, mg of rna was incubated with ng of rnase a (ambion) for min at room temperature. to cleave double stranded rna, rna was incubated with . u of rnase v (ambion) for min at room temperature. after treatments, rna was purified using trizol or precipitation/inactivation buffer according to the manufacturer's specifications. llc-mk cells were transfected with ng or indicated doses of dvg and genomic rna using lipofectamin (invitrogen). at hours post transfection, the cells were harvested and total rna was isolated using trizol according to the manufacturer's specifications. infected ifnb-yfp cells were collected at h post-infection. reporter mice were sacrificed days post-infection. lungs were collected and dissociated with collagenase (roche), followed by suspension on . m edta and rbc lysis buffer. single cell suspensions were then incubated with cd /cd fcblock for min at c, followed by incubation with biotinylated mouse anti-cd . . washed cells were incubated with anti-biotin microbeads (miltenyi) for min and passed through a magnetic column for negative selection. cd cells were sorted based on yfp expression using a facs vantage se sorter. statistical analyses were performed as indicated in each figure. graphpad prism version . for windows, graphpad software, san diego california usa, www.graphpad.com, was used for analysis. genes ncbi id numbers. tuba b: ; rps : ; ifnb: ; ifnl : ; illb: ; il b: ; tnf: ; il- , . text s supporting materials and methods. the text includes in detail descriptions of the procedures, as well as specific material and methods and references for figures included as supporting information. (docx) preference of rig-i for short viral rna molecules in infected cells revealed by next-generation sequencing -triphosphate rna is the ligand for rig-i lengthdependent recognition of double-stranded ribonucleic acids by retinoic acidinducible gene-i and melanoma differentiation-associated gene rig-imediated antiviral responses to single-stranded rna bearing -phosphates rig-i detects viral genomic rna during negative-strand rna virus infection recognition of triphosphate by rig-i helicase requires short blunt doublestranded rna as contained in panhandle of negative-strand virus cutting edge: stealth influenza virus replication precedes the initiation of adaptive immunity antagonism of the interferon-induced oas-rnase l pathway by murine coronavirus ns protein is required for virus replication and liver pathology defective viral particles and viral disease processes the origins of defective interfering particles of the negative-strand rna viruses incomplete forms of influenza virus defective interfering rnas: foes of viruses and friends of virologists detection of defective genomes in hepatitis a virus particles present in clinical specimens hepatitis b defective virus with rearrangements in the pres gene during chronic hbv infection in vivo and in vitro expression of defective hepatitis b virus particles generated by spliced hepatitis b virus rna characterization of hepatitis c virus deletion mutants circulating in chronically infected patients mechanisms associated with the generation of biologically active human immunodeficiency virus type particles from defective proviruses defective interfering viral particles in acute dengue infections sequence analysis of in vivo defective interfering-like rna of influenza a h n pandemic virus the characteristics required for a sendai virus preparation to induce high levels of interferon in human lymphoblastoid cells interferon induction by viruses. xix. vesicular stomatitis virus-new jersey: high multiplicity passages generate interferoninducing, defective-interfering particles activation of the beta interferon promoter by unnatural sendai virus infection requires rig-i and is inhibited by viral c proteins mda participates in the detection of paramyxovirus infection and is essential for the early activation of dendritic cells in response to sendai virus defective interfering particles a novel role for viral-defective interfering particles in enhancing dendritic cell maturation differential type i ifn-inducing abilities of wild-type versus vaccine strains of measles virus sendai virus infection induces efficient adaptive immunity independently of type i interferons cytokine-independent upregulation of mda in viral infection sendai virus defective-interfering genomes and the activation of interferon-beta nucleotide sequences that affect replicative and transcriptional efficiencies of sendai virus deletion mutants prevention of death in semliki forest virusinfected mice by administration of defective-interfering semliki forest virus defective interfering viruses: modulators of infection protection of mice from lethal influenza: evidence that defective interfering virus modulates the immune response and not virus multiplication protection of three strains of mice against lethal influenza in vivo by defective interfering virus defective interfering influenza a virus protects in vivo against disease caused by a heterologous influenza b virus defective interfering virus protects elderly mice from influenza melanoma differentiation-associated gene (mda ) is involved in the innate immune response to paramyxoviridae infection in vivo quasispecies diversity determines pathogenesis through cooperative interactions in a viral population the v proteins of paramyxoviruses bind the ifn-inducible rna helicase, mda- , and inhibit its activation of the ifn-beta promoter ) mda- , but not rig-i, is a common target for paramyxovirus v proteins the paramyxovirus, sendai virus, v protein encodes a luxury function required for viral pathogenesis c and v proteins of sendai virus target signaling pathways leading to irf- activation for the negative regulation of interferon-beta production newcastle disease virus v protein is a determinant of host range restriction the sendai paramyxovirus accessory c proteins inhibit viral genome amplification in a promoter-specific fashion longer and shorter forms of sendai virus c proteins play different roles in modulating the cellular antiviral response persistent infection. i interferon-inducing defective-interfering particles as mediators of cell sparing: possible role in persistent infection by vesicular stomatitis virus role of defective interfering particles of sendai virus in persistent infections comparative study of rabies virus persistence in human and hamster cell lines characterization of west nile virus persistent infections in genetically resistant and susceptible mouse cells. i. generation of defective nonplaquing virus particles characterization of a cell culture persistently infected with the da strain of theiler's murine encephalomyelitis virus persistent infection of some standard cell lines by lymphocytic choriomeningitis virus: transmission of infection by an intracellular agent human cytomegalovirus persistent infection in a human central nervous system cell line: production of a variant virus with different growth characteristics defective interfering particles of human parainfluenza virus type are associated with persistent infection in cell culture establishment of respiratory syncytial virus persistence in cell lines: association with defective interfering particles singlereaction genomic amplification accelerates sequencing and vaccine production for classical and swine origin human influenza a viruses the authors wish to thank dr. laurence eisenlohr for hd influenza virus, luis muñ oz for technical support, and the flow cytometry and cell sorting resource laboratory and the dna sequencing facility at the university of pennsylvania. we also thank drs. thomas moran, christopher basler, and benjamin tenoever (icahn school of medicine at mount sinai), and marco colonna (washington university) for providing invaluable reagents. conceived and designed the experiments: kt wk cbl. performed the experiments: kt wk xml ys ed ma mw. analyzed the data: kt wk cbl. wrote the paper: kt wk cbl. key: cord- -ob igsv authors: kizziah, james l.; manning, keith a.; dearborn, altaira d.; dokland, terje title: structure of the host cell recognition and penetration machinery of a staphylococcus aureus bacteriophage date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: ob igsv staphylococcus aureus is a common cause of infections in humans. the emergence of virulent, antibiotic-resistant strains of s. aureus is a significant public health concern. most virulence and resistance factors in s. aureus are encoded by mobile genetic elements, and transduction by bacteriophages represents the main mechanism for horizontal gene transfer. the baseplate is a specialized structure at the tip of bacteriophage tails that plays key roles in host recognition, cell wall penetration, and dna ejection. we have used high-resolution cryo-electron microscopy to determine the structure of the s. aureus bacteriophage α baseplate at . Å resolution, allowing atomic models to be built for most of the major tail and baseplate proteins, including two tail fibers, the receptor binding protein, and part of the tape measure protein. our structure provides a structural basis for understanding host recognition, cell wall penetration and dna ejection in viruses infecting gram-positive bacteria. comparison to other phages demonstrates the modular design of baseplate proteins, and the adaptations to the host that take place during the evolution of staphylococci and other pathogens. introduction staphylococcus aureus is a gram-positive bacterium and opportunistic pathogen that colonizes the nasal cavities of � % of the population, increasing the risk of pathogenic infection, especially in the clinical setting [ ] . almost , cases of s. aureus blood stream infections occurred in the united states in , with , associated deaths [ ] . s. aureus resistant to methicillin (mrsa) and other antibiotics have become a major public health concern. most resistance and virulence factors in s. aureus are encoded on mobile genetic elements (mges), including bacteriophages (phages) and genomic islands [ , ] . given the rarity of genetic conjugation loci and restriction against transformation, transduction via bacteriophages is considered the main mode of horizontal gene transfer of mges in s. aureus [ ] . some staphylococcal bacteriophages carry virulence factor genes in their genomes. many phages are also capable of transferring unrelated genetic material through generalized transduction. furthermore, certain "helper" phages are involved in highly specific, high frequency mobilization of s. aureus pathogenicity islands (sapis) and similar genetic elements [ , ] . the underlying mechanisms of horizontal gene transfer and the ability of a particular phage to transfer genetic material to a new host are therefore central to genetic diversification and the emergence of novel pathogenic strains in s. aureus. bacteriophages of many firmicutes, including s. aureus, interact with wall teichoic acid (wta), a variable carbohydrate polymer present on the surface of most gram-positive cells [ ] . most strains of s. aureus have a unique type of wta that is distinct from that of other (coagulase-negative) staphylococci (cons) and generally blocks infection by cons-specific phages [ ] . a key determinant of host specificity is provided by the phage tail tip complex (ttc) or more elaborate baseplate, specialized structures at the tips of phage tails that are usually the first point of contact between the phage and its host [ , ] . enzymatic activities associated with ttcs and baseplates degrade the cell wall, ultimately leading to penetration of the plasma membrane and ejection of the encapsidated dna into the cell. tailed, double-stranded dna phages (order caudovirales) are divided into three families based on the structures of their tails: long and flexuous (siphoviridae); long and contractile (myoviridae); or short (podoviridae) [ ] . ttcs and baseplates from these diverse groups vary greatly in complexity, but tend to share a common set of structural modules, combined with a variable set of host interaction proteins, including tail fibers and receptor binding proteins. the best-described bacteriophage baseplate structures are those of the lactococcal siphoviruses tp - , tuc , and p , and the escherichia coli myovirus t , for which various structures have been produced [ , ] . in contrast, little structural information is available for baseplates from staphylococcal bacteriophages. phage α is a typical staphylococcal siphovirus with a . kbp genome and a nm icosahedral capsid, attached to a nm long flexuous tail that is capped by an ornate baseplate [ ] [ ] [ ] . α is one of the best-described s. aureus phages, and is closely related to ϕeta and other phages involved in specifying bacterial pathogenicity [ ] . α serves as the mobilizing phage for several sapis [ ] . the tail and baseplate gene cluster in α includes open reading frames (orfs) - , which are part of the large late operon (orfs - ) that encodes all structural proteins, the terminase proteins involved in dna packaging, and the lysis proteins ( fig a and b) . here, we have determined the cryo-em structure of the α baseplate at . Å resolution, allowing most of the baseplate proteins and part of the tail to be modeled in atomic detail. this is the first high-resolution structure of a baseplate from a staphylococcal siphovirus and of a siphovirus tail. the α baseplate structure has several unique features, including the presence of three tail fiber/receptor binding proteins, but also displays striking similarities to other phage baseplates, indicating a mix-and-match strategy for baseplate evolution across the phages of the firmicutes. our structure contributes to a broader understanding of the diversity of the cell recognition and penetration machinery of siphoviruses infecting gram-positive bacteria and how these structures access the cell interior and trigger dna ejection during infection. α tails were produced using an α lysogen (strain st ) with a deletion of the first amino acids of the scaffolding protein (gp ), which results in failure to form capsids and accumulation of tails in the lysate (fig c) [ ] . initial cryo-em datasets of up to � , baseplate particle images were collected with a philips cm microscope on so- film or using an fei titan krios microscope equipped with a direct electron de- detector, and reconstructed with sixfold (c ) symmetry to . Å resolution (fsc . ) using eman and eman [ ] . a larger dataset was subsequently collected with the titan krios and de- detector, and processed with motioncor , gctf and relion- [ ] , yielding a final c reconstruction at . Å resolution (fsc . ) from , particle images (fig d- f , s and s figs). a reconstruction of the same data with application of only threefold (c ) symmetry was generated with relion- [ ] and reached . Å resolution (fig g, s and s figs) . the baseplate is Å wide and Å tall and consists of six peripheral structures surrounding a core that represents a continuation of the tail (fig a and b ). the tail itself is Å wide and consists of � hexameric rings with an inter-ring spacing of Å, enclosing a Å wide lumen (fig b) . three of the tail rings are embedded within the baseplate itself. the peripheral structures consist of two large rings surrounding the embedded part of the tail, and six trefoiled, club-shaped features, about Å wide and Å long, connected to the baseplate core via six kinked stems. at lower cutoff level, six fibers can be seen to extend from the lower ring and wrap around the outside of the baseplate, terminating in globular densities near the baseplate tip (fig c) . at least seven tail rings can be discerned, and a rod-like protrusion extends from the tip of the baseplate. additional, noisy densities, disconnected from the rest of the baseplate, appear below this protruding rod. d class averages that represent end-on views of the baseplate (fig e) suggested the presence of six thin fibers spread out radially in the plane of the ice, but these features were not observed in the d reconstruction. orf , encoding gene product (gp) , was previously identified as the major tail protein (mtp) of α. likewise, gp could be identified as the tape measure protein (tmp), typically table ) [ , ] . other orfs could be assigned by comparison with the closely related phage ϕ [ ] , for which hhpred analysis [ ] had identified homologs in the pdb structure database. these orfs include the distal tail protein (dit, gp ), which comprises the baseplate "hub", the tail-associated lysin (tal, gp ), comprising the tip of the tail, and the receptor binding protein (rbp, gp ) (fig a and b ; table ). the n-terminal part of ϕ gp , corresponding to α gp , was shown to have some similarity with upper baseplate protein (bppu) of lactococcal phage tp - [ , ] . the crystal structure of ϕ rbp (gp ), which is % identical to the α rbp, was recently determined [ ] . crystal structures have also been determined for bacillus phage spp dit [ ] and the baseplates of lactococcal phages p and tp - , which include dit, tal, bppu, and receptor binding proteins that are not similar to the ϕ rbp [ , ] . we carried out hhpred analysis on α proteins gp , gp , gp , gp , gp , gp , gp and gp , which identified additional homologies compared to ϕ , due to server improvements [ ] and the presence of more structures in the database ( table ). most importantly, gp was identified as a tail fiber (fibl) with coiled-coil structure, while gp was predicted as a collagen-like fiber protein (fibu). gp encodes a predicted cell wall hydrolase (hyd) that is % identical to ϕ gp , which was previously shown to exhibit muralytic activity [ ] . gp was the only putative baseplate protein that could not be identified in the baseplate reconstruction, presumably because it is disordered and/or present at low occupancy, consistent with previous ms analysis [ ] . atomic models for mtp (gp ), dit (gp ), rbp (gp ), and parts of tal (gp ), fibl (gp ), fibu (gp ) and tmp (gp ) were built into either the c or c baseplate reconstructions, using i-tasser models as starting points [ ] , complemented with real-space refinement in phenix [ ] . the rbp, fibl and fibu models were further improved by refinement into focused reconstructions (see methods). in total, polypeptides comprising different proteins ( table ) were included in the final model ( fig d- f, s fig) . the fsc curves between the final models and the corresponding reconstructions are shown in s fig. the major tail proteins of siphoviruses, tail tube proteins of myoviruses, and the hcp tubeforming proteins of type secretion systems (t ss) share a common fold, known as the tail tube fold [ ] . in addition, the tail tube fold has been identified in various baseplate proteins, tail terminator proteins, and the t capsid assembly protease, reflecting their evolutionary relationship [ ] . hhpred analysis of α gp yielded > % probability matches to major tail proteins of several siphoviruses, including spp gp . and λ gpv [ , ] , and lower probability matches to two t ss tube-forming proteins ( table ). six copies of mtp were built into each of the two most well defined tail rings closest to the baseplate. of the residues of mtp, residues - were modeled in the lower ring (denoted mtp l ) and - into the upper ring (mtp u ) (figs e and a). each mtp subunit includes two four-stranded β-sheets folded into a β-sandwich and flanked by an α-helix ( fig a) . in a complete tail ring, one of these β-sheets forms a -stranded, highly negatively charged β-barrel- Å in diameter-lining the tail lumen, presumably providing a slippery tube for the dna during infection (figs b and b ). this luminal β-sheet contains an extended insertion loop between β and β (the "stacking loop") that reaches across the � Å space that separates tail rings and inserts into a pocket between two subunits in the adjacent ring ( fig a) . the equivalent loop in the major tail proteins of phages λ, t and spp was shown to play an essential role in tail polymerization [ ] [ ] [ ] . an additional inter-ring contact is mediated by the c-terminus of mtp (the "c-arm"), which extends in the opposite direction to the stacking loop and fills a crevice on the exterior surface of the adjacent subunit ( fig a) . no equivalent contact has been seen in other systems. the major tail proteins of phages λ, spp and t instead have an additional c-terminal immunoglobulin (ig)-like domain [ ] [ ] [ ] . overall, the tail tube domain of α mtp differs from table . hhpred analysis of α baseplate protein sequences. the most relevant hits are shown for each protein, with matched residues, pdb id and chain identifier, hhpred probability (%), e-value and percent sequence identity in matched region. those of λ gpv and spp gp . with cα rmsd values of . Å and . Å, respectively, between the most well-matched residues (s table, the "distal tail protein" (dit) constitutes the hub of the baseplate, and is highly conserved between ttcs and baseplates of siphoviruses [ ] . hhpred matched α gp to the dit proteins of several phages with � . % probability, including spp gp . , tp - orf and p orf (table ) [ , , ] . dit forms a connection between the sixfold symmetric tail and the threefold symmetric tail tip (tal, see below), and provides an attachment site for the six rbp trimers (fig d- f ). six copies of dit were built into the hexameric ring of density in the baseplate core just below mtp l (figs e and a) . dit consists of two domains: an n-terminal domain (ntd, residues - ) with a tail tube fold, and a c-terminal domain (ctd, residues - ) with a galectin-like fold (fig a) . both dit domains are conserved between α and spp , tp - and p [ , , ] (s d- s f fig and s table) . the interior surface of the dit ntd forms a β-barrel that constitutes a continuation of the tail lumen (figs b and b ). the dit ntd lacks a c-arm equivalent to that of mtp; instead, an extended insertion loop (the "tail binding loop") between β and β of dit fits into the same crevice on mtp l that the c-arm of mtp l occupies on mtp u (fig a and c ). this topologically distinct dit loop contains a triplet of residues (y , r , f ) that matches a corresponding triplet in the mtp c-arm (y , r , f ), a striking case of convergent evolution that has not been observed in other systems ( fig c) . the dit ctd has an insertion (the "rbp binding loop", residues - ) that serves as the attachment site for the rbp trimer (fig d and e ) that is not present in spp and tp - . several aromatic residues in this loop insert into hydrophobic invaginations in the three-helix bundle that makes up the n-terminal end of rbp ( fig e, the dit ntd includes a stacking loop equivalent to that of mtp-previously described as the "belt extension" in spp [ ] -that interacts with tal ( fig a, f and g ). the ctds from all six dit subunits form a "crown" that grips the outside of the tal trimer ( fig f, see below) . the interaction between dit and tal is primarily hydrophobic in nature (fig g) . hhpred analysis matched the ntd of α gp , consisting approximately of residues - , to several proteins related to the gp baseplate hub protein from e. coli phage t (table ) [ ] . these proteins are trimers that contain two tail tube folds, resulting in a quasi-c structure. the ctd of gp , comprising residues - , matched various esterases and lipases of the gdsl-hydrolase family and the sgnh-hydrolase subfamily [ ] (table ) , suggesting a the surface is colored from - (red) to + (blue) kcal/(mol � e) according to the color bar. (c) superposition of mtp u onto mtp l (gray). mtp l (tan) shifted by the same amount then superimposes on dit (red). the expanded view shows the superposition of the dit tail binding loop (red) with the mtp c-arm (tan). the side chains of the triplet of residues conserved between the c-arm of mtp (y ,r ,f ) and the tail binding loop in dit (y ,r , f ) are shown in stick representation and labeled. (d) detail of the interaction between dit (red) and the rbp coiled-coil stem region (purple), showing the rbp binding loop in the dit ctd. (e) same view as d with rbp shown as a van der waals surface colored from most hydrophilic (tan) to most hydrophobic (purple), according to the kyte-doolittle scale. the rotated view shows the n-terminal end of the rbp trimer and its interaction with the rbp binding loop, with side chains shown in stick representation. (f) surface representation of dit (red) and tal (pink), viewed from the side. (g) surface representations of dit (top) and tal (bottom) rotated ˚in opposite directions to show the interacting surfaces. one subunit each of dit (red), tal (pink) and tmp (green) is shown in solid color, the rest are colored by hydrophobicity as in e. https://doi.org/ . /journal.ppat. .g role in cell wall degradation. for consistency with what was previously proposed for ϕ [ ] , we will refer to this protein as tal, for "tail-associated lysin," although its enzymatic activity has not yet been established. the tal ntd was initially fitted as a c trimer into the cap-like structure at the bottom of the baseplate, directly below the dit hexamer in the c symmetric reconstruction (fig a) . due to the quasi-sixfold nature of the tal trimer, it matched quite well overall, but much of the density was blurred due to the superposition of non-identical tal orientations. to obtain a reconstruction with only threefold symmetry imposed, we first subtracted all density not associated with tal from the baseplate images. these signal-subtracted images were then subjected to symmetry expansion and d classification to group together consistently oriented tal trimers within the larger c asymmetric unit. these orientations were used to generate a reconstruction of the whole baseplate with c symmetry, which reached a resolution of . Å ( fig g, s and s figs) . in this reconstruction, the density corresponding to the cap and the rod was well resolved (fig a- c ) and allowed modeling of residues - of tal (fig d and e) . the disorganized density beyond the rod was presumed to correspond to the tal ctd (residues to ), but was not interpretable even in the c reconstruction ( fig c) . the tal ntd (residues - ) consists of two tail tube domains (tt and tt ) that form a quasi-sixfold adapter to the dit hexamer (figs f, g, d and e). the two domains merge into a continuous -stranded β sheet on one side (fig e) . the two domains are separated by an insertion (ins ) with a fold related to the type secretion system protein escc (pdb id: gr ). the second tail tube domain has an additional domain (ins ) inserted in a position that is topologically equivalent to the β -β stacking loop in mtp (fig e) . residues - form an α-helix that extends into the tail lumen. in the tal trimer, these helices form a twisted tripod that occludes the opening of the lumen (fig d and f) . residues - from the three subunits make up an α-helical coiled coil that forms the rod and extends to the unresolved ctd (fig d and f ). the tal fold is similar to proteins from systems as distant as e. coli phage mu, listeria monocytogenes egd-e prophage a and an e. coli t ss, with rmsds ranging from . Å to . Å between equivalent cα atoms (s g-s i fig and s table) . after accounting for the plug and rod helices, there were three additional, well-ordered αhelical densities forming a second tripod interlaced with that of the tal plug ( fig f) . this density matched the c-terminal residues - of tmp. the tmp triplet fit into a hydrophobic patch on tal (fig g and h ). the localization of the tmp c-terminus at the baseplate agrees with previous research on other phages [ ] . additional density attributable to tmp could be seen inside the tail lumen, but was not interpretable in terms of the atomic structure ( fig b) . α gp is % identical in amino acid sequence to gp of the closely related phage ϕ , for which the crystal structure was previously determined [ ] . gp was previously identified as the primary receptor binding protein for ϕ , with binding affinity for wta, and was thus denoted rbp [ ] . rbp is a trimeric protein consisting of four domains: an n-terminal α-helical coiled-coil "stem" domain (residues - ) that is folded by ˚at a central hinge; a fivebladed β-propeller "platform" domain (residues - ) that was previously suggested to bind to the glcnac residues of wta on the host cell surface [ , ] ; and two highly similar c-terminal "tower" domains (residues - ; s table) , each consisting of a bent β-sheet superposed by an α-helix (fig a) . six such trimers surround the baseplate core, making up the bulk of the observed peripheral structures (fig d) . as expected, the α rbp is very similar to ϕ gp [ ] (fig b, s j fig; s table) . the greatest difference between the α and ϕ rbps is in the orientation of the stem domains relative to the platform and tower domains ( fig b) . as noted above, the n-termini of the coiled-coil domains from each rbp trimer connect to the rbp binding loop of dit (fig d and e) . the rbp hinge interacts with a ring formed by the fibl trimers (figs d and d, see below) . deletion of orf led to a complete loss of peripheral structures and of infectivity, although tails still assembled normally (s fig). the structure of rbp is remarkably similar to the "tail fiber" (gp ) of the distantly related staphylococcal podovirus p [ ] (fig c, s table) , presumably reflecting specificity for s. aureus surface structures. however, in p gp , the stem domain is less bent at the hinge compared to α ( ˚vs. ˚) (fig c) . in addition, since p lacks a dit equivalent, the gp stem domain is instead attached to the dodecameric portal protein. consequently, p has twelve gp trimers, compared to the six rbp trimers in α. gp is highly modular, with hhpred matches to several distinct phage protein structures ( table ). the first residues matched the n-terminal ig-like domain and α-helical coiledcoil domain of tp - bppu [ ] . the central part of gp (residues - ) matched several α-helical coiled-coil structures at lower probability (� . %; table ). residues - were matched to the tower domains from ϕ rbp, with lower probability matches of residues - and - to a similar domain from a pseudomonas aeruginosa r pyocin fiber protein ( table ). the c-terminal residues of fibl ( - ) matched the two c-terminal domains of the gp receptor binding protein from s. aureus phage k (pdb id: m f), which comprise an rbp-like tower domain and a distinct β-sandwich domain [ ] . because of the fibrous coiled-coil domain and its formation of the lower of the two exterior baseplate rings, the protein was designated fibl, for lower tail fiber. six trimers of the n-terminal ig-like domain of fibl were built into the lower of the external rings surrounding the baseplate (figs d- f and d- f). the two proximal subunits (p and p ) from each trimer contribute to the ring itself, while the third, distal subunit (d) is attached on the outside of the ring. each subunit in the trimer contributes an α-helix to the subsequent coiled-coil domain that extends to residue for subunit p , which contributes the top α-helix, and to and for the p and d subunits, respectively (fig e) . this organization is similar to that of the bppu protein of tp - , except that in this phage, one of the α-helices in the coiled coil originates from a subunit in the adjacent trimer [ ] (s l fig). due to the differing disposition of the n-terminal domains, the residues of the α-helical domains are shifted relative to one another, inducing asymmetry within the coiled coil (fig d) . the fibl ring is not closely associated with either the dit or mtp hexamers. although residues and of one of the fibl subunits approach the β -β loop of mtp u by � Å, most of mtp u is separated from the fibl ring by � - Å (fig f) . however, the bottom of the iglike fold of fibl associates closely with the hinge domain of the rbp stem region (fig d and e ). this is consistent with the observation that deletion of the gene encoding fibl (orf ) led to a complete loss of peripheral structures, including rbp (s fig), suggesting that the interaction between fibl and rbp is co-stabilizing. a similar phenotype was observed when the corresponding gene (orf ) was deleted in ϕ [ ] . beyond residue � , the density for the fibl coiled-coil and c-terminal domains was attenuated, likely due to flexibility in the coiled-coil domain (fig a) . at lower cutoff level, however, fibl density could be seen to wrap around the peripheral structures (fig c) . a new reconstruction was made from modified images created by subtracting the density (fig g) . this reconstruction reached . Å resolution (s and s figs). in this map, fibl could be seen as an elongated structure with a globular "knee", extending beyond the tips of the rbp trimers, terminating in two additional globular densities. while this map was not fully interpretable in terms of the atomic structure of the proteins, models for the remaining domains of fibl based on the hhpred predictions were rigid body fitted into the density and connected to provide a complete pseudo-atomic model for fibl (figs f and h ). in this model, the three coiled-coil α-helices continue with no twist between residues - . this is followed by an rbp-like tower domain that was fitted into the knee in the middle of the fiber, followed by another, shorter coiled-coil region. at the c-terminus of fibl, the predicted phage k-like tower and β-sandwich domains matched the disordered densities at the bottom of the baseplate (fig g and h ). like fibl, gp was predicted to include a bppu-like n-terminal ig-like domain ( . % probability; table ). however, gp lacks the α-helical coiled-coil portion of fibl. instead, residues - were predicted to match various collagen-like triple helix structures (� . % probability), suggesting that the protein forms a collagen-like fiber. the remaining residues did not match any known structure. an i-tasser model for the gp ntd matched the density corresponding to the upper external ring that makes up the peripheral structures of the baseplate. we designated this protein fibu, for upper fiber protein. despite the similarity between the ntds of fibu and fibl ( % sequence identity and rmsd = . Å; s k fig and s table) , the two proteins could be unambiguously distinguished based on bulky side chains (s f and s g fig) . based on its predicted collagen-like domain and similarity to fibl, fibu was expected to form trimers. however, there was only density for twelve copies of fibu in the upper ring, equivalent to the proximal (p and p ) subunits of fibl (fig e and i ). like fibl, fibu does not make close contacts with either mtp or dit, but both fibu subunits contact the distal subunit of fibl ( fig d and e ). fibu was not essential for infectivity, and deletion of the orf gene did not affect the structure of the rest of the baseplate (s fig). no density was observed for the predicted fibrous portion of fibu beyond residue ( fig c) . however, d class averages representing top views of the baseplate showed six thin fibers extending nm radially from the baseplate and terminating in a nm knob (fig e) . these features might correspond to fibu fibers interacting with the air-water interface when the baseplate is oriented in the plane of the ice. when the baseplate is oriented laterally (fig d) , the fibu fibers might be disordered and thus not observed in the reconstruction. we have determined the structure of the bacteriophage α baseplate at . Å resolution, allowing atomic models to be built for the major tail protein, part of the tape measure protein, and all of the major baseplate-associated proteins except gp , the predicted cell wall hydrolase, the c-terminal domain of tal, and the c-terminal part of fibu (gp ). this is the first high-resolution structure of a baseplate from a staphylococcal siphovirus. while the α baseplate shares features with other bacteriophages, the structure is unlike those of the well-octadecamer (colored as in e) and the mtp hexamer underneath (tan). (g) isosurface (cutoff . σ) of a reconstruction made from signal-subtracted images excluding density corresponding to mtp, dit, tal, rbp and fibu. fibl density is blue. additional density in the center (gray) probably corresponds to tmp. (h) model for the complete fibl protein trimer built into the density from g. the lower panel is colored as in e, with structural elements labeled (cc, coiled coil; ϕk is the phage k gp -like domain). (i) top view of the dodecameric fibu ring, colored as in e. https://doi.org/ . /journal.ppat. .g described baseplates of e. coli phage t and lactococcal phages p and tp - [ , ] . in particular, the presence of three separate tail fiber/receptor binding proteins and their organization into external rings surrounding the tail are unique features of the α baseplate. the dit hexamer constitutes the hub of the baseplate, forming an adaptor between the sixfold symmetric tail and the threefold symmetric tal protein, as well as the attachment point for the six rbp trimers. our identification of the c-terminus of tmp associated with tal supports the assumption that tail assembly starts from a nucleus containing dit, tal, and tmp. the first mtp ring is added through interactions involving the mtp stacking loop and the dit tail binding loop (fig ) . additional mtp subunits are added through interactions of stacking loops and c-arms until the full length of the tmp is reached [ ] . the peripheral structures are not required for this process, since deletions of the genes encoding rbp, fibl or fibu had no effect on tail formation (s fig). the additions of rbp and fibl to the hub appear to be interdependent, whereas fibu is probably the last protein to be added to the baseplate. rbp (gp ) is the main receptor binding protein of α, and is essential for infectivity. the virtually identical rbp of ϕ was shown to bind wta [ ] , a polymer present in the cell wall of most gram-positive organisms. wta serves as receptor for many phages, but is highly variable between different species. most strains of s. aureus have wta that is distinct from that of coagulase-negative staphylococci (cons) such as staphylococcus epidermidis, and is made from ribitol phosphate repeating units modified by glcnac [ , ] . however, some pathogenic s. aureus strains belonging to the st lineage express an altered form of wta that is more similar to that of cons, resulting in altered phage susceptibility and immune system evasion [ ] . host specificity is reflected in the structure of the receptor-binding structures of the infecting phages. the distantly related s. aureus-infecting podovirus p has a "tail fiber" protein (gp ) that is remarkably similar to the α rbp [ ] ( % sequence identity), suggesting that this structure is conserved among phages that infect s. aureus. in contrast, phage andhra, which is structurally very similar to p , but infects s. epidermidis [ ] , has a completely different predicted rbp structure based on a β-helical architecture. similarly, s. aureus phage ϕ , which infects the st lineage of s. aureus, lacks an rbp-like protein, but has a tail fiber protein (gp ) that, by hhpred analysis, resembles parts of fibl, including an n-terminal ig-like domain, a coiled-coil domain, a tower domain, and a c-terminal domain similar to the phage k tail fiber protein, gp (s fig). a striking feature of the α baseplate is the presence of two tail fiber proteins, fibl and fibu, in addition to rbp. these proteins are probably involved in host interactions, although it is not known whether they bind to wta or other components of the staphylococcal cell wall. although fibl was essential for α infectivity, this could be because deletion of orf also caused loss of rbp (s fig). fibu was not essential, and deletion of orf had no effect on either infectivity or baseplate integrity (s fig). the two c-terminal domains of fibl are similar to those of phage k gp (s fig), a large-genome myovirus that infects s. aureus strains belonging to several lineages [ ] . thus, while fibl and fibu may not be strictly required for infection of rn under standard laboratory conditions, they could facilitate adsorption under certain conditions through low-affinity, reversible binding to surface structures (fig a) . fibl and fibu could also be involved in binding divergent s. aureus strains that express altered wta. the presence of multiple copies of receptor binding complexes is a common theme among phages of gram-positive bacteria. α has six trimers of rbp, while p has twelve [ ] , and lactococcal phage tp - has trimers of its bppl receptor binding protein [ ] . presumably, the presence of multiple copies of the receptor binding proteins increases the avidity of the low-affinity interaction with wta and surface polysaccharides. in contrast, phages like λ and spp that use protein receptors-a high affinity interaction-generally do not have multiple receptor binding protein complexes. the conservation of the fibu and fibl ntds with proteins from many phages suggests that this fold is optimized for making rings for the attachment of various structures to phage tails [ ] . receptor binding is only the first step in the infection process that is mediated by the baseplate (fig a) . once bound, enzymatic activities associated with the baseplate are used to degrade the cell wall peptidoglycan and penetrate the cell wall. this process most likely requires conformational changes in the baseplate in order to expose the hydrolase domains of tal and hyd (fig b) . we previously observed that treatment of α with heat or low ph led to clustering of baseplates, presumably due to exposure of hydrophobic sequences resulting from a conformational change [ ] . the observed conformational differences in the stem between the α and ϕ rbps and the phage p tail fiber (fig b and c ) suggest that the hinge might constitute a pivot point that allows the rbps to rotate. this movement might assist in orienting the baseplate perpendicular to the cell wall. similarly, in p , reorientation of its orf receptor binding protein occurred as a consequence of a ca + -triggered conformational change in dit [ ] . straightening of the rbp stem might also lead to release of fibl, which is bound to the rbp hinge (fig d) . once the peptidoglycan layer is degraded and tal reaches the plasma membrane, the αhelices in the plug and rod must be removed to permit extrusion of the pore-forming tmp (fig c) [ ] . this conformational change is fundamentally different from the opening of the p baseplate [ ] . membrane penetration has to be signaled to the capsid in order to initiate dna release and must be tightly regulated to avoid premature dna ejection. other studies have revealed no apparent conformational change in the tail tube associated with the infection process in siphoviruses [ ] . even in the myoviruses, tail contraction itself is not the signal that leads to ejection [ , ] . our observation of tmp α-helices associated with tal resolves this conundrum: upon release by tal, the tmp α-helices might act as a "pull cord" that drags the dna along during injection (fig c) . most temperate bacteriophages with long tails (myoviruses and siphoviruses) share common features in structural and genomic organization, reflecting their common evolutionary origin [ ] . according to the "modular theory" of phage evolution, phage genomes evolve by exchanging modular units consisting of protein domains, as opposed to whole genes or transcriptional units. in accordance with this principle, the tail and baseplate proteins in α are based on a set of conserved modules that is found in a wide variety of phages. in α, mtp, dit and tal all incorporate the tail tube fold, one of the most highly conserved bacteriophage structural modules [ ] . in contrast, the peripheral structures are far less conserved between α and other phages, presumably reflecting different adsorption strategies between bacteriophages of different hosts. nevertheless, the proteins that make up the peripheral structures share common domains with proteins from several bacteriophages, reflecting the modular evolution of these proteins. modules such as ig-like n-terminal domains, rbp tower domains and phage k tail fiber ctds are repeated across phages from a broad range of hosts (s fig) . our study provides a structural basis for understanding host specificity and the infection process in staphylococcal bacteriophages. since bacteriophages are the primary mediators of horizontal gene transfer in staphylococci, this process is critical to the dissemination of virulence factors and the evolution of pathogenicity in s. aureus and other pathogens. the ability to infect a variety of strains and species will also be important for the development of effective therapeutic strategies utilizing phages. α tails were produced by mitomycin c induction of s. aureus strain st , which is an α lysogen with a deletion of residues - from the scaffolding protein, gp [ ] . the tails were purified from the lysate by peg precipitation and cscl gradient purification, as previously described. cryo-em samples were prepared on nickel quantifoil r / grids, as previously described [ ] . an initial cryo-em data set consisting of images was collected on so- film on a philips cm feg microscope. the films were scanned with a nikon ed film scanner at dpi, corresponding to . Å/pixel, and used to generate the initial model for reconstruction. high-resolution data was collected on an fei titan krios microscope equipped with a direct electron de- detector at the secm consortium at florida state university. a smaller dataset of micrographs and a larger dataset of , micrographs were collected at , x magnification, corresponding to . Å/pixel in the specimen, and with typical defocus of . - . μm. an initial c starting model was generated from , particles picked from the film data, then used to reconstruct and refine , particles picked from the smaller fsu dataset, using eman . and eman . [ ] . the resulting . Å resolution (fsc . ) reconstruction was in turn low-pass filtered and used as an initial model in the reconstruction of the larger fsu dataset in relion- . [ ] . , particles were picked from the , micrographs following frame alignment with dose weighting using motioncor and ctf correction using gctf, all from within relion. after extensive d and d classification, the final reconstruction was calculated from , particles using the gold standard approach and assuming c symmetry throughout the reconstruction process. following mask generation and map sharpening in relion, the overall estimated resolution reached . Å (fsc . ) (fig g and s fig) , ranging from . Å in the baseplate core to > . Å in peripheral parts of the fibers (s fig). generation of the c reconstruction used for modeling tal and dit required separation of the non-identical orientations of the c tal trimer superimposed as a result of the initial assumption of c symmetry. the c reconstruction was segmented in ucsf chimera [ ] and a mask generated from all non-tal density was used in subtracting the non-tal density from the particle images using relion- . [ ] . the resulting signal-subtracted images were c symmetry-expanded using relion_particle_symmetry_expand to create three copies of each particle where the tal trimer, regardless of initial orientation, was superimposed with the r otated non-identical orientation. masked d classification in relion- . of the expanded dataset using the tal density from the c reconstruction as a reference yielded two good classes related by a ˚rotation and excluding particles with suboptimal tal density. the more populated class was cleaned of duplicate particles, the orientations were transferred to the original, unsubtracted dataset, and the particles were reconstructed assuming c symmetry using relion- . with the c baseplate reconstruction as an initial model. following post-processing, the resolution of the c reconstruction reached . Å resolution (fsc . , fig g) . focused reconstructions for rbp and the n-terminal parts of fibl and fibu were done in the same way, but without symmetry expansion and with the application of c symmetry to optimize the resolution of each for atomic model refinement. reconstruction of signal-subtracted images lacking all non-fibl density (fig g) in relion- . allowed assembly of a full-length fibl pseudo-atomic model by rigid-body fitting matched structures from hhpred to the density. these models were manually mutated to the fibl sequence and joined together and to the refined fibl ntd structure (fig h) . the fsc curves and resmap analysis for the various reconstructions are shown in s and s fig, respectively. the initial model for rbp was adapted from the ϕ rbp (pdb id: efv) [ ] . initial models for the remaining proteins were generated using i-tasser [ ] . those best matching the baseplate reconstruction were fitted into the density using ucsf chimera [ ] . changes to the protein sequence, extension of the models, and manual adjustments to the protein backbone, including local real-space refinement with secondary structure, torsion angle, rotameric, and ramachandran restraints, were made in coot [ ] . the positions of large aromatic and basic side chains, as well as predicted secondary structures, were compared to the baseplate protein sequences and initial models to confirm the identity of proteins in the baseplate density and to correctly place the models during manual adjustment. the models were iteratively refined using real-space refinement in phenix [ ] , followed by manual correction and local realspace refinement in coot guided by geometry reports from molprobity [ ] and emringer [ ] . all models except tal and tmp were initially refined into the c reconstruction. the final dit, tal and tmp models were refined in the c reconstructions, while rbp, fibu and fibl were further refined in their respective focused reconstructions. all reconstructions were autosharpened in phenix for the final stages of model building. simulated annealing, rigid body fitting, and morphing were included in early cycles of real-space refinement in phenix. secondary structure restraints and non-crystallographic symmetry restraints generated from the models by phenix were included throughout. the model statistics for each protein are listed in s table and the quality of the fit (fsc) is shown in s fig. model and map visualization was done in ucsf chimera [ ] . for each refinement, the gold-standard half-map fsc plots for the appropriate maps, calculated in relion (green curve) and in phenix (red curve) are shown. the blue curve represents the corresponding model-to-map fsc plots, calculated in phenix. (tif) the predicted structure of the ϕ tail fiber is also shown. domains are labeled: cc, coiled coil; p, β-propeller platform domain; t, tower domain; k, phage k gp ctd; ig, immunoglobulin-like domain; βh, beta-helix; col, collagen-like helix. protein lengths and relevant residue numbers are indicated. (tif) s table. comparison of proteins. the α reference protein and residues selected are listed in columns and , the matched proteins and residues in columns and . (unless otherwise indicated, α proteins are implied.) the first rmsd value is between cα atoms in all residues considered equivalent by sequence alignment. the number or residues used is listed. the second rmsd value is after pruning to remove atom pairs that are more distant than the indicated cutoff level. the number of residues compared is listed. the percentage is the fraction of equivalent residues used in the pruned set compared to the unpruned set. all data were generated using the matchmaker function in ucsf chimera. (pdf) s table. reconstruction and modeling statistics. (pdf) pathogenesis of methicillin-resistant staphylococcus aureus infection vital signs: epidemiology and recent trends in methicillin-resistant and in methicillin-susceptible staphylococcus aureus bloodstream infections-united states staphylococcus aureus genomics and the impact of horizontal gene transfer mobile genetic elements of staphylococcus aureus phages of staphylococcus aureus and their impact on host evolution pirates of the caudovirales the floating (pathogenicity) island: a genomic dessert the wall teichoic acid and lipoteichoic acid polymers of staphylococcus aureus wall teichoic acid structure governs horizontal gene transfer between major bacterial pathogens long noncontractile tail machines of bacteriophages contractile tail machines of bacteriophages bacteriophage taxonomy: an evolving discipline structures and host-adhesion mechanisms of lactococcal siphophages the complete genomes of staphylococcus aureus bacteriophages and alphaimplications for the specificity of sapi mobilization competing scaffolding proteins determine capsid size during mobilization of staphylococcus aureus pathogenicity islands cleavage and structural transitions during maturation of staphylococcus aureus bacteriophage α and sapi capsids single-particle refinement and variability analysis in eman processing of structurally heterogeneous cryo-em data in relion new tools for automated high-resolution cryo-em structure determination in relion- transducing particles of staphylococcus aureus pathogenicity island sapi are comprised of helper phage-encoded proteins an essential role for the baseplate protein gp in phage adsorption to staphylococcus aureus a completely reimplemented mpi bioinformatics toolkit with a new hhpred server at its core structure of the phage tp - . mda baseplate suggests an alternative host adhesion mechanism structure of the host-recognition device of staphylococcus aureus phage ϕ crystal structure of bacteriophage spp distal tail protein (gp . ): a baseplate hub paradigm in grampositive infecting phages structure of lactococcal phage p baseplate and its mechanism of activation the peptidoglycan hydrolase of staphylococcus aureus bacteriophage plays a structural role in the viral particle capsid size determination by staphylococcus aureus pathogenicity island sapi involves specific incorporation of sapi proteins into procapsids i-tasser server for protein d structure prediction real-space refinement in phenix for cryo-em and crystallography phages have adapted the same protein fold to fulfill multiple functions in virion assembly a common evolutionary origin for tailed-bacteriophage functional modules and bacterial machineries bacteriophage spp tail tube protein self-assembles into β-structure-rich tubes the phage lambda major tail protein structure reveals a common evolution for long-tailed phages and the type vi bacterial secretion system bacteriophage t tail tube structure suggests a trigger mechanism for siphoviridae dna ejection functional and structural dissection of the tape measure protein of lactococcal phage tp - structure and genome ejection mechanism of staphylococcus aureus phage p genome of staphylococcal phage k: a new lineage of myoviridae infecting gram-positive bacteria with a low g+c content sapi dna is packaged in particles composed of phage proteins a novel staphylococcus podophage encodes a unique lysin with unusual modular design ubiquitous carbohydrate binding modules decorate lactococcal siphophage virions bacteriophages and their genomes visualizing density maps with ucsf chimera tools for macromolecular model building and refinement into electron cryo-microscopy reconstructions molprobity: all-atom structure validation for macromolecular crystallography emringer: side chain-directed model and map validation for d cryo-electron microscopy we are grateful to john spear and duncan sousa at florida state university for assistance with the high-resolution data collection at the southeastern consortium for microscopy of macromolecular machines (secm ). we are also grateful to dr. alasdair c. steven for access to the em facility at the national institute of arthritis and musculoskeletal and skin diseases (niams), where the initial data collection was carried out, to cynthia m. rodenburg for technical assistance at the uab cryo-em facility, to dr. josé penadés for providing the α baseplate deletion mutants, and to dr. pavel plevka for sharing the atomic coordinates for phage p . conceptualization: terje dokland. key: cord- - v xl s authors: trus, ivan; udenze, daniel; cox, brian; berube, nathalie; nordquist, rebecca e.; van der staay, franz josef; huang, yanyun; kobinger, gary; safronetz, david; gerdts, volker; karniychuk, uladzimir title: subclinical in utero zika virus infection is associated with interferon alpha sequelae and sex-specific molecular brain pathology in asymptomatic porcine offspring date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: v xl s zika virus (zikv) infection during human pregnancy may lead to severe fetal pathology and debilitating impairments in offspring. however, the majority of infections are subclinical and not associated with evident birth defects. potentially detrimental life-long health outcomes in asymptomatic offspring evoke high concerns. thus, animal models addressing sequelae in offspring may provide valuable information. to induce subclinical infection, we inoculated selected porcine fetuses at the mid-stage of development. inoculation resulted in trans-fetal virus spread and persistent infection in the placenta and fetal membranes for two months. offspring did not show congenital zika syndrome (e.g., microcephaly, brain calcifications, congenital clubfoot, arthrogryposis, seizures) or other visible birth defects. however, a month after birth, a portion of offspring exhibited excessive interferon alpha (ifn-α) levels in blood plasma in a regular environment. most affected offspring also showed dramatic ifn-α shutdown during social stress providing the first evidence for the cumulative impact of prenatal zikv exposure and postnatal environmental insult. other eleven cytokines tested before and after stress were not altered suggesting the specific ifn-α pathology. while brains from offspring did not have histopathology, lesions, and zikv, the whole genome expression analysis of the prefrontal cortex revealed profound sex-specific transcriptional changes that most probably was the result of subclinical in utero infection. rna-seq analysis in the placenta persistently infected with zikv provided independent support for the sex-specific pattern of in utero-acquired transcriptional responses. collectively, our results provide strong evidence that two hallmarks of fetal zikv infection, altered type i ifn response and molecular brain pathology can persist after birth in offspring in the absence of congenital zika syndrome. introduction zika virus (zikv) infection during human pregnancy may lead to fetal death, brain lesions, in utero growth restriction, and microcephaly in newborns resulting in severe life-long impairments [ ] [ ] [ ] [ ] . critically, the majority of congenital infections in humans is subclinical [ , ] and is not associated with easily identifiable brain lesions or birth defects. deleterious and less severe delayed neurodevelopmental, motor, and neurosensory abnormalities in apparently normal at birth human offspring have been described later within one-two years of life [ , ] . potentially detrimental life-long health outcomes in asymptomatic offspring evoke high concerns [ ] [ ] [ ] [ ] [ ] . thus, animal models addressing sequelae in offspring may provide valuable information. in a pigtail macaque model, maternal zikv inoculation during gestation resulted in substantial brain lesions and silent brain pathology (i.e., periventricular t -hyperintense foci and loss of fetal noncortical brain volume, injury to the ependymal epithelium with underlying gliosis, and loss of late fetal neuronal progenitor cells) in fetuses, even in the absence of microcephaly [ , ] . two very recent studies in immunocompetent mouse models reported neurocognitive disorders and neurobehavioral deficits in offspring affected with mild congenital zikv infection [ , ] . these pioneering studies provided critical information regarding outcomes of mild congenital zikv infection in mouse offspring. although, in these models, zikv induced clinical disease with reduced fetal birth weight, postnatal growth impediments, and neurobehavioral deficits. thus, models reproducing subclinical in utero infection and long-term silent health sequelae (e.g., molecular pathology which is difficult to identify with diagnostic tests in clinical settings) in offspring in the absence of congenital zika syndrome are not reported. while postnatal zikv infection in macaque infants resulted in altered emotional reactivity to acute stress [ ] , the evidence is still lacking for the cumulative impact of subclinical in utero zikv infection and postnatal environmental insults on health sequelae in offspring. this knowledge is important because secondary insults during postnatal life can unmask consequences of in utero acquired silent pathology [ , ] . pigs are relevant to model human in utero zikv infection [ ] [ ] [ ] and associated immunopathology and brain pathology in offspring because both species have similar physiology, genetics, immunity [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , fetal brain development and postnatal brain growth [ ] [ ] [ ] [ ] . we and others have recently developed a fetal pig model which reproduces key aspects of in utero zikv infection in humans with persistent infection in the fetal brain, fetal membranes, and placenta [ ] [ ] [ ] . similarly to human and mouse infections [ , ] , outcomes of infection in the porcine model depend on the gestational stage. zika virus inoculation at the early stage of fetal development ( gestation days, gd; the total duration of porcine gestation is days) resulted in fetal death [ ] . in contrast, fetuses infected at the mid-stage of development ( gd) did not show brain lesions and days later [ , ] . congenital zikv infection in mice increased in utero levels of type i ifns [ , ] , which was suggested to play a role in fetal demise [ ] . in our porcine model studies, subclinical persistent in utero infection in mid-gestation also increased interferon alpha (ifn-α) levels in fetal blood plasma and amniotic fluid, while ifn-α was below the detection limit in all control fetuses [ ] . in addition, levels of ifn-α positively correlated with zikv titers in fetuses. interestingly, while fetuses did not have pathology or lesions, they showed persistent infection and dysregulation of more than genes in their brains [ ] . in the present study, to establish subclinical in utero infection, we exposed porcine litters to zikv at mid and late gestation, when similarly to humans, the fetal pig brain has a growth spurt [ , , , ] . we defined whether subclinical in utero infection imposes ifn-α sequelae and molecular brain pathology in offspring that did not have clinical signs of congenital zika syndrome. we also tested whether prenatal exposure to subclinical zikv infection and postnatal social stress have a cumulative impact on immune responses and behavior in offspring. to induce subclinical in utero infection, we directly inoculated two conceptuses (a fetus with fetal membranes; on average pigs have - fetuses) from three sows with tcid /fetus of the zikv prvabc strain at - gd (s video; fig a and b; s fig) . litters with sham-inoculated conceptuses from three sows were used as controls. mothers did not show clinical signs. all pregnant pigs were synchronized and delivered at term ( - days) . control and experimental litters contained . % and . % dead newborns, respectively (s a table; p = . ), which is in line with usual rates of fetal mortality in pigs [ , ] . the number of weak piglets was also similar in both groups (s a table, p = . ). in utero zika virus exposure did not significantly affect cranium diameter in piglets (p = . ; s a table) . while body weights at birth were lower in the zikv group (zikv group: . ± . kg, control group: . ± . kg, p = . ) (fig c) , body weight gain was not affected (p = . ) (fig c and d ). brain weights in the zikv group (fig e) had a slightly wider (p = . ) distribution (zikv group: coefficient of variation . %, control group: coefficient of variation . %). brain to body weight ratio (fig f) was also not affected (p = . ). placental samples collected at birth and offspring brains did not have histopathological lesions (fig a- d) . to confirm in utero infection, we demonstrated high loads of zikv in amniotic membranes and placenta from all three zikv-exposed litters (fig i; s b table) . we also detected zikv rna by in situ hybridization in the placenta and by rt-qpcr targeting the negative strand of zikv rna in the placenta and amniotic membranes (fig e and f ; s b table) . these results confirm zikv transmission between siblings and productive, persistent infection in fetal membranes and placenta. all samples from control mothers and piglets were negative for zikv-specific igg antibodies (abs) (s c table) . in utero zikv exposure caused maternal infection as indicated by virus-specific igg ab in maternal plasma (fig j; s d table) . most probably, maternal infection was transient because maternal blood plasma samples were free for zikv rna at all five sampling time-points as determined by rt-qpcr (s b table) . moreover, in our previous studies, where we induced more severe in utero infection with higher viral doses, maternal endometrium and lymph nodes were free from zikv [ , , ]. next, we determined whether zikv replicated in fetuses and persisted in offspring. zikv-specific igg abs were detected in a porcine uterus has multiple fetuses (on average pigs have - fetuses) with each fetus possessing individual amniotic membrane and placenta. two fetuses in each pregnant pig were directly inoculated (i) with zikv. see s video for ultrasound-guided inoculation. afterward, zikv spreads (ii) between siblings and causes productive infection in ( ) amniotic membranes, ( ) placenta, and ( ) fetal brains of directly inoculated and trans-infected not-manipulated fetuses [ ] [ ] [ ] ]. (c) body weight, (d) body weight gain, (e) brain weight, and (f) brain/body weight ratio in control and zikv-exposed offspring. solid lines represent mean values. brains from offspring were collected at necropsy. see s a table for histology, viral loads, and zikv-specific ab responses. hematoxylin-eosin staining in the placenta (sampled at birth) (a: control sow # ; b: zikv-inoculated sow # ) and neonatal brain (sampled at euthanasia, days) (c: control piglet # , sow ; d: piglet # from zikv-inoculated sow ). zikv-specific in situ hybridization in the placenta (sampled at birth) from control (e) and zikv (f) litters. positive cells were found in a sample from pig # . an immunoperoxidase monolayer assay (ipma) to detect and quantify zikv-specific igg ab in porcine blood plasma. blood plasma from control (g) and zikv-exposed (h) offspring (sampled at birth). (i) zikv rna loads in amniotic membrane and placenta determined by rt-qpcr. solid lines represent mean values. the dotted line represents the limit of detection (lod). see s b table for individual values. zikv-specific ab in offspring blood plasma detected by ipma (j). all samples from the control litters were negative. black dots-ab titers in maternal blood. offspring blood at birth was collected before first colostrum feeding. offspring were subdivided into two subgroups based on zikv-specific serological status at birth: negative for ab at birth-"n" and positive for ab at birth-"p." solid lines represent mean values. the dotted line represents lod. see s c table for blood plasma from a subset of newborns in all three exposed litters (fig j; s c table) . in pigs, maternal abs do not pass to porcine fetuses through the placenta [ ] ; however, it is being transferred passively to offspring via colostrum. blood plasma samples from all newborns were collected before first colostrum ingestion, and high ab titers demonstrated productive fetal infection and subsequent in utero ab responses (fig j; s c table) . two zikvspecific rt-qpcr assays did not show viral rna in the blood plasma, cerebrum, and cerebellum from all exposed and control piglets (including stillborn and weak piglets) (s b table) . altogether, we induced subclinical persistent in utero infection which did not cause readily identifiable clinical pathology and productive infection in offspring. subclinical zikv infection in porcine conceptuses increases concentrations of ifn-α in amniotic fluids and fetal blood at days after inoculation [ , ] . here, we defined whether subclinical in utero infection imposes ifn-α sequelae in affected offspring. within days after birth, ifn-α (fig a) as well as il- β, il- , il- , il- , il- , il- , il- a, tgf-β, tnf, ifn-β, and ifn-γ levels (at birth) remained below or at the detection limit in control and zikv-exposed piglets. the same levels in both offspring groups suggest that maternal ifn-α (which could be transferred with milk within the first days of life) equally affected offspring in both groups during the nursing period. two zikv piglets, however, showed an increase in ifn-α levels already at days (at weaning-separation of mother from offspring) (fig a) . while piglets from both control and zikv litters had detectable ifn-α levels at days, % of zikv piglets showed considerably increased levels of ifn-α in their blood plasma (fig a; s c table) . next, we analyzed ifn-α responses in offspring subdivided into two subgroups based on zikv-specific serological status at birth: negative for ab at birth-"n" and positive for ab at birth-"p," (fig j; s c table) . remarkably, the increased ifn-α levels were mostly attributed to the p subgroup with high zikv-specific ab titers at birth (fig b) . ifn-α levels in the p subgroup were significantly higher than in the control group and n subgroup, indicating that ifn-α increase may correlate with the serological status in offspring at birth. maternal ifn-α likely did not affect increased ifn-α responses in zikv offspring at days because maternal blood ifn-α levels in zikv litters were lower or equal to that in control litters (p � . ) (the same was observed in colostrum, p = . ; s d table) and did not change significantly throughout the study (s fig) . moreover, the ifn-α increase in offspring was detected at days, eleven days after separation of piglets from mothers. in addition, maternal ifn-α is unlikely to affect fetuses and offspring as it does not cross through the human [ ] or porcine placenta [ , ] . all of other eleven tested cytokines did not show the increase (fig c; s e table) , suggesting specific ifn-α pathology. we do not know whether lower levels of il- β, il- , il- , il- , il- , il- , il- a, tnf, and ifn-γ in zikv offspring at days (fig c; s e table) were caused by subclinical in utero zikv infection or maternal cytokine background (s fig) . maternal blood levels of these cytokines were lower in the zikv group (although the difference was not statistically significant) (s fig) that could potentially contribute to the lower cytokine levels in offspring. previously described markers of maternal immune activation which may affect fetal health, il- [ ] and il- a [ ] in the zikv group were lower or equal to that in control litters (p � . ); these data are in agreement with findings in pregnant women with acute zikv infection [ ] and mice [ ] . also, il- and il- a levels in zikv group did not change significantly throughout the experiment (s overall, these data suggest that subclinical in utero zikv infection, without active maternal infection and changes in maternal cytokines, may specifically affect ifn-α response in offspring. we performed a mixing test (s video) on control and affected piglets at days of age to identify whether subclinical in utero zikv infection and social stress have synergistic effects on cytokine responses in offspring. after the mixing test, piglets in the control group showed a slight, up to a -fold decrease ( . ± . ) in blood plasma ifn-α levels in comparison to the levels before the mixing test ( fig a; s c table) . in contrast, piglets affected with subclinical in utero infection showed dramatic, up to a -fold decrease ( . ± . ) in blood plasma ifn-α levels (fig a) . this abrupt decrease in peripheral ifn-α was demonstrated by a considerable proportion of zikv-affected offspring and did not depend on the initial ifn-α level before stress induction. for example, zikv-affected offspring with exceptionally high ifn-α levels before mixing test showed similar or even lower ifn-α levels as in other groupmates after the mixing test (s c table, sow # , piglet # ; sow # , piglets # and # ; sow # , piglets # and # ). also, the decrease was observed not only in offspring with increased ifn-α levels. zikv-affected piglets which initially had ifn-α levels comparable to control piglets also showed a dramatic decrease after the mixing test (s c table: sow # , piglet # ; sow # , piglet # ; sow # , piglets # , # and # ). next, we analyzed ifn-α responses in offspring subdivided into two subgroups based on zikv-specific serological status at birth (fig b; s c table) . zikv offspring in both p and n subgroups showed a statistically significant decrease in blood plasma ifn-α levels (fig b) . in sharp contrast, stress did not induce a significant decrease in other tested cytokines ( . - . mean fold change, fig c; s e table) . no difference in ifn-α shutdown was observed between serological (n and p; p = . ) and sex (p = . ) subgroups. our findings reveal synergistic interactions between subclinical in utero zikv infection and postnatal stress in promoting pathological ifn-α responses in affected offspring. we sought to characterize whole genome expression in the prefrontal cortex (pfc) of clinically normal offspring affected by subclinical in utero zikv infection. brains from zikvaffected offspring and control offspring with no history of in utero infection were sampled at days after birth and analyzed using rna-seq (s c table) . on a global transcriptional level, gene expression differed considerably between pfc samples from zikv-affected and control offspring (s a and s b fig; s a table) . functional set enrichment of gene ontology (go) biological processes also showed significant effects in brains of zikv offspring (fig ; s b table) . specifically, genes with altered expression in the pfc of virus-affected offspring were positively enriched for processes related to cell death ( go pathways related to apoptosis and necrosis; false discovery rate (fdr)-adjusted p < . , s b table) , cytokine responses and immunity ( go pathways; fdr-adjusted p < . , s b table) and organ/tissue morphogenesis, development, and regeneration ( go pathways; fdr-adjusted p < . , s b table) . whereas a large set of biological processes involved in neuronal function, i.e., synaptic transmission, gabaergic signaling, calcium ion regulation, cerebral cortex neuron differentiation, and others, were negatively enriched ( go pathways; fdr-adjusted p < . , s b table) (fig ) . more stringent analyses of go biological pathways related to neuronal and cell death pathways with fdr-adjusted p < . is represented in fig . interestingly, "response to type i interferon" (fdr-adjusted p = . ), "positive regulation of type i interferon production" (fdr-adjusted p = . ), "regulation of type i interferon production" (fdr-adjusted p = . ) and "response to interferon beta" (fdr-adjusted p = . ) go processes were positively enriched in the pfc of affected offspring (s c fig; s b table) . another upregulated biological process was "response to corticosteroid" (fdr-adjusted p = . ) (s d fig; s b fold decrease of ifn-α level after the mixing test. (c) fold decrease of other tested cytokines after the mixing test. offspring subgroups: n-negative for endogenous zikv-specific ab at birth; p-positive for endogenous zikv-specific ab at birth. the dotted line represents loq. for negative samples (below loq), fold change was calculated using loq value as a baseline. solid lines represent means. see raw data in s c and s e table for individual values. https://doi.org/ . /journal.ppat. .g table) , which is in line with previously reported dysregulation in genes related to physiological stress responses in porcine fetuses [ ] . in support of the observed altered transcriptional profile of corticosteroid-responsive genes, fetuses showed significantly elevated in utero cortisol levels during persistent zikv infection [ ] . to find out whether transcriptional and hormonal in utero cortisol disbalance associated with subclinical infection imposes sequelae in offspring, we measured cortisol concentrations in hair, a well-established test to assess chronic stress throughout the lifespan [ ] . the cortisol levels were higher in zikv piglets (s e fig; s f table) providing considerable support of transcriptional findings in affected offspring. a previous study in rhesus macaques demonstrated that even moderate increase in mean hair cortisol levels ( . times) is indicative of chronic stress [ ] . the difference in porcine offspring, however, was not statistically significant (mean- . times; p = . ). thus, the relation between subclinical in utero zikv infection and chronic stress in offspring remains to be further confirmed. next, we generated gene sets compiled from malacards (http://www.malacards.org) and previous publications [ ] (s c table) linked to the following clinical disorders associated with congenital zika syndrome in human fetuses and neonates: microcephaly, epilepsy, dysphagia, clubfoot, and arthrogryposis. additionally, we collected gene sets from malacards for guillain-barré syndrome, schizophrenia, attention deficit-hyperactivity disorder, psychotic disorder, anxiety disorder, mood disorder, and learning disability (s c table) [ ] . like in the previous fetal rna-seq study [ ] , gene set enrichment analysis (gsea) showed that zikv-affected offspring were negatively enriched in the schizophrenia gene set (fdr-adjusted p = . ) (s d and s e table) . to determine whether offspring with the distinct zikv-specific serological status at birth have molecular pathology in the brain, we compared rna-seq data between n and p subgroups. offspring in both subgroups showed a similar number of downregulated and upregulated genes (fig a; s f and s g table) . a considerable number of upregulated and downregulated go processes (fig b; s h-s j table) , including neuronal and behavioral processes (fig c; s j table) , were also shared between n and p subgroups, which is likely reflective of the similar gene expression profiles. collectively, this analysis suggests that subclinical in utero zikv infection may cause similar molecular pathology in the brain of offspring both positive and negative for zikv-specific ab at birth. next, we analyzed molecular pathology in female and male offspring affected with subclinical in utero zikv infection. female and male offspring showed a distinct transcriptional signature in the pfc (fig a; s k and s l table) . the considerably larger number of upregulated pathways in female offspring represented by developmental, differentiation, morphogenesis, cell migration, transcription, catabolic, cell adhesion/junction, signaling, and tissue remodeling processes (fig b) , may signify more intensive compensatory responses to sequelae of in utero infection. while females and males shared enriched biological processes (s m table) , considerably larger loss of affected neuronal and behavior pathways (fdr-adjusted p < . ) was observed in males (fig c) . enrichment analysis of significantly altered processes also attributed neuronal and behavioral pathways to male offspring (fig b and fig d) . this suggests a greater loss of neuronal function in males, which is in a strong agreement with more prominent zikv-induced neurocognitive pathology in male mouse offspring [ ]. although humans and pigs have different placentation types (hemochorial and epitheliochorial, respectively) that prevents studies on mechanisms of virus and ab transmission from mother to fetus, a fetal placental mesenchyme in pigs has all major cell types found in the human fetal side of the placenta (chorionic plate) and performs the same fundamental functions [ ] [ ] [ ] . similar to replication in the human placenta [ ], zikv replicates in the porcine fetal placental mesenchyme [ , ] . also, we have recently demonstrated that similar to human placental infection [ ] , zikv infection in the porcine placental mesenchyme is associated with the increased number of cd -positive cells [ ] . thus, to provide independent support for the sex-specific pattern of transcriptional responses to zikv infection, we profiled the whole genome expression in placental samples persistently infected with zikv (s b table) . we observed that on a global transcriptional level, gene expression signature in the infected fetal placental samples also exhibited the considerable sex-specific pattern (fig a; s n -s p table) . after correcting gene expression data for sex the principal component analysis showed that while female and male control samples were grouped close to each other, zikv-infected samples had a strong sex-specific separation indicating a strong dependency of zikv-induced transcriptional changes on animal sex. zika virus infection may cause discordant clinical outcomes in human dizygotic twins, ranging from severe disease to asymptomatic infection, and different whole-genome transcriptional responses in neuronal progenitor cells of twins [ , ] . in accordance, among females, we found three low-responders which grouped very closely with control samples (fig a; s n table) . other three samples were high-responders (fig a; s o table) . non-responder female samples were from the same litter (# ). interestingly, both low-and high-responder subsets had the high placental viral loads (low-responders: . - . log zikv rna copies/g, high-responders: . - . log zikv rna copies/g; s b table) . the number of differentially expressed genes in zikv-positive placental samples was considerably higher than in zikv-positive brain samples, which is in agreement with ongoing productive placental infection (fig i; s b table) . the high number of altered genes in both female and male samples is also in agreement with in vitro rnaseq studies in placental cells [ ] and in vivo studies in human cells [ ] . the higher number of affected genes in samples from males (fig b and c ; s o and s p table) might suggest a stronger potential for pathological outcomes in males than in females [ ] . a large set of affected biological processes (fdr-adjusted p < . ) represented blood vessel and endothelial development, proliferation, migration, and morphogenesis (fig d, s q table) . this is in agreement with studies in mice [ ] and rhesus macaques [ ] where zikv-induced vascular pathology was described. additionally, biological pathways related to actin, extracellular matrix, and syncytium formation were enriched (s q table) . collectively, rna-seq results in the brain of affected offspring and virus-infected placental samples, which both did not show histopathology and lesions, provide strong evidence for silent sex-specific molecular pathology. next, we tested whether molecular pathology in the brain of affected offspring is associated with altered behavior in the normal environment and during stress. we did not observe differences between animal groups in the regular environment, in a pen with mother and littermates ( days after birth- . ± . % of active control piglets; . ± . % of active zikv piglets), after maternal removal ( days after birth- . ± . % of active control piglets; . ± . % of active zikv piglets), or one day after maternal separation ( days after birth- . ± . % of active control piglets; . ± . % of active zikv piglets). then, we tested responses under stressful conditions using the mixing test. mixing unfamiliar piglets often results in aggressive fighting to establish a dominance hierarchy [ ] . using this behavioral pattern, we compared fighting in control and zikv offspring (s video). the percentage of initiated and won fights table) . go biological vascular processes significantly altered (fdr-adjusted p < . ) in the placenta with persistent zikv infection. see raw data in s q table for (individually for males and females) were compared against the expected value ( % versus %) in control versus zikv groups. the percentage of fights initiated by zikv male piglets (mean . %) was more than twice higher than the percentage of fights initiated by control male piglets (mean . %) (p = . ). (fig ; s g table) . won fights in males were evenly distributed between control and zikv groups ( . % versus . %; p = . ). in contrast, control females demonstrated more aggressive behavior than zikv females as represented by . % of initiated fights (p = . ) and . % won fights (p = . ) (fig ; s g table) . altogether, the lack of behavioral differences between control and affected groups in the regular environment and distinct behavioral patterns between groups during the mixing test suggest that subclinical in utero zikv infection and altered gene expression in the pfc might affect behavior in offspring in the stressful environment. we addressed a question of whether subclinical in utero zikv infection may pose health sequelae in offspring in the absence of congenital zika syndrome. there are two key findings from this study. first, subclinical in utero zikv infection was associated with abnormal ifn-α responses in apparently healthy offspring under normal environmental conditions and during social stress. second, offspring affected with subclinical in utero infection showed the profoundly altered transcriptional signature in the brain free for zikv and lesions. here, like in previous mouse [ , ] and non-human primate zikv studies [ ], we used in utero inoculation because maternal inoculation in pigs does not cause fetal infection [ ] . mixing tests were performed between control and zikv-affected offspring to induce social stress and assess behavior. see raw behavioral data in s g table. https://doi.org/ . /journal.ppat. .g however, the present study in offspring clearly demonstrates that subclinical, relatively isolated, zikv infection of the fetal-placental unit, without active maternal infection and changes in maternal cytokines, may cause long-term silent immunopathology and brain molecular pathology in offspring. this is the important finding suggesting that in addition to well-recognized maternal immune activation [ ], rationale combinatory therapeutic interventions against congenital infections and long-term sequelae should also target specific fetal immunopathology. among others, zikv-agitated in utero ifn-α responses of the fetal and placental origin [ , , ] can be considered as therapeutic targets. based on our previous fetal studies and present findings in offspring, we suggest a link between the fetal ifn-α pathways reprogrammed during in utero zikv infection [ , ] and altered ifn-α responses in offspring (figs and ) . the hypothesis of fetal origins of adult disease was first described by david barker, who proposed that disruptions to the in utero environment during fetal development program increase risks for disease during adulthood [ ] . subsequently, many studies confirmed that adverse effects during fetal development may program postnatal immune and neurological pathology which can be transferred across generations [ - ]. our data also suggest age-specific ifn-α responses in the porcine model of subclinical in utero zikv infection. during fetal development, ifn-α levels in fetal blood and amniotic fluids are increased for several weeks after in utero inoculation [ ] , with a subsequent drop to undetectable levels at around days after inoculation (s fig). afterward, offspring show elevated levels of ifn-α at around - days after birth (s fig). in support of our findings, the age-specific nature of in utero acquired neurodevelopmental, behavioral, cognitive, and neuroimmune defects has been experimentally demonstrated [ , , [ ] [ ] [ ] [ ] [ ] . for example, immune activation during mouse pregnancy causes transiently elevated brain cytokine levels in offspring at birth, decreased levels postnatally, and then elevated levels during adulthood [ ] . presumably, maturation of the immune and endocrine systems is required to trigger prenatally acquired neurodevelopmental pathology in offspring [ ] [ ] [ ] . factors determining the age-specific nature of the in utero acquired zikv-induced systemic immunopathology remain to be defined. while cerebrum and cerebellum were negative for zikv, we cannot fully exclude persistent infection in offspring because lymphoid tissues were not tested; however, in our previous study newborn piglets inoculated with . tcid zikv intracerebrally, intradermally, or intraperitoneally cleared the virus from lymphoid tissues within seven days [ ] . additional studies, are required for comprehensive testing of multiple organs for persistent zikv infection in offspring affected with subclinical in utero infection and its connection with altered ifn-α responses. a secondary insult during postnatal life may be necessary to unmask the silent consequences of in utero immune activation [ , ] . here, we used the mixing test, a validated approach to model social confrontation in pigs [ ] [ ] [ ] [ ] . mixing a pair of unfamiliar individuals in a new environment induces stress, to which animals respond with physiological changes. the test induces cumulative stress from the separation of littermates, confrontation with a novel environment (a mixing chamber), confrontation with the unfamiliar conspecific and aggression (fight)-induced stress (s video). possible effects of sex, body weight, and familiarity with the environment, which may influence aggressive/social behavior and outcomes of the test [ ] , were excluded by mixing piglets of the same sex and similar weights in unfamiliar mixing chambers. we demonstrated that combined exposure to subclinical prenatal zikv infection and postnatal social stress induces synergistic pathological effect promoting abrupt ifn-α shutdown in affected offspring. further attempts to better understand silent pathology and develop alleviative interventions in zikv-affected offspring should take into account synergistic interactions of multiple environmental insults. the ifn-α increase and ifn-α shutdown apparently have different, yet unknown, mechanisms as indicated by different responses between n and p subgroups in the normal environment (fig b) and during stress (fig b) . abrupt nature of both phenomena, however, is most probably attributed to changes in peripheral blood cells. because blood monocytes and dendritic cells are the primary sources of ifn-α in humans [ , ] and pigs [ ] , these cells should be studied in terms of in utero acquired zikv-induced ifn-α pathology. we also suggest studying hematopoietic stem cells in affected fetuses and offspring since excessive ifn-α may impact proliferation and reconstituting ability in hematopoietic stem cells and stem cell niche [ ] [ ] [ ] . interestingly, in contrast to the altered molecular signature in the brain and placenta, the increase of ifn-α in porcine offspring blood did not have the sex-specific nature, suggesting that brain/placenta-specific phenomenon and described ifn-α immunopathology in blood most probably have different mechanistic background. to our knowledge, type i ifn profiles in human fetuses and offspring affected with zikv infection were not addressed, yet. however, it has been shown that acute zikv infection is associated with increased ifn-α levels in the blood sera of pregnant women [ ] . human infections with other related flaviviruses (dengue virus and west nile virus) were also associated with increased ifn-α levels in the blood plasma/sera [ , ] . the relevance of ifn-α sequelae identified in porcine offspring affected with subclinical in utero zikv infection to sequelae in humans remains to be established. if zikv-induced ifn-α sequelae persist in human offspring, it might have dramatic health consequences. excessive ifn-α levels were previously associated with severe viral infection [ ] , immune dysfunction [ ] , major depressive disorder [ , ] , and dementia [ ] . importantly, cognitive impairment, severe neurological sequelae, seizures, prefrontal hypometabolism, and psychiatric syndromes were described in adult and pediatric patients treated with therapeutic doses of ifn-α [ ] [ ] [ ] [ ] [ ] [ ] . affected ifnα responses during stress, specifically, the stress-induced ifn-α shutdown, might also influence the susceptibility of offspring to other infections. interestingly, we recently demonstrated that similar to zikv infection [ , ] , porcine circovirus (a dna virus of circoviridae family which causes pathology and death in fetuses) in utero infection is associated with consistently increased fetal ifn-α levels (pcv -positive fetuses- . ± . pg/ml of blood plasma; pcv -negative-below the detection limit). this proof of concept study is in agreement with a hypothesis that type i ifns is a common culprit of severe congenital viral infections and associated grave complications in fetuses [ ] . in addition, we suggest that in utero inflammation induced by subclinical torch infections [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , including zikv, may evoke type i ifn pathology in offspring which can be targeted to reduce long-term sequelae. in agreement with previous histological, magnetic resonance imaging, and computed tomography screening of porcine fetal brains [ , ] , subclinical in utero zikv infection at mid and late gestational periods did not cause brain lesions in offspring. however, we found profound transcriptional changes in brains of offspring which did not show clinical signs of congenital zika syndrome. we do not know whether two hours-long social stress which was induced two days before a necropsy could affect transcriptional signature in brains of control and zikv offspring. although, the considerable number of tested brain samples, the obvious difference of the whole genome expression between brains of control and zikv offspring, and independent confirmation of the altered transcriptional profile in placental samples, clearly indicate effects of subclinical in utero infection. offspring brains were negative for zikv, suggesting that transcriptional changes in the pfc may be a persistent representation of earlier zikv replication in their brains during subclinical in utero infection. we believe that zikv caused brain infection in at least some offspring during their in utero fetal life and was subsequently cleared. in support, first, zikvspecific abs in newborns detected at birth (p subgroup) (fig j) postulated productive infection in developing fetuses during in utero life and most probably in fetal brains because fetal brain is the target for zikv in the porcine model [ , ] . second, when porcine litters inoculated at the mid of gestation, zikv replicates in the fetal brain for at least days [ , ] and cleared at days [ ] . the virus clearance from fetal brains of rhesus monkeys has been also suggested [ ] . third, affected type i ifn processes (fig ) and "defense response to virus" (fdr-adjusted p = . ) and "response to virus" (fdr-adjusted p = . ) (s b table) in brains of offspring, strongly suggest previous infection in the fetal brain. finally, in offspring of the n subgroup, the lack of virus-specific abs at birth (fig j) does not rule out earlier subclinical fetal and fetal brain infection because zikv can persist in brains of porcine fetuses negative for abs [ , ] . even if zikv offspring in the n subgroup did not have virus replication in their internal organs during in utero life, most of them had infection in their individual amniotic membranes and placenta collected at birth (fig i; s b and s c table) . fetuses and fetal membranes form a conceptus, a fetal-fetal membrane unit, because fetal, amniotic membrane, and placental blood circulations are intimately interconnected. replicating in individual amniotic membranes and placenta during persistent in utero infection, zikv may distantly affect the developing fetal brain. this was previously described in herpesvirus and malaria infections in mice and humans [ , ] , where pathogens did not reach the fetus, but the inflammatory process in the placenta affected the normal fetal development. the role of immune responses to zikv at the maternal-fetal interface in birth defects has been also suggested [ ] . in strong agreement, numerous biological processes, including vascular processes, were affected in the porcine placenta persistently infected with zikv (fig d) . also, offspring of both n and p subgroups had the altered whole-genome expression signature in brains (fig ) . in the fetal pig model of zikv infection, the cumulative negative effects originated in the developing fetal brain, fetal membranes, and placenta are also possible. alternative animal model approaches are required to discriminate the effects of local zikv replication in the fetal brain and placenta on health in offspring. using rna-seq, we defined sex-specific transcriptional changes in brains of affected porcine offspring, and subsequently confirmed sex-specific responses in infected placental samples. it is well-recognized that male fetuses are more vulnerable in affected pregnancies, with more adverse long-term outcomes occurring after birth [ , ] . accordingly, zikv-affected male piglets demonstrated more dramatic molecular pathology both in the brain and placenta. in the brains, most affected biological processes related to neuropathology were represented by male offspring. in the placenta, a higher number of differentially expressed genes was showed by male offspring (fig b and c) . our findings are in agreement with a study in mice, where male offspring affected with mild zikv infection showed higher risks of developing neurocognitive disorders [ ] . moreover, in a recent study on the prospective human cohort of zikv-exposed children, male gender was identified as a potential predictor of delayed developmental alterations [ ] . observed behavioral differences between groups during social stress also had a sex-specific trend (fig ) . zika virus-affected male offspring showed more aggressive social behavior than affected female offspring as represented by a higher percentage of initiated and won fights (fig ) . altered behavior conveyed by zikv-affected porcine offspring during stress is in high agreement with findings in macaques [ ] , where postnatal zikv infection of infants resulted in altered functional connectivity between brain areas involved in emotional behavior and arousal functions, as well as in distinct alterations in the species-typical emotional reactivity to acute stress. it is difficult to compare our sex-specific data in pigs with the macaque model study due to different experimental approaches [ ] . while zikv-affected offspring showed the trend to behavioral differences (fig ) , the statistical significance was not attained in both female and male animals, which is not surprising in the context of subclinical in utero infection and silent pathology. delayed neurodevelopmental abnormalities identified at the only second year of life have been described in the most recent study on the prospective human cohort of zikv-exposed children [ ] . thus, to further confirm and better understand behavioral sequelae in the porcine model, its functional connection to molecular pathology in the brain and placenta, and the relevance of the model to study delayed childhood neurodevelopment described in affected human offspring [ ] , future experiments should utilize the larger sample size, the longer observational periods, and a broader panel of standardized methods for behavioral and cognitive research in pigs [ ] [ ] [ ] [ ] [ ] [ ] . collectively, our results provide strong evidence for long-term silent immunopathology and sex-specific brain molecular pathology in porcine offspring and novel insights into pathogenesis of subclinical in utero zikv infection. we also demonstrated that subclinical prenatal zikv exposure and postnatal social stress may cumulatively induce immunopathology in affected offspring. these findings should encourage further efforts to better understand silent pathology in fetuses and offspring, monitor zikv-affected human cohorts, and develop strategies to prevent and alleviate long-term sequelae. we used low-passage, contemporary, asian-lineage zika virus (zikv) strain prvabc [genbank: ku . ] isolated from human serum specimen (puerto rico, ) [ ] . after two passages on c / cells, cell culture media containing zikv was centrifuged ( , g, min, + ˚c), and the supernatant was collected. media from virus-negative c / cells was used for mock-inoculation. the absence of mycoplasma contamination in all inoculums and cell cultures was confirmed using lookout mycoplasma pcr detection kit (sigma-aldrich). animal experiments were performed in strict accordance with the canadian council on animal care guidelines for humane animal use. all animal protocols were approved by the university of saskatchewan's animal research ethics board. six, pregnancy-matched landracecross pigs, were obtained from a high-health status herd free for porcine reproductive and respiratory syndrome virus and porcine parvovirus (viruses which can cause fetal infection in pigs). to exclude porcine circovirus (pcv ) in utero infection (another pig virus which can cause infection in fetuses) we tested blood plasma collected from all newborns at birth, before feeding colostrum, for pcv antibodies (ab) [ ] . all samples were negative for anti-pcv ab. pregnant pigs were housed at the vaccine and infectious disease organization-international vaccine centre (vido-intervac) level facilities. pregnant pigs were randomly assigned into control (three animals) and zikv (three animals) groups and housed in identical, but isolated rooms. housing conditions and diet were the same for all sows and offspring. in utero inoculation was performed at - gestation days (gd) (the total duration of porcine pregnancy is - days) as previously described [ ] with some modifications. briefly, to establish subclinical infection, we manipulated only two conceptuses per pregnant pig. two conceptuses from three experimental pigs (pig # , # , # ) were inoculated intraperitoneally + intra-amniotic (ip+ia; μl+ μl) with tcid (tissue culture infectious dose with % endpoint) of zikv. two conceptuses from three control pigs (pigs # , # , # ) were inoculated with virus-free media. for precise inoculation, we used an ultrasound-guided technique which verifies fetal viability before and after injection by visualizing heart beating (s video). two fetuses close to the uterine bifurcation (s fig) were directly inoculated. this fetal location was selected in order to maximize the virus spread within the uterus. fetuses in the only one uterine horn were inoculated to reduce manipulation. we collected blood samples (in sterile edta tubes with vacutainer blood-sampling system (bd)) from mothers at - and gestation days (at and days post-fetal inoculation), on the day of parturition, and and days after parturition. after blood centrifugation ( , g, min, + ˚c) plasma was aliquoted and immediately frozen (- ˚c). colostrum was collected at delivery day, centrifuged ( , g, min, + ˚c), and immediately frozen at - ˚c. births were monitored closely. rejected after birth placental tissues and individual amniotic membranes surrounding each newborn were collected and immediately frozen on dry ice or preserved in % buffered formalin. at birth (day ), we tagged piglets, recorded gender, and measured cranium, body dimensions and body weight. after birth, piglets were monitored for days for clinical signs by veterinarians. placental, amniotic membrane and brain tissues from stillborn and weak piglets were tested for zikv. weak piglets were euthanized at day after birth and excluded from the study (s a table) . mothers were separated from piglets at days after birth. piglets were weighed at birth and at days after birth. blood from piglets was collected at (immediately after birth, before the first colostrum uptake), , , , and days after birth. blood was collected in sterile edta tubes with vacutainer blood-sampling system (bd) by puncture of the vena cava. after blood centrifugation ( , g, min, + ˚c) plasma was aliquoted and immediately frozen (- ˚c). piglets were euthanized at days of age. animals were euthanized by licensed veterinarians with an anesthetic overdose followed by exsanguination: after injecting the anesthetic, complete unconsciousness was confirmed by loss of pedal and palpebral reflexes and pigs were rapidly exsanguinated to ensure a quick death. this method minimizes animal distress, and is consistent with the recommendations of the panel on euthanasia of the american veterinary medical association and approved by the university of saskatchewan's animal research ethics board. at euthanasia, we weighted piglets. brains were removed, weighed, and preserved in liquid nitrogen (left hemisphere) or formalin (right hemisphere) within - minutes after euthanasia. to avoid the effects of circadian rhythms on gene expression in the brain, animals from both groups were sampled at the same time within a short period. cameras were mounted above the home pens with mothers and offspring for continuous video recording. behavior of each piglet was recorded to determine whether in utero zikv infection affects individual activity in a regular environment, i.e., in a home pen in the presence of their mother (at days after birth, the whole day), during a novel situation in a familiar environment, i.e., after removal of the mother from the home pen (the second half ( : pm - : am) of the st day), and at a later time point (at days, the whole day) as previously described [ ] . we analyzed video recordings and quantified the activity of the whole litter using sampling at -min intervals. the number of lying down, and sitting events (passive behavior), and the number of standing, walking, playing, interacting, and running events (active behavior) was registered for each litter [ ] . the number of active piglets was divided by litter size and expressed as a percentage at each moment of observations [ ] . the mixing test [ ] [ ] [ ] [ ] was performed at days after birth to determine effects of subclinical in utero zikv infection on blood cytokine levels and aggressive behavior in a stressful environment-during a social confrontation with an unfamiliar piglet. we used following criteria to form mixing pairs: (i) a piglet from the zikv group was mixed with a piglet from the control group; (ii) males were mixed with males, females with females; (iii) the piglets' body weight differences did not exceed kg. mixing pairs were individually marked with numbers on their backs, relocated to the mixing room and simultaneously released into individual unfamiliar mixing pens ( . m x . m). cameras were mounted above each pen. the room was left, and videos were recorded for min, as most fights between unfamiliar pigs occur during the first few hours after mixing [ , , ] . then immediately after the mixing test, we collected blood samples as described above. we used video recordings to score fighting activities (s video). a fight, i.e., a 'bout' of fighting, was defined as a period of time lasting at least s during which (i) two pigs showed close physical contact and (ii) five or more head knocks or bites were shown by one or both pigs. a fight was deemed to have ended when aggressive acts ceased after the retreat of one or both pigs and pigs were staying separated for s or more. for every fight, we scored the initiator, i.e., the pig that first bites or head knocks. in addition, we recorded the winner and loser of every fight. the pig that first stopped fighting, retreated, turned away from its opponent or tried to flee was considered to be the loser of the fight. from these observations, we calculated the ratio of initiated fights and the ratio of winners within the group. three blinded investigators performed analyses of behavioral data (s g table) . observations were compared (initiators: fleiss' kappa = . , winners: fleiss' kappa = . ) and averaged both in males and females [ ] . rna extraction, rt-qpcr assays, serology, bio-plex assay, cortisol assay, histology, and zikv-specific in situ hybridization were performed as previously described [ , , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the sandwich elisa (lsbio ls-f ) to quantify porcine ifn-β in blood plasma samples was performed as per the manufacturer's instructions. all details on sample testing are provided in s appendix. rna was isolated using trizol (thermo fisher scientific) lysis and extraction, and then cleaned using total rna purification kit (norgen biotek). rna was assessed on a bioanalyzer and all samples had rna integrity above . . complementary dna (cdna) libraries for sequencing were prepared using nebnext ultra ii directional rna library prep kit for illumina with rrna depletion (new england biolabs). libraries were sequenced on a novaseq as paired-end reads using the base read kit. over the whole experiment, there was an average of million paired-end reads per sample. sequencing data were mapped and quantified using the pseudo alignment method of kallisto [ ] . a sequence database of coding and non-coding transcripts was generated from ensembl sus scrofa . . the count table was assembled using tximport in r and normalized using edger then converted into a normal distribution using the voom function and differential expression was calculated using linear and bayes models as part of the r package limma. gene set enrichment was done using human annotation of gene ontology using the r function camera. confirmation of sex in placental samples was accomplished using the expression of the y chromosome and expression of the x chromosome gene kdm a as it escapes x inactivation and gives a higher signal in females. correction for sex was done directly in the linear model by setting sex as a variable in the model. before visualization corrected expression values were generated using the r function remove batcheffects for data represented in heatmaps (generated using the r package pheatmap) or principal component plots (generated using the r function princomp and the ggplot package). the set enrichment results from camera were graphed in cytoscape using the enrichment-map plugin. all networks were generated using a jaccard + overlap with a cutoff of . and a combined constant of . . sub-networks were discovered using glay cluster and annotated using the wordcloud plugin of the top words with a bonus of for word co-occurrence. gene expression data are provided in s table. an accession number for rna-seq data is prjna in ncbi bioproject. we used graphpad prism software (graphpad software inc., san diego, ca, usa) for statistical analyses. results were considered significantly different when p < . . all data were expressed as mean ± standard deviation (m ± sd). all raw animal data for this study are provided in s table. prevalence of stillborn and weak piglets was compared using pearson's chi-squared test with yates' correction for continuity. body and brain weights and body dimensions were compared with mann-whitney u-test. maternal cytokines levels between control and zikv groups and within groups were compared with bonferroni-corrected repeated measures analysis of variance (ranova). ifn-α levels in offspring blood plasma sampled at birth, , , and days after birth were compared between control and zikv groups with mann-whitney u-test. unpaired student's t-test was used to analyze cortisol in hair samples. ifn-α levels in blood plasma sampled before and after the mixing test were compared between control and zikv groups by anova with tukey's post-test. we also assessed fold change between ifn-α levels before and after the mixing test within n and p subgroups and in sex subgroups-mann-whitney u-test. in the mixing test, within each pair, we quantified a percentage of fights initiated and a percentage of fights won by control piglet or zikv piglet. the percentage of initiated and won fights-individually for males and females-were compared against the expected value ( % versus %) in control versus zikv groups with two-sided wilcoxon signed-rank test. circles represent data from individual sows. boxes represent the highest and lowest observations. a horizontal line inside the box is the mean. an asterisk ( � ) represents a statistically significant difference (p < . ) between control and zikv groups. an arrowhead (▲) represents statistically significant difference within groups, versus day . dpi-days post-inoculation, gd-gestation days. the dotted line represents loq. see raw data in s d table for genes with fdr-adjusted p < . . x and y axes represent sample identification and genes, respectively. # , # , and # -control litters; # , # , # -zikv litters. see raw data in s a table for individual gene values. vir-alpcr-represents viral loads in amniotic membranes (s b table) (c) enrichment plots of gene sets of "response to type i interferon" (fdr-adjusted p = . ), "positive regulation of type i interferon production" (fdr-adjusted p = . ), "regulation of type i interferon production" (fdr-adjusted p = . ) and "response to interferon beta" (fdr-adjusted p = . ) go processes (s b table) . (d) enrichment plot of gene sets of "response to corticosteroid" go process (fdr-adjusted p = . ) (s b table) . (e) chronic cortisol in offspring hair collected at necropsy. whiskers denote % confidence interval. see raw data in s f table for individual values. (tif) s fig. kinetics of ifn-α in the blood of zikv-affected porcine fetuses and offspring. ifnα levels (m±se) were measured in the blood plasma of zikv-affected and control porcine fetuses and offspring. data for the fetal period (at gestation days, gd) were compiled from our published study [ ] , where virus-infected and control fetuses were tested. data for gd are from study where virus-infected and control fetuses were tested (s c table) . elevated ifn-α levels at gd were significantly higher in zikv infected fetuses (p = . , mann-whitney test) [ ] . (tif) s skills attained by infants with congenital zika syndrome: pilot data from brazil zika virus infection in pregnant women in rio de janeiro preliminary report of a case-control study. the lancet infectious diseases early growth and neurologic outcomes of infants with probable congenital zika virus syndrome adverse birth outcomes associated with zika virus exposure during pregnancy in são josé do rio preto, brazil children born to mothers with rash during zika virus epidemic in brazil: first months of life delayed childhood neurodevelopment and neurosensory alterations in the second year of life in a prospective cohort of zikv-exposed children the aftermath of zika: need for long-term monitoring of exposed children zika virus infection during pregnancy and effects on early childhood development stress in puberty unmasks latent neuropathological consequences of prenatal immune activation in mice distinct alterations in motor & reward seeking behavior are dependent on the gestational age of exposure to lps-induced maternal immune activation microencephaly in fetal piglets following in utero inoculation of zika virus zika virus causes persistent infection in porcine conceptuses and may impair health in offspring persistent zika virus infection in porcine conceptuses is associated with elevated in utero cortisol levels selected physiologic compatibilities and incompatibilities between human and porcine organ systems comparison of mucus glands in the tracheobronchial tree of man and animals the immune system of the respiratory tract in pigs structural and functional annotation of the porcine immunome an in-depth comparison of the porcine, murine and human inflammasomes; lessons from the porcine genome and transcriptome advances in porcine genomics and proteomics-a toolbox for developing the pig as a model organism for molecular biomedical research advances in swine biomedical model genomics the pig: a model for human infectious diseases prenatal and postnatal growth and development of the central nervous system of the pig the pig as a model of developmental immunology differential gene expression in endometrium, endometrial lymphocytes, and trophoblasts during successful and abortive embryo implantation longitudinal field study to assess sow level risk factors associated with stillborn piglets impermeability of pig placenta for antibodies experimental study of transplacental passage of alpha interferon by two assay techniques in vivo study of interferon-alpha-secreting cells in pig foetal lymphohaematopoietic organs following in utero tgev coronavirus injection maternal immune activation alters fetal brain development through interleukin- the maternal interleukin- a pathway in mice promotes autism-like phenotypes in offspring biomarkers and immunoprofiles associated with fetal abnormalities of zikv-positive pregnancies hair cortisol as a biological marker of chronic stress: current status, future directions and unanswered questions analysis of endogenous cortisol concentrations in the hair of rhesus macaques quantitative changes of sialoadhesin and cd positive macrophages in the implantation sites and organs of porcine embryos/fetuses during gestation maternal immune activation causes age-and region-specific changes in brain cytokines in offspring throughout development immunoadolescence: neuroimmune development and adolescent behavior widespread alterations in the synaptic proteome of the adolescent cerebral cortex following prenatal immune activation in rats prenatal immune activation causes hippocampal synaptic deficits in the absence of overt microglia anomalies in-vivo rodent models for the experimental investigation of prenatal immune activation effects in neurodevelopmental brain disorders effects of stress throughout the lifespan on the brain, behaviour and cognition peri-pubertal maturation after developmental disturbance: a model for psychosis onset in the rat adolescent development, hypothalamic-pituitary-adrenal function, and programming of adult learning and memory neonatal pigs are susceptible to experimental zika virus infection individual coping characteristics, aggressiveness and fighting strategies in pigs effects of butafosfan on salivary cortisol and behavioral response to social stress in piglets repeated social defeat in female pigs does not induce neuroendocrine symptoms of depression, but behavioral adaptation cortisol administration to pregnant sows affects novelty-induced locomotion, aggressive behaviour, and blunts gender differences in their offspring human monocytes represent a competitive source of interferon-α in peripheral blood the nature of the principal type interferon-producing cells in human blood porcine peripheral blood dendritic cells and natural interferon-producing cells interferon alpha (ifn) treatment of bone marrow stroma inhibits haematopoesis ifnα activates dormant haematopoietic stem cells in vivo ifnα-mediated remodeling of endothelial cells in the bone marrow niche interferon and interferon-induced chemokine expression is associated with control of acute viremia in west nile virus-infected blood donors high levels of interferon alpha in the sera of children with dengue virus infection pathogenic potential of interferon αβ in acute influenza infection interferon-alpha, immune activation and immune dysfunction in treated hiv infection type i interferon signaling genes in recurrent major depression: increased expression detected by whole-blood rna sequencing a longitudinal study evaluating the effects of interferon-alpha therapy on cognitive and psychiatric function in adults with chronic hepatitis c a potential role for interferon-α in the pathogenesis of hiv-associated dementia seizures during alpha interferon therapy cognitive impairment in patients with chronic hepatitis treated with interferon alpha (ifnα): results from a prospective study letter to the editor: further evidence of neurological sequelae associated with interferon therapy in the pediatric population interferon alpha (ifnα) and psychiatric syndromes: a review prefrontal cortical hypometabolism during low-dose interferon alpha treatment neuropsychiatric adverse effects of interferonalpha: recognition and management treatment of subclinical congenital toxoplasmosis by sulfadiazine and pyrimethamine continuously during year: apropos of cases auditory and visual defects resulting from symptomatic and subclinical congenital cytomegaloviral and toxoplasma infections relationship between the production of interferon-alpha/ beta and interferon-gamma during acute toxoplasmosis human cytomegalovirus induces the interferon response via the dna sensor zbp subclinical transplacental parvovirus b infection: an increased fetal risk? subclinical congenital rubella infection associated with maternal rubella vaccination in early pregnancy asymptomatic herpes simplex virus type (hsv- ) infection among pregnant women in turkey protective efficacy of multiple vaccine platforms against zika virus challenge in rhesus monkeys type i interferon production during herpes simplex virus infection is controlled by cell-type-specific viral recognition through toll-like receptor , the mitochondrial antiviral signaling protein pathway, and novel recognition systems viral infection of the placenta leads to fetal inflammation and sensitization to bacterial products predisposing to preterm labor epidemiology and burden of malaria in pregnancy placental inflammatory response to zika virus may affect fetal brain development boys live dangerously in the womb delayed growth, motor function and learning in preterm pigs during early postnatal life the pig as a model animal for studying cognition and neurobehavioral disorders. current topics in behavioral neurosciences social support modulates stress-related gene expression in various brain regions of piglets mapping the amphetamine-evoked dopamine release in the brain of the gö ttingen minipig prepulse inhibition of the acoustic startle reflex in pigs and its disruption by d-amphetamine the d-amphetamine-treated gö ttingen miniature pig: an animal model for assessing behavioral effects of antipsychotics. psychopharmacology (berl) phylogeny of zika virus in western hemisphere unusual outcome of in utero infection and subsequent postnatal super-infection with different pcv b strains influence of resources on pig aggression and dominance effects of straw and unfamiliarity on fighting between newly mixed growing pigs measuring nominal scale agreement among many raters detection of zika virus by sybr green one-step realtime rt-pcr one-step rt-pcr for detection of zika virus acute and chronic neurological consequences of early-life zika virus infection in mice peripheral regulation of stress and fear responses in pigs from tail-biting pens intermittent suckling causes a transient increase in cortisol that does not appear to compromise selected measures of pigletwelfare and stress effects of oxytocin administration on the response of piglets to weaning hair cortisol concentration as a noninvasive measure of long-term stress in free-ranging grizzly bears (ursus arctos): considerations with implications for other wildlife near-optimal probabilistic rna-seq quantification we thank vido-intervac animal care technicians and veterinarians for the help with animal experiments. zikv was provided by the division of vector-borne diseases, centers for disease control and prevention (fort collins, colorado, usa). jan erickson made drawings in fig b and s fig. published as vido-intervac manuscript series number . key: cord- -lpf lpky authors: chen, yongwen; wu, shengxi; guo, guoning; fei, lei; guo, sheng; yang, chengying; fu, xiaolan; wu, yuzhang title: programmed death (pd)- -deficient mice are extremely sensitive to murine hepatitis virus strain- (mhv- ) infection date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: lpf lpky the inhibitory receptor programmed death- (pd- ) has the capacity to maintain peripheral tolerance and limit immunopathological damage; however, its precise role in fulminant viral hepatitis (fh) has yet to be described. here, we investigated the functional mechanisms of pd- as related to fh pathogenesis induced by the murine hepatitis virus strain- (mhv- ). high levels of pd- -positive cd (+), cd (+) t cells, nk cells and macrophages were observed in liver, spleen, lymph node and thymus tissues following mhv- infection. pd- -deficient mice exhibited significantly higher expression of the effector molecule which initiates fibrinogen deposition, fibrinogen-like protein (fgl ), than did their wild-type (wt) littermates. as a result, more severe tissue damage was produced and mortality rates were higher. fluorescence double-staining revealed that fgl and pd- were not co-expressed on the same cells, while quantitative rt-pcr demonstrated that higher levels of ifn-γ and tnf-α mrna transcription occurred in pd- -deficient mice in response to mhv- infection. conversely, in vivo blockade of ifn-γ and tnf-α led to efficient inhibition of fgl expression, greatly attenuated the development of tissue lesions, and ultimately reduced mortality. thus, the up-regulation of fgl in pd- -deficient mice was determined to be mediated by ifn-γ and tnf-α. taken together, our results suggest that pd- signaling plays an essential role in decreasing the immunopathological damage induced by mhv- and that manipulation of this signal might be a useful strategy for fh immunotherapy. although liver transplantation has emerged as an effective therapeutic approach for treating fulminant virus hepatitis (fh), mortality rates associated with fh remain very high worldwide [ ] . the recent development of a mouse fh model, based upon infection with the murine hepatitis virus strain- (mhv- ), has provided insights into mechanisms underlying the disease pathogenesis and resulted in some novel treatment strategies [ ] . mhv- is a single-stranded, positive-sense rna virus that belongs to the coronaviridae family. in inbred laboratory mice, the virus produces strain-dependent disease profiles that depend on the infection route, age, genetic background, and immune status of the host. for example, balb/c, c bl/ and dba/ mice develop acute fulminant hepatitis, while c h mice develop a mild chronic disease and mice of the a strain exhibit no evidence of hepatitis [ , ] . in contrast to the resistant a strain mice, fh induced by mhv- in susceptible mice is characterized by the presence of sinusoidal thrombosis and hepatocellular necrosis [ , ] . these pathological findings occur concomitantly with expression of fibrinogen-like protein (fgl ), a virus-induced procoagulant molecule in the sinusoidal lining cells in the liver. fgl has the capacity to promote fibrinogen deposition and subsequently directly induce the coagulation cascades by the expression of procoagulant activity (pca) [ ] . thus, up-regulation of fgl is an essential component of the lethal effects of mhv- -induced fh. programmed death (pd)- is an inhibitory receptor expressed on activated t cells, b cells and myeloid cells. pd- -deficient mice (pdcd / ) develop various spontaneous autoimmune diseases, including glomerulonephritis and dilated cardiomyopathy, indicating that this receptor plays a critical role in maintenance of peripheral tolerance [ ] . pd-l (b -h ) and pd-l (b -dc), two immunoregulatory molecules belonging to the b superfamily, were identified as ligands for pd- , engagement of pd- with its ligands mediates negative signaling events via recruitment of phosphatases, such as shp- , and dephosphorylation of some effector molecules involved in downstream t cell receptor (tcr) signaling [ , ] . pd- signaling has also been shown to modulate the balance between antimicrobial immune defense and immune-mediated tissue damage. for example, pd- -deficient mice develop more severe hepatocellular injury than their wild-type (wt) littermates in response to adenovirus infection [ ] . in a herpes simplex virus (hsv) stromal keratitis mouse model, blockade of pd- signaling led to increased hsv- -specific effector cd + t cell expansion, ifn-c production, and exacerbated keratitis [ ] . a functionallysignificant high level of pd- expression has been found to be maintained by exhausted cd + t cells in mice chronically infected with lymphocytic choriomeningitis virus (lcmv), in primates exposed to simian immunodeficiency virus (siv), and in humans suffering from infection with human immunodeficiency virus (hiv), hepatitis b or c virus (hbv or hcv), or human tlymphotropic virus (htlv). however, blockade of the pd- /pd-ls pathway efficiently restored the virus-specific effector functions of the exhausted t cells, and lead to substantially reduced in viral load [ , , , , ] . the pd- signal is also known to play a key role in the chronicity of infections with bacteria (helicobacter pylori and schistosoma mansoni) [ , ] , pathogenic fungus (histoplasma capsulatum) [ ] , and parasitic worms (taenia crassiceps) [ ] . it appears that a number of pathogenic microorganisms exploit the pd- signal in order to evade host immune responses and to establish persistent infection. although the influence of pd- signal activity has been studied in several infection models, there are no data available concerning the role of this pathway in fh. to this end, we used the mhv- induced mouse fh model to demonstrate that pd- signaling acts to limit the immunopathological damage during disease progression. furthermore, our findings suggested that enhanced pd- signaling might represent a useful immunotherapeutic strategy for treating fh. pd- expression has been previously described as being induced on specific cell subsets in response to viral or bacterial infection [ ] . thus, we first determined the status of pd- expression at h after mhv- infection ( pfu) by immunohistochemical techniques. pd- -positive cells were observed in tissues from the thymus, spleen, lymph nodes and liver. cellular expression was localized to the cell membrane and in the cytoplasma while was completely absent from the nuclear compartment. pd- -positive cells were distributed throughout the medulla and cortex of the thymus and lymph nodes. in the spleen, pd- -positive cells were restricted to the germinal center under normal conditions, but extended to the red pulp after infection. in infected liver, more pd- -positive cells were present in the portal and parenchymal areas, as opposed to the relatively low presence of pd- -positive cells in only the portal area in phosphate-buffered saline (pbs) treated-mice (fig. a) . the amount of pd- -positive cells in the different organs of infected and control mice were counted and compared, results showed that the number of positive cells was significantly higher in infected mice (fig. b) . furthermore, facs analysis revealed that pd- expression was enhanced on multiple subsets of immune cells, including the cd + and cd + t cells, nk . + nk cells and cd + macrophages (fig. c) . pd- positive cells were also observed in the lung, heart and kidney, however, the numbers of pd- positive cells in these tissues did not significantly increase in response to mhv- infection (fig. s ). to investigate the potential role of pd- signaling in regulating fh tissue pathology, organs from mhv- infected pd- -deficient (pd- ko) and wt mice were assessed for morphological differences. small and discrete foci of necrosis with sparse polymorphonuclear leukocyte infiltration were observed in liver tissues from pd- -deficient mice after h of infection. in contrast, wt mice exhibited normal liver architecture at this time point. slight liver damage became apparent in wt mice after h of infection, meanwhile, the damaged areas of pd- -deficient mice had enlarged and confluent necrosis had become evident. by h of infection, the damaged region in pd- -deficient mice had extended throughout the entire liver, while wt mice suffered much less damage and up to % of their liver tissue remained normal at this time point ( fig. a) . likewise, higher levels of alanine aminotransferase (alt) and aspartate aminotransferase (ast) were observed in serum from pd- -deficient mice after h of infection (fig. b ). more interestingly, pd- -deficient thymus, spleen and lymph node tissues infected with mhv- for h exhibited severely disrupted architecture, loss of cellularity, and the presence of substantial amounts of karyorrhectic/apoptotic cell bodies. the histology of these organs from infected wt mice at h was relatively normal (fig. c ). in conjunction with the apparent tissue necrosis, higher levels of cell apoptosis were also evidenced in the organs from pd- -deficient mice by tunel staining (fig. d ). the architecture of other organs, including the heart, kidney and lung was relatively normal and only rare apoptosis events were observed in these tissues after infection (fig. s ). in all, these results demonstrated that pd- deficiency led to enhanced pathological damage by mhv- in the liver, spleen, lymph node and thymus, where higher levels of pd- -positive cells were found after infection. the earlier and increased organ damage suffered by pd- deficient mice infected with mhv- instigated our monitoring of the mortality rates of pd- -deficient mice and their similarlyinfected ( pfu) wt littermates. as shown in fig. , all of the pd- -deficient mice died within four days after infection, while the principal characteristic of fulminant viral hepatitis (fh) induced by the murine hepatitis virus strain- (mhv- ) is severe hepatocellular necrosis, which is mediated by the fibrinogen-like protein (fgl ), a molecule that has the capacity to promote fibrinogen deposition and activate the coagulation cascades. here, we report that mhv- infection of program death- (pd- )-deficient mice results in tissue damage throughout multiple organs, including the liver, spleen, thymus and lymph nodes. the liver damage, in particular, occurred earlier and was more severe in pd- -deficient mice than in their wild type (wt) littermates. further investigation determined that mhv- infection was associated with high levels of ifn-c and tnf-a in the damaged organs of pd- -deficient mice. conversely, intraperitoneal injection of a combination of anti-ifn-c and anti-tnf-a blocking mabs led to inhibition of fgl expression, greatly attenuated tissue lesions and reduced mortality. our results demonstrate that pd- signaling controls immunopathological damage following mhv- infection, indicating that manipulation of the pd- signal might represent a useful strategy for fh immunotherapy. % of the wt mice survived up to the end of the -day survey period (p = . ). these data indicated that pd- is likely a critical factor that controls mhv- -mediated tissue damage and mortality. to understand the mechanisms of pd- deficiency-mediated tissue damage and mortality, we performed a comparative genome-wide microarray analysis (nimblegen) of genes expressed in liver tissues of pd- -deficient and wt mice after h of mhv- infection. the most notable finding was pronounced upregulation ( . -fold) in the liver of pd- -deficient mice of the fgl transcripts (fig. a) , the protein product of which has been demonstrated to induce lethality of mhv- -induced fh [ ] . in addition, the enhanced fgl expression was confirmed by quantitative (q)pcr, results revealed a . -fold and . -fold higher level was present in liver from wt and pd- -deficient mice, respectively, after h of mhv- infection, as compared to their uninfected controls. moreover, its level in pd- -deficient liver was . -fold higher than that in the wt group at this time point (fig. b) . immunohistochemistry was used to show that fgl -positive cells were present in necrotic liver tissues in pd- deficient mice at h after mhv- injection. the protein expression was found to be enhanced rapidly upon infection, and the highest level occurred at h post-infection. however, occasional fgl -positive cells were detected in the livers of wt mice at h post-mhv- infection and these cells were also present, and slightly enhanced in number, at both the h and h time point (fig. c ). western-blot was used to verify the higher fgl protein level in the livers of pd- -deficient mice, as compared to wt littermates after h of infection (fig. d ). fgl has the capacity to induce fibrinogen deposition, which then activates the coagulation cascades and finally induces procoagulant activity. therefore, the expression of fgl and fibrinogen deposition in damaged liver tissues was measured. dual fluorescent staining evidenced that substantial fibrinogen deposition occurred in the fgl -positive damaged liver tissue (fig. e ). likewise, the level of fibrinogen deposition was more robust in livers from pd- -deficient mice that in livers from wt littermates, at both the h and h time points (fig. f) . to determine whether fgl -mediated pca activity was also involved in inducing damage in the other organs of pd- -deficient mice, the expression of fgl was analyzed in the thymus, spleen and lymph nodes. immunohistochemistry evidenced that fgl positive cells were also present in these organs. in thymus and lymph nodes, fgl -positive cells were detected in both the medulla and cortex. in spleen, however, the positive cells were only found in the red pulp. again, the expression of fgl appeared to be restricted to the cell membrane and cytoplasma. the distribution of fgl -positive cells in pd- -deficient mice had not changed after h of mhv- infection, but the number of positive cells in the examined organs was enhanced significantly and the levels of expression were much stronger (fig. a ). in addition, the transcription of fgl in the spleen of pd- -deficient mice was also significantly increased in response to infection (fig. b) . meanwhile, higher levels of fibrinogen deposition were found in the spleen and lymph node tissues of pd- -deficient mice (fig. c) . moreover, the level of fgl present in serum, as measured by elisa, was found to increase rapidly after infection, and the level in pd- -deficient mice was significantly higher than that in wt littermates (fig. d ). to clarify the source of fgl , fluorescent dual staining was performed on spleen tissues and results demonstrated that fgl was principally associated with cd c-positive dendritic cells (dcs), cd -positive macrophages and cd -positive endothelial cells (fig. e ). all of these results indicated that the absence of pd- signaling can result in enhanced fgl expression, consequently inducing stronger fibrinogen deposition and more severe tissue necrosis in pd- deficient mice following mhv- infection. we further examined whether fgl secretion was regulated by pd- directly or indirectly. fgl /pd- dual fluorescent staining was performed and results indicated that fgl and pd- were not co-expressed on the same cells in the liver, thymus, spleen or lymph nodes (fig. a) . previous studies have shown that the secretion of fgl can be triggered by the pro-inflammatory factors ifn-c and tnf-a [ , ] . on the other hand, the production of ifn-c and tnf-a by activated t cells, nk cells and macrophages can be inhibited by the pd- signal [ ] . therefore, we compared the status of ifnc and tnf-a in pd- -deficient and wt mice in response to mhv- infection. qpcr revealed that the transcription of the ifn-c gene in liver was significantly higher in pd- -deficient mice than in wt mice at h post-mhv- infection (fig. b) . in pd- -deficient spleen, the transcription of both ifn-c and tnf-a was found to be rapidly enhanced upon mhv- exposure (fig. c ). facs analysis indicated that ifn-c secretion from nk cells, but not from cd + t cells, in the liver was much higher in pd- deficient mice at h after mhv- infection (fig. d ). the fact that ifn-c and tnf-a both have the capacity to initiate fgl expression may explain why higher fgl expression was observed in the pd- -deficient mice. to further demonstrate that ifn-c and tnf-a were responsible for the observed fgl up-regulation in mhv- infected pd- deficient mice, pd- -deficient mice were infected with mhv- and simultaneously treated with a combination injection of anti-ifn-c and anti-tnf-a blocking mabs. the expression of fgl was measured by qpcr and protein detected by immunohistochemistry. the transcription of fgl mrna (fig. a ) and its protein levels (fig. b) were completely inhibited by h after injection of anti-ifn-c and anti-tnf-a mabs, as compared to the control rat igg isotype antibodies-treated group. moreover, the tissue necrosis (fig. c ) and liver damage (as indicated by alt and ast levels) (fig. d ) in pd- -deficient mice were also significantly reduced, thus the mhv- -mediated mortality rates were decreased as well (fig. e ). the pd- signaling is best known for its ability to inhibit or dampen the immune response. most of the evidence for this function, however, comes from models of tolerance or chronic infections [ , , , ] . although some studies have indicated that this signal might also participate in regulating acute infections [ , , , ] , its functions in this disease condition are much less clear. here, we used a mouse fh model mediated by mhv- infection to describe the effects of pd- in this disease process. firstly, pd- was found to be significantly up-regulated on t cells, macrophages and nk cells within the thymus, spleen, lymph nodes and liver in response to mhv- infection. to determine the exact role of pd- in the pathogenesis of fh, pd- deficient mice were used to establish an infection model. interestingly, mhv- -induced liver damage in pd- -deficient mice occurred rapidly and the lesion area was much larger than in their wt littermates. we then extended our investigation to the thymus, spleen and lymph nodes, where increased pd- -positive cells were observed post-infection. surprisingly, severe tissue necrosis and substantial apoptosis was observed in these organs of pd- -deficient mice at h after mhv- infection. in contrast, these organs from wt mice exhibited relatively normal histology, a finding in agreement with previously reported results [ ] . taken together, these results suggested that pd- deficiency promoted expansion of the pathological damage from the liver to the lymph organs, including the spleen, lymph node and thymus in this fh model, thereafter, the absence of pd- was associated with higher mortality rates in response to mhv- infection. murine fh induced by mhv- is a recognized and validated model for studying host resistance/susceptibility to human hepatitis virus, and several studies have shown that balb/c or c bl/ mice have an innate susceptibility to the infection [ , ] . fgl has been proposed as a critical mediating factor of lethality in the mhv- -induced fh mice due to the fact that it has the capacity to induce fibrinogen deposition, which in turn activates the coagulation cascades and induces procoagulant activity [ ] . to clarify whether the tissue necrosis we observed in pd- -deficient mice following infection was also mediated by fgl , the expression of fgl was analyzed. results showed that the expression of fgl was principally associated with cd -positive endothelial cells, cd -positive macrophages and cd c-positive dcs. surprisingly, significantly higher levels of fgl were observed after infection in all of pd- -deficient organs, including the liver, thymus, spleen, lymph nodes, and serum than that in those from wt littermates. in addition, increased fibrinogen deposition was observed in the organs of pd- -deficient mice. although we currently have no direct data to evidence that fgl directly mediates the mortality of our pd- -deficient mice, data from other researchers have clearly shown that fgl promoted mouse mortality in response to mhv- infection [ , , , ] . considering this, our results strongly indicate that the mortality of pd- -deficient mice post-mhv- infection is due to the higher level of fgl secretion and increased fibrinogen deposition. indeed, it has been reported that both fgl and pd- are expressed on t cells, macrophages, and dcs, and that targeted deletion of fgl or pd- leads to impaired t cell activity, and these events are related to the development of autoimmune diseases [ , , ] . we here also observed pd- expression as being enhanced on t cells (both cd + and cd + t cells). it was reasonable to propose that the expression of fgl may have been directly regulated by pd- signals. unexpectedly, our fgl /pd- dual staining showed that pd- -positive cells in the liver, thymus, spleen and lymph nodes did not co-express fgl , indicating that the expression of fgl was not directly regulated by pd- . on the other hand, the expression of fgl is believed to be induced by ifn-c and tnf-a [ , ] , while pd- signaling has the capacity to inhibit ifn-c and tnf-a secretion from pd- -positive immune cells [ ] . therefore, we evaluated and compared the status of ifn-c and tnf-a in both pd- -deficient and wt mice. definitively, the transcription of ifn-c and tnf-a genes was rapidly enhanced post-mhv- infection in pd- -deficient mice, as compared to wt controls. in particular, a higher level of ifn-c was observed in nk cells but not in cd + t cells of pd- deficient liver post-mhv- infection, indicating that the pd- signal can inhibit ifn-c secretion from nk cells under such condition. conversely, injection of a the combination of anti-ifnc and anti-tnf-a blocking mabs was able to successfully inhibit fgl mrna transcription and protein expression, resulting in reduced tissue damage and significantly protecting against mhv- -mediated mortality in these mice. these results demonstrated that up-regulation of fgl in pd- -deficient mice after mhv- infection was controlled, at least partially, by ifn-c and tnf-a. recently, the secretion of fgl from naturally occurring cd + foxp + regulatory t cells (tregs) was demonstrated and it was reported that deficiency of treg-produced fgl resulted in increased effector t cell proliferation [ ] . more interestingly, levy and colleagues showed that the frequency of fgl + tregs was higher in lymphoid tissues of mhv- infected mice, and treatment with fgl -specific antibodies reversed mhv- induced liver injury and mortality in vivo. these findings demonstrated that fgl is an important effector cytokine of tregs that contributes to mhv- -induced fh [ ] . pd- signaling has also been described as participating in regulation of treg differentiation and function [ , ] . in our study, we also analyzed the status of foxp + cells in both pd- -deficient and wt controls. however, the number of foxp -positive cells in the liver, spleen, lymph node or thymus was not significantly different between pd- -deficient mice and their wt littermates after h of mhv- infection (fig. s ) . therefore, foxp + cells are unlikely to be involved in the mortality of pd- -deficient mice. however, the functional status of these tregs (for example, the level of fgl secretion) in pd- -deficienct mice requires further investigation, and such studies are in progress in our lab. in conclusion, we have determined that pd- signaling can limit the immunopathological damage induced by mhv- infection in a mouse fh model. our results suggest that enhancing the pd- signal by an immunotherapeutic approach might be a useful treatment for fh. all experiments were approved by and conducted in accordance with the guidelines of the animal care and use committee of the third military medical university. all efforts were made to minimize animals' suffering. mice pd- -ko-n (strain: balb/cj) mice were kindly provided by prof. t. honjo (department of immunology and genomic medicine, kyoto university, japan). the wt control mice were purchased from the animal center of beijing university school of medicine. all mice were maintained in micro-isolator cages and housed in the animal colony at the animal center, third military medical university, standard laboratory chow diet and water was supplied ad libitum. mice were used in experimental analysis at age of six weeks and at an average weight of g (range: , g). mhv- was kindly provided by prof. q. ning (institute of infectious disease, tongji hospital of tongji medical college, wuhan, china). the virus was plaque-purified and then expanded in murine l cells. virus-containing supernatants were collected and stored at - uc until use. mice were intraperitoneally (i.p.) injected with pfu/mouse in a total volume of ml. in some experiments, pd- -deficient mice were infected with mhv- ( pfu) and simultaneously treated with a infection was analyzed by immunohistochemistry. blue color indicates nuclear dapi staining. scale bar = mm. magnification . ns: not significant different. *p, . , ** p, . . doi: . /journal.ppat. .g combination injection of anti-ifn-c ( mg/mouse per day, clone: r - a , ebioscience, san diego, ca, usa) and anti-tnf-a ( mg/mouse per day, clone: mp -xt , ebioscience) mabs, tissues were isolated for hematoxylin and eosin (h&e) staining to detect damage, and for fgl mrna transcription measured by qpcr (see below). serum alt and ast levels were measured by an au automatic biochemistryanalyzer (olympus, japan). in order to monitor the mortality, anti-ifn-c and anti-tnf-a blocking mabs or rat igg control mabs were injected everyday for a total of days. paraffin-embedded tissue blocks were cut into mm slices which were mounted on polylysine-charged glass slides. endogenous peroxidase activity was blocked by exposure to . % h o for min. antigen retrieval was performed in a citrate buffer the expression of pd- on immune cells (cd , cd , nk and macrophages) from different organs was assessed by flow cytometry (facsaria cytometer; becton dickinson, germany). briefly, cell suspensions of liver, spleen, blood and thymus tissues were washed and resuspended in pbs. cells were then incubated for min at room-temperature in the dark using primary antibodies (pe-pd- , fitc-cd , fitc-cd , fitc-nk . and fitc-cd . ebioscience). to analyze the source of ifn-c in the liver, pd- -deficient and wt mice were treated with mhv- ( pfu). after h, liver tissues were isolated and mechanically homogenized, lymphocytes were collected thereafter. cells were then treated with brefeldin a solution (bfa) for h, and fitc-nk . , fitc-cd or pe-ifn-c mabs (ebioscience) were added and the solution incubated for an additional h. for each analysis, cells were evaluated. flow cytometric data were analyzed with cellquest pro software. the microarray experiment was performed under contact by kangcheng co. ltd. (shanghai, china). briefly, total rna was isolated by trizol from liver tissue of pd- -deficient and wt mice treated with pfu mhv- for h. rna concentration was measured on the nd- spectrophotometer (nanodrop, wilmington, de, usa) and quality evaluated by denaturing gel electrophoresis. samples were then amplified and labeled using a nimblegen one-color dna labeling kit and hybridized using the nimblegen hybridization system (roche applied science, shanghai, china). after hybridization and washing, the processed slides were scanned by the axon genepix b microarray scanner. three independent experiments were performed, and for each test and control sample, two hybridizations were carried out by a reverse fluorescent strategy. only genes whose alteration tendency was concordant between both microarray assays were selected as differentially expressed genes. total rna from the liver and spleen of wt and pd- -deficient mice was isolated by trizol (invitrogen, carlsbad, ca, usa), according to the manufacturer's instructions. rna samples were quantitated by measurement of optical density at nm. total mrna ( mg) was reverse-transcribed to cdna using the revertaid h minus first strand cdna synthesis kit (fermentas china, shenzhen city, china), in accordance with the manufacturer's instructions. qpcr was performed to quantitatively analyze the gene transcription levels of fgl , ifn-c and tnf-a genes. the primers for fgl were: sense -tggacaacaaagtgg-caaatct- and anti-sense -tggaacacttgccatc-caaa- . the primers for ifn-c were: sense -tcaagtgg-catagatgtggaag- , and anti-sense -cgcttatg-ttgttgctgatgg- . the primers for tnf-a were: sense -cacgctcttctgtctactgaac- and anti-sense -atctgagtgtgagggtctgg- . the primers for b-actin (internal control) were: sense -cactatcggcaatgag-cggttcc- and anti-sense -cagcactgtgttggca tagaggtc- . the qpcr was performed at uc for s followed by cycles of uc for s, uc for s, and uc for s. the specificity of pcr product was examined by a dissociation curve, and results were analyzed by the ddct method [ ] . the expression of fgl in liver from mhv- infected ( h) pd- -deficient mice or their wt littermates was determined by western-blot; the protocol has been described previously [ ] . the serum fgl level from mice infected with or without mhv- was detected by using the mouse fgl elisa kit (cat: e mu; uscn life science inc., wuhan, china) and following the manufacturer's instructions. all results shown are representative of at least three separate experiments. unpaired student's t-test (two-tailed) or the mann-whitney test was used for comparison of two groups where appropriate. kaplan meier curve with log-rank test (graphpad prism . software) was used to analyze the mortality rate. pvalue , . was considered as statistically significant. its protein expression in the liver, spleen and lymph node from pd- -deficient mice after h of mhv- infection in the presence of ifn-c and tnf-a mabs or rat igg isotype control antibodies was detected by qpcr and immunohistochemistry, respectively. (c) the ifn-c and tnf-a mabs treatment resulted in decreased damage to the liver, spleen, lymph node and thymus after h of mhv- infection. (d) reduced fgl level by ifn-c and tnf-a mabs treatment resulted in reduced liver damage (indicated by ast and alt levels). (e) pd- -deficient mice were infected with mhv- ( pfu) and simultaneously treated with ifn-c and tnf-a blocking mabs (n = ) or rat igg control (n = ), the survival rate was monitored for a total of days. p = . , . was considered significantly different. one representative of three experiments that yielded similar results is shown. magnification . ns: not significantly different. *p, . and **p, . . n = /group. doi: . /journal.ppat. .g figure s the number of foxp -positive cells was not changed significantly in pd- -deficient mice after mhv- infection. foxp -positive cells in the liver, thymus, spleen, and lymph nodes between pd- -deficient and wt mice at h after mhv- infection were detected by immunofluorescence staining (left). statistical analysis of the number of foxp -positive cells in the indicated organs (right). blue color indicates nuclear dapi staining. scale bar = mm. ns: not significantly different. found at: doi: . /journal.ppat. .s ( . mb tif) viral hepatitis in the liver transplant recipient pattern of disease after murine hepatitis virus strain infection correlates with macrophage activation and not viral replication induction of monocyte procoagulant activity by murine hepatitis virus type parallels disease susceptibility in mice acquired immunity of a/j mice to mouse hepatitis virus infection: dependence on interferon gamma synthesis and macrophage sensitivity to interferon gamma the fgl / fibroleukin prothrombinase contributes to immunologically mediated thrombosis in experimental and human viral hepatitis pd- and its ligands in tolerance and immunity pd- inhibits t-cell receptor induced phosphorylation of the zap /cd zeta signalosome and downstream signaling to pkctheta shp- and shp- associate with immunoreceptor tyrosine-based switch motif of programmed death upon primary human t cell stimulation, but only receptor ligation prevents t cell activation pd- inhibits antiviral immunity at the effector phase in the liver cd ) inhibits the development of herpetic stromal keratitis (hsk restoring function in exhausted cd t cells during chronic viral infection enhancing sivspecific immunity in vivo by pd- blockade upregulation of pd- expression on hiv-specific cd + t cells leads to reversible immune dysfunction dysfunction and functional restoration of hcv-specific cd responses in chronic hepatitis c virus infection programmed death- -induced interleukin- production by monocytes impairs cd (+) t cell activation during hiv infection expression of b -h on gastric epithelial cells: its potential role in regulating t cells during helicobacter pylori infection schistosoma mansoni worms induce anergy of t cells via selective up-regulation of programmed death ligand on macrophages the pd- /pd-l costimulatory pathway critically affects host resistance to the pathogenic fungus histoplasma capsulatum role of the programmed death- pathway in the suppressive activity of alternatively activated macrophages in experimental cysticercosis novel strategies to eliminate persistent viral infections cytokineinduced hepatic apoptosis is dependent on fgl /fibroleukin: the role of sp / sp and stat /pu. composite cis elements intact type immunity and immune-associated coagulative responses in mice lacking ifn gamma-inducible fibrinogen-like protein role of pd- in regulating acute infections pd- expression by macrophages plays a pathologic role in altering microbial clearance and the innate inflammatory response to sepsis recall responses by helpless memory cd + t cells are restricted by the upregulation of pd- pd- on dendritic cells impedes innate immunity against bacterial infection a virus related to that causing hepatitis in mice (mhv) expression of the fgl and its protein product (prothrombinase) in tissues during murine hepatitis virus strain- (mhv- ) infection fulminant hepatic failure in murine hepatitis virus strain infection: tissue-specific expression of a novel fgl prothrombinase the novel cd +cd + regulatory t cell effector molecule fibrinogen-like protein contributes to the outcome of murine fulminant viral hepatitis characterization of human fibroleukin, a fibrinogen-like protein secreted by t lymphocytes targeted deletion of fgl leads to impaired regulatory t cell activity and development of autoimmune glomerulonephritis pd-l negatively regulates cd +cd +foxp + tregs by limiting stat- phosphorylation in patients chronically infected with hcv pd-l regulates the development, maintenance, and function of induced regulatory t cells analysis of relative gene expression data using real-time quantitative pcr and the (-delta delta c(t)) method triptolide inhibits b -h expression on proinflammatory factor activated renal tubular epithelial cells by decreasing nf-kappab transcription we thanks prof. t honjo kindly give us the pd- ko mice. mhv- virus was provided by prof. q ning and she also gave the research some invaluable suggestions. conceived and designed the experiments: yc cy. performed the experiments: yc sw gg lf sg xf. analyzed the data: yc cy yw. wrote the paper: yc yw. key: cord- -qn xp v authors: striker, rob; mehle, andrew title: inhibitors of peptidyl proline isomerases as antivirals in hepatitis c and other viruses date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: qn xp v nan viruses have small genomes with limited coding capacity. a common strategy by which viral genomes maximize their coding capacity is to express multifunctional proteins that promiscuously interact with various cellular partners to perform an array of essential functions. these interactions often involve flexible, and in some cases intrinsically disordered, viral domains or entire proteins that assume distinct conformations only upon binding cellular partners (see review, [ ] ). viral coding capacity is further enhanced by relying on host factors and protein folding machinery to access different conformations and functions. these disordered peptide regions can be computationally recognized by features such as glycine, serine, and proline residues in contexts that are not conducive to b-strands or a-helices (reviewed by [ ] ). bioinformatic analysis was used to predict the rigidity of proteins encoded by nearly , genomes from archaea, bacteria, eukaryotes, and viruses. this analysis suggests that almost all genomes with greater than % of their encoded residues in a predicted disordered state are viral genomes [ ] . thus, disordered proteins are enriched in the viral proteome and are common features to a large number of viruses. flexible viral proteins and/or domains interact with the cellular folding machinery, including proline isomerases. while proline is traditionally thought of as being a rigid amino acid that can ''kink'' the polypeptide chain, prolines can slowly rotate between two energetically similar configurations, cis or trans. this rotation is only fast enough to be physiologically relevant when facilitated by proline isomerases (rotamases) such as mammalian cyclophilins [ ] . at least four structurally distinct classes of cellular proline isomerases exist in bacteria and eukaryotes, and some viruses encode their own proline isomerase [ , ] . identification of the host isomerases exploited by viruses and the viral proteins that require them to perform essential viral functions for replication in culture, or more importantly, in animals, presents an obvious antiviral strategy. whether or not inhibition of host proline isomerases could be an antiviral strategy for hepatitis c virus (hcv) was a subject of debate for several years. cyclophilins are the most-studied proline isomerases and likely the least discriminating in terms of substrate choice. cyclophilins were discovered as a target of the immunosuppressive drug cyclosporine. cyclosporine, as well as immunosuppressant tacrolimus, both inhibit the adaptive immune response by inhibiting distinct classes of peptidyl proline isomerases; cyclosporine inhibits cyclophilins while tacrolimus inhibits fk -binding proteins (fkbps) [ ] . a decade ago two groups showed that self-replicating hcv rnas (replicons) are dependent upon hcv nonstructural proteins and are inhibited by cyclosporine, but not tacrolimus [ , ] . this led to the proposal that hcv replication requires cyclophilins, but not fkbps. this conflicted with observations by many clinical scientists. from until around , when tacrolimus began supplanting cyclosporine, hcv-positive transplant patients received cyclosporine to prevent organ rejection, but this treatment did not simultaneously cure their hcv infection. then and now, hcv is not only the most common reason for a liver transplant but also a common reason for needing a second liver transplant because immunosuppression (or the associated higher viral load that may arise from increased replication in the transplanted liver) clearly accelerated hcv-mediated disease in the liver graft. thus, defining a role for proline isomerases during hcv infection in patients was confounded by the fact that any antiviral benefit from cyclosporine must overwhelm its immunosuppressive effect. replicon data provided critical evidence for the role of cyclophilins during hcv infection in culture, yet cyclosporine and tacrolimus appeared to be ''equally bad'' for hcv-infected solid organ transplant patients; thus, controversy persisted [ ] . there is now no longer a question that cyclophilin a plays a crucial role during hcv replication and cyclophilin inhibitors possess potent anti-hcv activity. using an innovative mousehuman chimera model, it was shown that hcv replicates to significantly lower levels in cyclophilin a-deficient animals than in mice with cyclophilin a [ ] . several cyclophilin inhibitors that retain anti-hcv activity but do not have the immunosuppressive properties of cyclosporine and tacrolimus have also been studied. these compounds maintain their antiviral activity, and at least one has reached late phase iii trials for hcv [ ] . whether there is a role for cyclophilin inhibitors in future cures of hcv remains unclear. multiple inhibitors that target viral enzymes and promote viral clearance in a high percentage of patients are being adopted [ ] . still, cyclophilin inhibitors may ultimately prove clinically useful for viral infections that resist new treatment regimes or useful when used in combination with existing therapies. hcv encodes only ten proteins, including structural and nonstructural (ns) proteins. cyclophilin inhibitors that reduce viral replication also block interactions between cyclophilin a and ns a, suggesting that this association is important during the viral life cycle and might be the relevant target of the antiviral activity of cyclosporine [ ] [ ] [ ] . an interaction between cyclophilin b and the viral polymerase ns b was also reported [ ] . selection of subgenomic replicons for cyclosporine resistance created mutations in both ns a and ns b [ ] . mutating specific ns b residues in isolation conferred low level or no resistance to cyclosporine [ ] , whereas mutations in ns a, the most prolinerich hcv protein, conferred the highest levels of resistance [ ] . these data suggest that ns a is the most important substrate of cyclophilins for hcv replication. additional work by multiple groups showed that cyclophilin a is the major, if not only, cyclophilin to play a role in the hcv life cycle [ , ] . ns a is approximately kd with three distinct domains separated by low-complexity sequences (figure ). approximately % of the amino acids in ns a are prolines. in addition, ns a has regions of high disorder, multiple reversible posttranslational modifications, and helical tendencies [ ] . ns a displays significant sequence variability across viral genotypes, yet it typically contains approximately pro-pro motifs. it is not clear if these conserved diprolines are biologically relevant cyclophilin ainteracting sites. mapping studies have implicated the residues in domain as critical for the effects of cyclophilin a and cyclophilin inhibitors during viral replication ( figure a , b) [ ] [ ] [ ] . nuclear magnetic resonance (nmr) studies indicate that much of domain is disordered [ ] . additionally, nmr studies show that several prolines in domain occupy both cis and trans configurations and may thus be substrates for isomerases [ ] . domain from genotype strains also contains a proline-rich insert ( figure b) . but, despite the presence of the proline-rich insert at least one genotype strain has less cyclophilin dependence [ ] , suggesting that specific proline context, rather than the number or percentage of proline residues may determine the importance of cyclophilin and whether cyclophilin inhibitors are an applicable antiviral strategy. within ns a domain , most evidence implicates a single proline (p ) and the tryptophan, aspartate, and tyrosine residues surrounding it in a warpdyn motif as being especially significant [ , , ] . the warpdyn motif itself is bracketed by additional proline residues (p[a/i]warpdynp). mutations conferring resistance to cyclophilin inhibitors map to the warpdyn motif, e.g., r w and d e [ ] . the d e mutation had little to no effect on the binding of ns a to cyclophilin a. however, even though this is a conservative change, the d e mutation appears to alter the local protein conformation. nmr spectra of a -amino-acid peptide that includes the prolines bracketing the warpdyn motif showed that the isomerization state of p exists in equilibrium with approximately % in the trans conformations. conversely, spectra collected on peptide containing the resistance mutation d e revealed that approximately % of p was now in the cis conformation. thus, mutations that confer resistance to cyclophilin inhibitors shift the cis:trans ratio of configurations in the motif, reducing dependence on the isomerase activity of cyclophilins [ ] . cyclophilin a has at least low-level affinity for multiple other stretches of domain , including two tripeptide alanine-hydrophobic residue-proline motifs [ ] that surround the warpdyn. additional mutations adjacent to the warpdyn motif arise in patients treated with cyclosporine. an atypical proline (p , which is the consensus amino acid in only % of genotype strains) downstream of the motif was detected in one patient prior to treatment that mutated to serine following exposure to cyclosporine [ ] . the ns a p variant possessed enhanced susceptibility to cyclosporine in replicon experiments that was lost upon mutation to serine, suggesting in at least this patient a figure . hcv ns a is a protein that is rich in both proline residues and disorder and that associates with cyclophilin a via domain . a) crystallographic model of domain of ns a (residues - , pdb zh ), which has a well-defined structure. interestingly, a similar, but alternate structure for domain with a completely different dimer interface (pdb fqm) has also been solved and it is currently unknown whether a single conformation or both best represent the intracellular state. b) linear representation of ns a. current evidence suggests the entire carboxy terminus is disordered, but it has traditionally been studied as seperate domains termed domain and [ ] . red bars represent diprolines. plot of ns a disorder prediction from iupred (iupred.enzim.hu/). doi: . /journal.ppat. .g concentration of cyclosporine was achieved in vivo that had an antiviral effect [ ] . these data identify critical regions in ns a that recruit and utilize cyclophilin a to participate in viral genome replication. despite the depth of information regarding ns a domain , our basic understanding of which viral prolines in hcv or other viruses require isomerases is limited. this is in part because only one other example has been investigated extensively-the association between cyclophilin a and capsid (ca) from the hiv gag protein [ ] . that interaction was captured in crystal structures, revealing that cyclophilin a binds a relatively flexible loop between structured parts of the viral ca ( figure ) [ ] . it is certainly premature to draw general conclusions about viralcyclophilin interactions when only two have been characterized to any depth, but some noteworthy similarities and differences can be made. the hiv ca loop contains a single glycine-proline motif, with the proline (p ) existing in both trans and cis conformations in different structures and in solution [ , ] . the lack of structural rigidity for this loop is exemplified by the multiple conformations detected in solution structures, the higher b-factor for this region of the protein in crystal structure without cyclophilin, or disorder and the lack of structural information in some models (figure ). the glycine-proline motif of ca has no obvious resemblance to the cyclophilin a interaction site in hcv ns a. however, these regions share several common properties: the flexible cyclophilin-interacting loop in ca is also bracketed by prolines, just as the pawarpdynp motif in hcv; residues , , and that surround the glycine-proline in ca influence viral susceptibility to cyclophillin inhibitors similar to mutations surrounding ns a p [ ] ; and the critical glycine-proline is amino-terminal to regions of ca that are actually more proline rich and predicted by bioinformatics analysis to be disordered, analogous to the positioning of the pawarpdynp motif in hcv ns a. the interaction of hiv ca with cyclophilin a leads to packaging of cyclophilin a into the viral particle. yet, cyclophilin a appears to function primarily in capsid uncoating [ ] , rather than particle assembly, and does not have a role in hiv genome replication as it does for hcv. while limited, this comparison provides common themes that may facilitate the identification of other viral proteins that rely on host proline isomerases for function and may thus be susceptible to intervention by blocking isomerization. the hiv gag protein also contains proline stretches termed proline-rich motifs (prms) [ ] , but they do not appear to be critical targets for cyclophilin a. while some proline content favors disorder, consecutive prolines impart rigidity. prms generally have proline content surpassing %, and neither viral nor human prms have yet been described interacting with cyclophilins specifically. new viral threats emerge much faster than rationally designed antivirals. the number of clinically useful antivirals remains limited, so a drug that works on multiple viruses would be welcomed. identifying viruses and viral proteins that depend on host proline isomerases is an appealing strategy. for example, at least some lethal coronaviruses are suppressed by cyclophilin inhibitors [ ] . unfortunately, merely identifying a proline-rich viral protein is not sufficient to predict interactions with isomerase or sensitivity to isomerase inhibitors. creative, systematic approaches are needed to determine which viral proteins contain prolines that are substrates for isomerases and access multiple conformations to perform critical viral functions. identifying host factors that regulate viral infection sequences and topology: intrinsic disorder in the evolving universe of protein structure orderly order in protein intrinsic disorder distribution: disorder in proteomes from viruses and the three domains of life peptidyl-prolyl cis-trans isomerases, a superfamily of ubiquitous folding catalysts structural, biochemical, and in vivo characterization of the first virally encoded cyclophilin from the mimivirus specific inhibition of hepatitis c virus replication by cyclosporin a cyclosporin a suppresses replication of hepatitis c virus genome in cultured hepatocytes the natural history of recurrent hepatitis c and what influences this completion of the entire hepatitis c virus life cycle in genetically humanized mice hepatitis c ns a protein: two drug targets within the same protein with different mechanisms of resistance cyclosporine inhibits a direct interaction between cyclophilins and hepatitis c ns a hcv resistance to cyclosporin a does not correlate with a resistance of the ns a-cyclophilin a interaction to cyclophilin inhibitors deb (alisporivir) inhibits hepatitis c virus replication by preventing a cyclophilin a induced cis-trans isomerisation in domain ii of ns a cyclophilin b is a functional regulator of hepatitis c virus rna polymerase sensitivity of hepatitis c virus to cyclosporine a depends on nonstructural proteins ns a and ns b cyclophilin a is an essential cofactor for hepatitis c virus infection and the principal mediator of cyclosporine resistance in vitro essential role of cyclophilin a for hepatitis c virus replication and virus production and possible link to polyprotein cleavage kinetics hepatitis c virus proteins: from structure to function suppression of viral rna binding and the assembly of infectious hepatitis c virus particles in vitro by cyclophilin inhibitors a conserved tandem cyclophilinbinding site in hepatitis c virus nonstructural protein a regulates alisporivir susceptibility phenotypic analysis of ns a variant from liver transplant patient with increased cyclosporine susceptibility structural insights into the catalytic mechanism of cyclophilin a crystal structure of human cyclophilin a bound to the amino-terminal domain of hiv- capsid structure of the amino-terminal core domain of the hiv- capsid protein correlation of naturally occurring hiv- resistance to deb with capsid amino acid polymorphisms proline-rich regions and motifs in trafficking: from escrt interaction to viral exploitation cyclophilins as modulators of viral replication we sincerely apologize to the many authors whose work was not cited because of space limitations. key: cord- -nn gj z authors: krzyzaniak, magdalena anna; zumstein, michael thomas; gerez, juan atilio; picotti, paola; helenius, ari title: host cell entry of respiratory syncytial virus involves macropinocytosis followed by proteolytic activation of the f protein date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: nn gj z respiratory syncytial virus (rsv) is a highly pathogenic member of the paramyxoviridae that causes severe respiratory tract infections. reports in the literature have indicated that to infect cells the incoming viruses either fuse their envelope directly with the plasma membrane or exploit clathrin-mediated endocytosis. to study the entry process in human tissue culture cells (hela, a ), we used fluorescence microscopy and developed quantitative, facs-based assays to follow virus binding to cells, endocytosis, intracellular trafficking, membrane fusion, and infection. a variety of perturbants were employed to characterize the cellular processes involved. we found that immediately after binding to cells rsv activated a signaling cascade involving the egf receptor, cdc , pak , and downstream effectors. this led to a series of dramatic actin rearrangements; the cells rounded up, plasma membrane blebs were formed, and there was a significant increase in fluid uptake. if these effects were inhibited using compounds targeting na(+)/h(+) exchangers, myosin ii, pak , and other factors, no infection was observed. the rsv was rapidly and efficiently internalized by an actin-dependent process that had all hallmarks of macropinocytosis. rather than fusing with the plasma membrane, the viruses thus entered rab -positive, fluid-filled macropinosomes, and fused with the membranes of these on the average min after internalization. rab was required for infection. to find an explanation for the endocytosis requirement, which is unusual among paramyxoviruses, we analyzed the fusion protein, f, and could show that, although already cleaved by a furin family protease once, it underwent a second, critical proteolytic cleavage after internalization. this cleavage by a furin-like protease removed a small peptide from the f subunits, and made the virus infectious. human respiratory syncytial virus (rsv) belongs to the paramyxoviridae, a family of enveloped viruses with a negativestranded rna genome. it is a ubiquitous human pathogen that causes severe respiratory tract infections affecting mainly children and the elderly worldwide. despite ongoing efforts, there are no available vaccines or treatments except passive immunoprophylaxis [ ] . a better understanding of virus/host cell interactions is critical for the development of new therapeutic strategies. rsv particles produced in tissue culture are heterogeneous in size and shape. some are rounded with a diameter of - nm, others filamentous with a length up to mm [ ] . the nucleocapsid is helical and contains in addition to the rna the nucleoprotein n, the viral polymerase l, its cofactor-phosphoprotein p, and the transcription processivity factor m - . the matrix protein m is believed to form a layer on the inside of the viral envelope [ ] . the lipid envelope is derived from the plasma membrane (pm) of the infected host cell, and contains three viral glycoproteins; the major attachment protein g, the fusion protein f, and a small hydrophobic protein sh. cell attachment of rsv is mediated by g and f, which bind to cellular glycosaminoglycans [ ] . that g and sh are not essential for replication in cell culture [ ] , indicates that the f protein can support both attachment and fusion. in vivo, rsv targets airway epithelial cells, and in the human mucociliary epithelium it infects ciliated cells from the apical surface [ ] . previous studies on rsv entry employing a lipid-dequenching assay suggested that rsv, as most other paramyxoviruses, fuses its membrane directly with the pm of target cells [ ] . that rsv entry is ph-independent is consistent with this view [ ] . on the other hand, kolokoltsov and coworkers concluded, that rsv uses clathrin-mediated endocytosis (cme) to infect hela cells because a targeted sirna screen revealed clathrin light chain, eps- , and ap- as important cellular factors in rsv infection [ ] . in a recent publication, san-juan-vergara et al. [ ] argued that in primary nheb cells rsv entry is a two-step process; rsv docks to cholesterol-rich pm domains facilitating hemifusion between the viral envelope and the pm followed by endocytosis and complete fusion in endosomes. to determine the pathway of rsv entry into hela and a cells, we developed quantitative fluorescence-activated cell sorting (facs) assays and complemented them with confocal microscopy to monitor cell binding of rsv, endocytosis, fusion, and infection. we tested the effects of inhibitors and other perturbants. our results indicated that rsv infected the cells by an endocytosismediated mechanism that fulfilled the criteria of macropinocytosis. after uptake into macropinosomes, a second proteolytic cleavage in f served as a 'cue' for penetration by membrane fusion. in our studies, we used a recombinant rsv strain called rgrsv that expresses gfp [ ] enabling us to quantify infection by facs. the virus was grown in hep- cells, and to minimize exposure to broken cells, harvested from the cell supernatant before cytopathic effects were observed. the quality of virus purified by gradient centrifugation was confirmed by sds-page (fig. a) . when the purified particles were examined by indirect immunofluorescence (iif) using antibodies to the f and the n proteins, we found three different particle populations. half of the particles represented intact virions because in addition to f (green) they contained n (red) (fig. b) . of these, % also stained with phalloidin (blue, pseudocolored white) indicating the presence of actin filaments as previously reported (fig. b arrowheads) [ ] . the remaining particles constituted capsid-free envelopes (vlps). they stained for f but not for n. since we did not detect free capsids that would stain only for n or p (data not shown), we used the presence of the capsid antigens to distinguish between intact rsvs and vlps. when purified virus preparations were incubated with hela cells at uc, immunoblotting after sds-page showed that more than half of the input n and p associated with the cells indicating that rsv binding in the cold was efficient (fig. c) . to measure infection, rsv was added to hela cells for h and infection was continued for additional - h before measuring gfp expression by facs (fig. d ). the fraction of cells respiratory syncytial virus (rsv) is a highly pathogenic paramyxovirus. we developed assays for rsv endocytosis, intracellular trafficking, membrane fusion, and infection. the results showed that rsv was rapidly and efficiently internalized, and that acid-independent membrane fusion occurred intracellularly after endocytosis. cell biological studies demonstrated that endocytosis was macropinocytic, and that it was required for infection. the process involved activation of the egf receptor and its downstream effectors including cdc , pak , and myosin ii. rsv induced transient actin rearrangements accompanied by plasma membrane blebbing, elevated fluid uptake, and internalization of intact rsv particles into large macropinosomes. expression of a dominant negative rab mutant but not rab decreased infection indicating that rsv penetration is intracellular, and takes place in rab positive macropinosomes before fusion with endolysosomal compartments. the reason why rsv, unlike most paramyxoviruses, depended on endocytic entry was found to be the need for activation of the f protein by a second proteolytic cleavage. it occurred after endocytosis, and involved most likely a furin-like, vacuolar enzyme. . gradient purifier rsv (, particles/ ml) was resolved on the sds-page, followed by the blue silver gel staining. (b) . after binding to a glass slide, purified rsv was stained with anti-f-af (green), anti-n-af (red), and phalloidin-af (pseudocolored white). the particles (n = ) were imaged with a confocal microscope and analyzed for colocalization by imaris. arrowheads show particles with all three stains. (c). equal volumes of the virus input (moi ), the cell bound virus lysates, and the unbound virus (sup) were resolved by a sds-page. western blots were developed with anti-p or anti-n rsv specific antibody. (left) representative western blots. (right) densitometry quantification of the p-and n-protein bands intensities for the virus input and cell bound virus samples. (d). hela cells were infected with rsv moi ( - ) for h at uc. virus inoculum was replaced with medium and the infection was carried for indicated times. the percentage of infected cells expressing gfp was measured by facs. doi: . /journal.ppat. .g expressing gfp increased with time and with increasing multiplicity of infection (moi). in cells infected at moi of , gfp expression was detected as early as h post-infection (hpi) ( % gfp positive cells) and after hpi % of the cells were infected. at a moi of , gfp expression was delayed by about h. to follow the fate of the cell-bound particles in the cold after warming to uc, iif with anti-f and anti-n antibodies was used. actin filaments were labeled with phalloidin to visualize cell boundaries. confocal z-stack image series in the orthogonal view revealed that after min virus particles containing n and f were present not only on the cell surface but also deep inside the cytoplasm ( fig. a) . this indicated that viral particles and vlps were endocytosed. after binding in the cold, cell-associated viruses and vlps can be removed from the cell surface by brief incubation with trypsin in the cold that does not affect cell attachment (fig. b , min) [ ] . we found that when cells after virus binding in the cold were incubated at uc, an increasing fraction of the cell-associated particles became trypsin resistant (fig. b , - min). quantitation using spot detection software imaris showed that after min, % vlp and % rsv-containing spots were, in fact, resistant to trypsin (fig. c ). that the total number of rsv-and vlp-containing spots decreased over time was probably caused by the accumulation of multiple particles in common endocytic vacuoles that represented single spots. of the anti-f stained spots, % stained for n indicating that they were intact viruses. to confirm that rsv was endocytosed in an intact form, it was important to determine whether the endocytosed particles also contained the viral lipid envelope. purified rsv was therefore labeled with a lipophilic fluorescent dye, dioc, which partitions into the viral membrane. it is fixable with formaldehyde, and can be quenched by the membrane-impermeable dye, trypan blue (tb) [ ] . after labeling, % of re-purified particles contained detectable dioc (data not shown). when added to cells and incubated at uc, the rsv-dioc particles were visible as discrete fluorescent spots, and of these some were quenched when tb was added (fig. a) . facs analysis showed that, % of the fluorescence was resistant to tb after min, and full resistance was reached in about min ( fig. b top) , indicating that internalization of rsv and vlps was rapid and complete. importantly, when the intracellular accumulation of f and n proteins was measured in parallel ( fig. b bottom), internalization of both antigens and dioc (fig. b top) followed similar kinetics. to monitor fusion of rsv with cellular membranes, we used a method developed by sakai and coworkers [ ] . in this case, rsv was labeled with two fluorescent lipids, r (red) and dioc (green). concentrations were used at which the r quenches the fluorescence signal emitted by the dioc. therefore, when allowed to bind to cells and viewed live by confocal microscopy, the labeled viruses were initially all red (fig. c, min) . however, after about min at uc, yellow and green intracellular spots became apparent increasing in numbers over time, because after fusion, the two lipids were diluted out and the green fluorescence of dioc was no longer quenched by r (fig. c) . some of the spots showed a ring-like fluorescence indicating that the dioc was localized in the limiting membrane of intracellular vacuoles. quantitative facs analysis showed that the dequenching of dioc became detectable already after min at uc (fig. d ). it reached a half maximal level at min, and plateaued after min. treatment of cells with tb during facs analysis revealed that more than % of the fluorescent dioc failed to be quenched by this membrane impermeable agent confirming that the dioc was localized in intracellular organelles. from the time course, it was apparent that the fusion events occurred on the average min after endocytosis. our interpretation of these results was that the virus particles and vlps that bound to the cell surface were endocytosed. endocytosis was rapid and efficient, and the internalized viruses accumulated in endocytic vacuoles. after a lag period, the viral envelopes underwent fusion with vacuolar membranes. to bring infection into the picture, cells with virus bound in cold were transferred to uc. at indicated times, they were placed on ice, incubated with trypsin to strip away surfaceattached rsv, re-plated and incubated for additional hours to allow infection to proceed and gfp to be expressed. facs analysis demonstrated that in cultures that had been incubated at uc before trypsinization, the fraction of gfp-expressing cells increased with time. maximum infection was reached within min, and the half time was around min (fig. e ). that the time course coincided perfectly with the time course of virus endocytosis (fig. b) implied that productive infection depended on endocytosis. if rsv entry and infection depended on endocytosis as indicated by our experiments, we expected perturbants that inhibit endocytosis to block internalization and infection. in the experiments that followed, endocytosis of rsv was quantified by measuring the amount of the incoming n protein that was trypsin resistant using facs analysis h after warming. infection was scored as a percentage of cells expressing gfp hpi. it is important to mention that the dose-dependence (fig. s ) and toxicity (data not shown) of each inhibitor was carefully determined. for clarity for most of the inhibitors we will present data at a single concentration where we found a strong effect but low cytotoxicity. since rsv has been reported to enter cells by cme in hela cells [ ] , we tested five cme inhibitors: chlorpromazine [ ] , pitstop- [ ] , and three inhibitors of dynamin- (dynasore, dyngo- a and dynole- - ) [ ] . none of them influenced rsv endocytosis, although, internalization of the well-characterized cme cargo protein transferrin (trf) was efficiently inhibited by all (fig. a ). with the exception of pitstop- , which was too toxic in the prolonged infection assay, none of the agents inhibited rsv infection (fig. b) . infection by semliki forest virus (sfv), a virus known to depend on cme, was efficiently blocked by all (fig. b ) [ ] . rsv infection has been reported to be insensitive to an increase in endosomal ph [ ] . this was confirmed by the lack of influence of bafilomycin a, ammonium chloride, and monensin on rsv infection (fig. c ). as expected all three agents blocked infection by sfv, which needs low endosomal ph to trigger fusion [ ] . the small reduction in rsv infection observed for ammonium chloride and monensin may reflect the importance of a balanced vacuolar environment for productive rsv infection. taken together, the results indicated that rsv endocytosis and infection did not depend on cme nor did it require acidification. when rsv was bound to hela cells in the cold and the cells warmed to uc, rapid and dramatic changes in cell shape and actin distribution were observed (fig. a ). the number of actin stress fibers decreased, the cells rounded up, and transient blebs filled with actin formed on the cell surface (fig. a, min) . these changes were clearly visualized by live cell imaging (movie s ). the cell morphology and actin distribution returned to normal within hpi. when the ratio of g (globular) and f (fibrous) actin was determined, it was found that min after addition of rsv the ratio of g to f actin was : compared to : in control cells . hela cells without (top) or with bound rsv (moi , . ) at uc (bottom) were transferred for min to uc before fixation. cells were processed for confocal microscopy with anti-f-af (green), anti-n-af (red), and phalloidin-af (blue), and zstack image series acquired. the orthogonal views of z-stack projections (pseudocolored white) were generated with imagej. (b). rsv (moi , ) was bound to hela cells at uc, unbound virus was removed and cells transferred to uc. at indicated times cells were placed on ice and treated with pbs or trypsin for min (pbs wash vs. trypsin treated). samples were processed for confocal microscopy as in (a). (c). quantification of the image series represented in (b). analysis was performed with imaris to detect spots with anti-f staining only (left) or anti-f and -n staining (right) at different times after warming the cells to uc. doi: . /journal.ppat. .g disruption of actin filaments with cytochalasin d and latrunculin a as well as filament stabilization by jasplakinolide were found to strongly reduce rsv infection, whereas sfv infection was enhanced (fig. c ). rsv endocytosis was also significantly decreased (fig. d ). inhibition of cdc (pirl ), inhibited rsv infection effectively, while inhibitors of rac (nsc ), rhoa (ct ) and its effector rock (y ) had only a moderate effect (fig. e ). both infection and endocytic uptake were reduced when some of cdc 's downstream effectors were inhibited including pak (ipa- ), n-wasp (wiskostatin), and moderately when arp / (ck- ) was targeted (fig. e , f). nocodazole and taxol that interfere with microtubules had no effect on rsv or sfv infection (fig. d) . these results demonstrated that actin and its regulators played a critical role during rsv endocytosis and infection. f-actin was transiently depolymerized, resulting in the formation of blebs. in addition, cdc , pak , and n-wasp were required for rsv internalization and infection. the formation of blebs, the involvement of actin, and the role of cdc and pak suggested that infectious entry of rsv occurred by macropinocytosis as recently shown for several other viruses [for reviews see [ , ] ]. one of the characteristic features of macropinocytosis is an elevation in the uptake of extracellular fluid [ ] . indeed, when serum-starved hela cells were exposed to rsv, we observed that the internalization of kda dextran-af , a soluble, fluorescent tracer added to the medium, increased by % and % at low and high moi, respectively, over mock treated cells (fig. a ). the elevation was significantly higher than in serum-stimulated cells. iif showed that majority of endocytosed viruses stained by anti-f and -p antibodies were localized in large, dextran-af filled, intracellular vacuoles ( fig. b ). macropinosome formation requires na + /h + exchanger (nhe) activity to modulate rho gtpases at the pm [ ] . inhibition of nhe by eipa (an amiloride derivative) has become one of the diagnostic criteria for macropinocytosis. eipa inhibited rsv internalization and infection by % (fig. c ). in addition, pretreatment of cells with eipa blocked the increase in fluid phase uptake induced by rsv (fig. d ). taken together, these results demonstrated that rsv induces macropinocytosis and uses it for virus endocytosis and infection. macropinocytosis is usually initiated by activation of receptor tyrosine kinases (rtks) or integrins, followed by the activation of a spectrum of cellular signaling factors [ ] . accordingly, we found that rsv infection was significantly decreased by two broad range protein kinase inhibitors; staurosporine (ser/thr kinases) and a multi-target protein tyrosine kinase inhibitor, genistein ( fig. c, d) . to test whether rtks were involved, we used a human phospho-rtk array comprising antibodies against different phosphorylated rtks. lysates from cells exposed to rsv for min, and lysates from mock-treated control cells were used as probes. the epidermal growth factor receptor (egfr) was the only rtk for which activation was detected; a five-fold increase in phosphorylation compared to control (fig. a ). when the egfr was depleted using sirna, greater than % reduction in infection was observed (fig. b ). we found, moreover, that egfr inhibitors (cas - - , iressa) significantly decreased rsv infection (fig. c ). inhibition of pi k (wortmannin, ly , pi- ), a downstream effector of egfr, also reduced infection (fig. c ). egfr inhibitors had little effect on sfv. that the pi k inhibitors boosted sfv infection was consistent with a distinct entry mechanism for this virus. in addition, inhibition of pkc (rottlerin, calphostin c) decreased rsv (fig. d ). although the effect on sfv was smaller, it suggested a role for pkc in the entry of both viruses. finally, since non-muscle myosin ii is thought to mediate closure of macropinosomes, we tested the effects of a myosin ii inhibitor (blebbistatin), and a myosin light chain kinase inhibitor, (ml- ) . serum starved hela cells were incubated with % fcs or purified rsv (moi , , ), at uc for h. the inoculum was replaced with medium containing kda dextran-af and transferred for min to uc before fixation. the mfi of af- measured by facs. (b). purified rsv (moi , ) was bound at uc to serum-starved hela cells. the input virus was replaced with medium containing kda dextran-af (green) and transferred to uc. at indicated times cells were fixed, permeabilized and stained with anti-f-af (red) and anti-p-af (blue) antibody. images represent a z-stack projection acquired with the same confocal microscope settings. (c). hela cells were pretreated with solvent (mock) or eipa at indicated concentration. (left) cells were infected with rsv (moi , ) at uc for hours before facs analysis of gfp expressing cells. (right) rsv (moi , ) was bound to the cells at uc followed by h of internalization at uc. cells were trypsinized, fixed and stained with anti-n-af antibody, and the mfi of af- measured by facs. (d). serum starved hela cells were pretreated with eipa at indicated concentration and incubated with purified rsv (moi , ) or no virus control at uc. the inoculum was replaced with medium containing kda dextran-af and eipa and transferred for min to uc. cells were fixed and the mfi of af- measured by facs. doi: . /journal.ppat. .g [ ] . both reduced rsv infection with little effect on sfv (fig. e) . all the inhibitors that decreased infection also reduced rsv endocytosis (fig. c-e bottom) . depending on the compound, rsv internalization was reduced by - %. none of the inhibitors affected rsv cell binding (fig. s ) . thus, we concluded that infectious rsv cell entry and endocytosis were associated with activation of egfr and its downstream signaling partners including pi k and pkc. combined with the requirement for myosin ii, these findings were consistent with productive rsv internalization by macropinocytosis. in addition, we performed a series of experiments in a cells (fig. s ). they revealed changes in actin morphology and polymerization after addition of rsv, and a role of egfr, nhe, cdc , pak , and other factors similar to hela cells in rsv infection. that internalization and infection were clearly dependent on the same cellular processes and factors in a cells indicated that entry by macropinocytosis was not hela cell specific. intracellular trafficking of macropinosomes is not well characterized, but it has been shown that like endosomes, they acidify and acquire rab followed by rab before fusing with endolysosomes [ ] . wild type (wt) gfp-rab and gfp-rab as well as various constitutively active (c/a) and dominant negative (d/n) mutants of the rabs were transiently expressed in hela cells. after min post warming, we observed that some of the incoming rsv colocalized with gfp-rab wt positive vacuoles (fig. a) . colocalization was even more evident in cells expressing the c/a gfp-rab mutant q l that exhibits enlarged rab -positive vacuoles that fail to undergo further maturation [ ] . there was no detectable colocalization with rab at this time. after min, some colocalization with gfp-rab wt was still observed. in cells expressing the d/n gfp-rab s n, we noted accumulation of f-and p-stained particles inside large vacuoles in the perinuclear space. colocalization of rsv with gfp-rab wt was also detected. to determine whether rab and rab played a role in infection, we infected cells expressing gfp-tagged constructs of rab , rab , and their mutants with rsv-a . to determine the fraction of infected cells among cells expressing gfp-tagged rabs, we stained the cells with anti-n-af . facs analysis revealed that the d/n -gfp rab (s n) was the only rab construct that caused a significant decrease in rsv infection when overexpressed (fig. b) . we confirmed this result by imaging of rrrsv expressing red fluorescent protein (fig. s ). together with our imaging data, these results indicated that rsv depends on rab gtp for infection but does not require rab . infectious penetration is thus likely to be determined during early stages of macropinosome maturation. it is noteworthy that expression of the c/a rab (q l), which is known to generate enlarged rab -containing endosomes and prevent endosome maturation and trafficking to lysosomes [ ] , did not affect infection. pretreatment of cells with pikfyve inhibitor (cas - - ) had no effect on the rsv infection while sfv infection was decreased by % (fig. c) . by generating ptdins( , )p , pikfyve is involved in the maturation of endosomes and macropinosomes [ ] . this suggested that full maturation of macropinosomes was not required for rsv. these results demonstrated that after pinching off from the pm, macropinosomes containing rsv acquired rab and later rab . maturation of macropinosomes involving rab was evidently a critical step in infection, whereas later stages in maturation coordinated by rab and pikfyve were not essential. since acidification of macropinosomes was not needed for infection, we speculated that rsv required some other intracellular cue to trigger fusion. the f is unique among paramyxovirus fusion proteins in having two cleavage sites for furin-like proteases generating in addition to f and f ( and kda, respectively) a soluble amino acid (aa) peptide (p ) [ , ] . the p peptide is located between f and f n-terminal to the fusion peptide in f (fig. d) . we hypothesized that removal of this peptide after endocytosis might be required to activate the f protein. experiments in which cells were pretreated with a membrane permeable furin inhibitor, dec-rvkr-cmk, prior to addition of rsv indicated that a protease was indeed involved. dec-rvkr-cmk treatment reduced infection by about % (fig. a) . that a membrane impermeable furin inhibitor, a-pdx, had no effect on infection suggested that the activating proteolysis did not occur on the cell surface. a broad range protease inhibitor leupeptin (serine, cysteine, threonine proteases) caused only a slight increase in infection. that the inhibition of infection by dec-rvkr-cmk involved the f protein, was confirmed using a recombinant virus strain (rsvdshdg) that lacks the sh and g glycoproteins [ ] . infection by this mutant virus was also blocked by dec-rvkr-cmk (fig. a) . when dec-rvkr-cmk inhibitor was applied h after the virus inoculation, there was no inhibition indicating that the critical proteolytic step coincided with entry (fig. b ). fusion assays with r /dioc labeled rsv and rsvdshdg revealed that dec-rvkr-cmk impaired viral fusion (fig. c ). its inhibitory effect was comparable to the effect of eipa, which blocked virus internalization (fig. c) . to confirm the presence of the p peptide in the purified virus, we used a targeted mass spectrometry approach based on selected reaction monitoring (srm) (fig. e) . as a negative control, we analyzed hep- cells extracts used to produce the virus. in trypsin digested virus preparations, we detected peptides corresponding aa - and - of f protein both spanning p peptide (fig. d) . neither peptide was present in hep- control samples. srm transitions of the targeted peptides are included as supporting information in table s . finally, sds-page was used to monitor changes in the f protein itself during entry. blotting with an anti-f antibody revealed a protein band in the isolated virus migrating with a molecular weight of kda, confirming that the f protein had been cleaved at least once already in the producer cells (fig. f) . when virus bound to cells in the cold were allowed cell enter for . h at uc, the mobility of the cell-associated f became faster ( kda) indicating that further processing had occurred. the reduction in size of around kda was consistent with the loss of p at the n terminus of f . importantly, in cells pretreated with dec-rvkr-cmk, the processing of the kda f protein did not occur. as expected, a-pdx and leupeptin did not influence the processing step. when the time course of f processing was followed, we observed that some processed f was detectable already min after cell warming (fig. g) . it peaked at min when the precursor was fully consumed. that the amount of f protein gradually decreased at later time points was probably due to lysosomal degradation explaining the decrease in band intensity in the endocytosis lanes in fig. f . . hela cells were pretreated with dec-rvkr-cmk, a -pdx or leupeptin at indicated concentration for h before experiment and inhibitors were continuously present during following steps of the experiment. cells were infected with rsv or rsvdgdsh for h before facs analysis of gfp expressing cells. (b). hela cells were infected with rsv or rsvdgdsh for h. virus inoculum was replaced with medium containing mm dec-rvkr-cmk and incubated for before facs analysis of gfp expressing cells. (c). versene detached hela cells were pretreated with solvent (mock), dec-rvkr-cmk or eipa at indicated concentrations and inhibitors were continuously present during following steps of the experiment. rsv-r /dioc or rsvdgdsh-r /dioc (moi , ) was bound to cells at uc. unbound virus was removed and cells were incubated at uc for h in the presence of inhibitor. cells were fixed and the mfi of dioc fluorescence measured by facs and normalized to mock no inhibitor controls. (d). rsv f protein ( aa) is proteolytically processed by furin like protease at the two sites (a aa- and b aa- ) to generate disulfide bonds linked f +f and small peptide p (aa sequence depicted above). at the n-terminus of f is a fp (fusion peptide) and at the c-terminus tm (transmembrane domain), numbers indicate aa position and red underlines specify peptide sequences detected in mass spectrometry. (e). proteomic analysis of hep- cells and purified rsv particles. the n-terminal sequence of the p peptide (f protein) was quantified by a targeted mass spectrometry based on the selected reaction monitoring (srm). representative srm peaks of peptides (left) fmnytlnnakktnvtlsk + and (right) elprfmnytlnnak + peptides, corresponding to the aa - and - of f protein, respectively. different srm transitions for a peptide shown in different colors (see supporting information table s ). the bar graphs show the results of the targeted peptide quantitation, presented as the sum of the areas of all the srm peaks for a given peptide. where no peptide peak was detectable, noise values were reported as a reference. rt retention time, and cps counts per second. (f). hela cells were pretreated or mock treated with dec-rvkr-cmk, a-pdx, or leupeptin at indicated concentration. rsv (input control in the first line) was bound for h in cold (b-binding) or after washaway unbound virus was internalized for . h at uc before processing (i-internalized). lysed cell samples were resolved by sds-page and blots were probed with anti-f antibody. (g). rsv was bound for h in cold to hela cells; unbound virus wash away and cells were placed at uc for indicated times before, lysis, sds-page, and processing for western blot probed with anti-f antibody. doi: . /journal.ppat. .g these results indicated that to become fusion competent and infectious, the f protein underwent a second, highly efficient cleavage by a furin-like convertase in an endocytic compartment. the time course indicated that the processing of f occurred soon after endocytosis preceding fusion by about min. to address whether our results applied to cell types infected by rsv in vivo, we tested polarized epithelial cells hbe o obtained from human bronchial biopsies [ ] . after days in culture, the distribution of the tight junction marker, zo- , showed that the cells had reached a polarized phenotype (fig. a) . after making certain that rsv could infect hbe o cells from the apical side (fig. a) , we tested the effects of nine diagnostic inhibitors previously used in hela cell experiments (fig. b, ce , c, c, a). they inhibited dynamin, macropinocytosis, and furin proteases. rsv infection was quantified by an image-based approach that detected the fraction of gfp-expressing cells. in agreement with our findings in hela cells, inhibition of dynamin by dynasore had no effect on rsv infection in hbe o cells; it even boosted infection. eipa and seven other inhibitors of macropinocytosis decreased infection in a dosedependent fashion indicating that macropinocytosis was involved in entry. the need for f processing was confirmed by dec-rvkr-cmk, which was found to decrease infection by as much as %. paramyxoviruses are generally thought to infect cells by fusing directly with the pm [ , ] . that paramyxovirus particles can also be endocytosed is, however, also clear. this has most recently been documented for sendai, nipah, rsv, newcastle disease viruses and for a lentivirus vector pseudotyped with measles virus glycoproteins [ , , , , , ] . which of the two pathways -fusion at pm or fusion after endocytosis -leads to infection is not clear. in our experiments, we found that intact rsv was rapidly and efficiently endocytosed with capsid, glycoproteins, and the lipid envelope intact. the rsv and capsid-free vlps accumulated within cytoplasmic vacuoles with a half time of about min followed by fusion in the vacuoles with the half time of around min. a sensitive fusion assay using r /dioc-labeled fluorescent viruses showed that fusion occurred intracellularly. no fusion of viruses with the pm was detected, and no formation of syncytia by fusion-from-without was observed even after exposing cells to high moi (data not shown). the significant delay between rsv internalization and fusion could at least in part be explained by the requirement for post-endocytic cleavage of f protein. perturbations that inhibited endocytic uptake caused a dramatic reduction in infection confirming a role for endocytosis in infection. eipa inhibited both endocytosis and infection by %, and a similar level of inhibition was observed for agents that interfered with actin dynamics, a variety of kinases, and myosin ii. the inhibitory effect of rab d/n expression was also consistent with a role for endocytosis in infectious entry. proteolytic activation of the f protein necessary for fusion and infection occurred in intracellular compartments. we concluded that rsv and vlps were efficiently endocytosed, that penetration by membrane fusion occurred in endocytic vacuoles, and that at least % of infection was caused by endocytosed viruses. that the endocytic mechanism responsible for the entry was macropinocytic was demonstrated by the following observations: observations satisfied all the main criteria currently used to define macropinocytosis [ , ] . when inhibitor studies were performed using polarized physiologically relevant epithelial cells (human bronchial epithelium cells, hbe o), infectious entry of rsv was found to depend on the actin cytoskeleton, on cell signaling, and on a furinlike protease activity as also observed in hela cells. the results indicated that infection of these polarized epithelial cells monolayers derived from human bronchial tissue involved macropinocytosis and proteolytic activation of the f protein. macropinocytosis is a clathrin-independent mechanism for the uptake of fluid and cell-associated particles within large, uncoated vesicles formed at the pm [ ] . in most cell types, it is transiently induced by the activation of rtks and downstream signaling factors [ ] . in recent years, several viruses have been shown to use it for infectious cell entry. as recently reviewed [ ] , the bestdescribed examples include large viruses such as vaccinia, ebola, adeno , and kaposi sarcoma-associated viruses. interestingly, nipah virus, a paramyxovirus of the henipavirus subfamily, also belongs to this group. it uses ephrinb a as a receptor, the phosphorylation of which is required for macropinocytic internalization and infection in cho-k and veroe cells [ ] . we found that egfr phosphorylation was activated by rsv, and that inhibitors such as iressa targeting this receptor blocked endocytosis and infection. it is noteworthy that iressa was only inhibitory when present during the first hour of virus cell contact confirming that its effect was entry-specific (data not shown). when the egfr was depleted using sirna, infection decreased only by % suggesting that other rtks may be able to compensate in long-term experiments. downstream effectors of egfr such as pi k and pkc were also important for rsv endocytosis and infection. infection has previously been shown to promote cell survival mediated by pi k/nfkb. in a cells, pi k activation and phosphorylation of its effector akt occurs within min after rsv addition [ ] . interestingly, it has also been demonstrated that rsv binding to nheb cells induces pkc-a phosphorylation and translocation to the pm, while inhibition of pkc-a, as confirmed here, blocks rsv uptake and infection [ ] . our results contradicted a previous report proposing that rsv entry in hela cells occurs by cme [ ] . the authors based their interpretation on hits such as clathrin and associated proteins in a targeted sirna silencing screen against factors involved in endocytosis. (the hits also included actin modulators such as pak but their function in entry was not addressed). however, since the read-out was infection after h, a role for cme in postendocytic steps in the rsv infectious cycle could not be excluded. in our experiments, we did not observe inhibition of rsv endocytosis or infection by five different agents that block cme: chlorpromazine, dynasore, pitstop- , dyngo- a, and dynol- - . importantly these agents efficiently inhibited sfv, a virus that enters via cme. that dynasore fails to inhibit rsv infection was also recently reported by others [ ] . while macropinosomes are still poorly characterized, there is evidence that they undergo a maturation process similar to that of endosomes involving acidification, association with rab and rab , and fusion with late endosomes or endolysosomes [ ] . we noted that some of the vacuoles containing rsv were in fact rab -and later rab -positive. over-expression of a d/n rab mutant inhibited infection suggesting that rsv penetration required passage through 'early' macropinosomes that contained rab . the lack of inhibition by rab mutants, a pikfyve inhibitor, and nocodazole, all known to inhibit vacuolar maturation, implied that macropinosome maturation beyond the rab positive stages was not necessary. finally, our results provided a likely molecular explanation for the endocytosis requirement exhibited by rsv. unlike other paramyxoviruses, the f protein in rsv has two activating cleavage sites [ ] . our mass spectroscopy analysis and western blots showed that while f in the isolated virus had been cleaved in the a-site (rarr) generating f and f , it had not been cleaved at the more c-terminal b site (kkrkr). the second cleavage occurred after endocytosis. inhibition of the second cleavage by dec-rvkr-cmk inhibited rsv fusion and infection. that dec-rvkr-cmk is a furin inhibitor suggested that the protease in question belonged to the furin family of convertases. the enzyme was evidently acid-independent, and active in early rab -positive macropinosomes. cleavage at the b site was most likely important because after removal the p peptide 'cap' from the n-terminus, the hydrophobic fusion peptide is rendered the most n-terminal sequence in f . in other class i viral fusion proteins, including other paramyxovirus f proteins, the fusion sequence is invariably n-terminal [ ] . in conclusion, we confirmed that rsv requires two cleavages in its f protein for infectivity and showed that the second cleavage occurs during cells entry. infectious entry depends on endocytosis, which the virus induces by transiently activating macropinocytosis. the virus most likely meets the enzyme that generates the second cleavage in rab positive macropinosomes, and fusion occurs after some delay in these vacuoles. in this respect, the virus resembles ebola and sars viruses, the fusion proteins of which are also activated within endocytic vacuoles by proteases [ , ] . it is interesting to note that the f of nipah virus, which has a single monobasic cleavage site in its f, is activated after endocytic uptake by cathepsin [ , ] . inhibitors of cathepsins block infection, and cathepsin double knock-out cells are not infected. the infectious entry of other paramyxoviruses (and other viruses that have phindependent membrane fusion) may thus be endocytosis-dependent and the mechanisms more complex than previously assumed. for rsv, it will now be important to analyze the molecular features of the entry process in more detail, to identify the protease(s), and to determine whether the intracellular route is relevant also in vivo. being inducible and highly regulated, the macropinocytic process may prove more amenable to inhibition than other endocytic mechanisms, and therefore more easily targeted by therapeutics. hela, a and hep- cells were obtained from the atcc and cultured in dmem supplemented with % fetal calf serum (fcs), mm hepes, % glutamax (invitrogen). transformed bronchial epithelial hbe o cells obtained from dr. d. gruenert [ , ] , were grown in rpmi medium supplemented with % fcs, % l-glutamine and % nea for at least days before infection. recombinant sfv-zsgreen stocks were kindly provided by dr. g. balistreri [ ] . rsv-a was purchased from atcc. recombinant rsv strains expressing gfp (rgrsv, rgrsvdshdg) or rfp (rrrsv) were kindly provided by drs. m. peeples and p. collins [ , ] . rsv was produced in hep- cells. virus was collected form cell culture supernatant. the inhibitors used included: pi- (alexis biochemicals), dynasore, dyngo- a,dynol- - , pitstop- (ascent scientific), pirl (chembridge), wiskostatin (enzo), cas - - , cas - - , dec-rvkr-cmk, ly , nsc , staur-osporine, taxol, wortmannin, y , a-pdx (calbiochem), bafilomycin a, blebbistatin, calphostin c, chlorpromazine, cytochalasin d, eipa, genistein, ipa- , jasplakinolide, latrunculin a, leupeptin, ml , monensin, nh cl, nocodazole, and rottlerin (sigma). antibody and fluorescent dyes that were used comprised: anti-n monoclonal mab - - and anti-rsv goat polyclonal ab (millipore), anti-f monoclonal ab (abcam), anti-p rabbit polyclonal ( -v biosciences, menlo park, usa), anti-zo- , goat anti-mouse, goat anti-rabbit, donkey anti-goat af-conjugated, kda dextran-af , phalloidin af-conjugated, r , dioc, and transferrin-af (molecular probes), goat anti-mouse, goat anti-rabbit hrp-conjugated (bio-rad). expression plasmids encoding gfp-tagged rab , rab and its mutants were kindly provided by dr. m. zerial (max planck institute, dresden, germany). hep- cells ( - % confluent) in t flasks were infected with rsv (moi . ) in ml serum free dmem-hepes medium (dmem, mm hepes) for h at uc, and then inoculum was replaced with complete medium (dmem, % fcs, % glutamax, mm hepes). after h, the cell supernatant was collected, clarified by centrifugation, aliquoted, snap frozen, and stored in uc for experiments that did not require purified virus. for virus purification the method of ueba [ ] was modified as follows. the pre-cleared cell supernatant was centrifuged ( . rpm, min, uc, sw ti rotor, beckman optima -k ultracentrifuge) through ml % w/v sucrose cushion in hbss-hepes buffer (hbss, mm hepes). pellets were gently washed, reconstituted in % sucrose and centrifuged in a , , % sucrose step gradient ( . rpm, min, uc, sw rotor, beckman optima -k ultracentrifuge). an opalescent virus band was collected from the and % interface, overlaid over a - % continuous sucrose gradient and centrifuged ( . rpm, h, uc, sw rotor, beckman optima -k ultracentrifuge). the virus fraction at about % sucrose was harvested, diluted in the hbss-hepes and pelleted by additional centrifugation ( . rpm, min, uc sw rotor, beckman optima -k ultracentrifuge). virus pellets were resuspended in hbss-hepes, snap frozen, and stored in uc. all rsv stocks were titered by infecting hep- cells with serial dilutions of the virus in well plates. infection was allowed to proceed for - h at uc. fixed cells were assessed by microscopy for gfp expression, or stained with rsv anti-n antibody (af- ) to detect infected cells of rgrsv or rsv-a respectively. protein assay (bio-rad) was used to measure the amount of protein. when the influence of pharmacological inhibitors was tested, cells were preincubated with a medium containing inhibitors for min at uc before virus binding or infection (except rho gtpase and protease inhibitors that required - h of preincubation, and pitstop- which was preincubated for min). inhibitory compounds at indicated concentration were continuously present during all of the steps of infection and internalization experiments. dioc ( mg) was added to freshly purified rsv ( mg) in ml of hbss-hepes buffer and incubated at room temperature while gently shaking for h. to remove unincorporated dye, virus was filtered through . mm syringe filter (millipore) and snap frozen as ml aliquots, stored in uc, and used within weeks. labeled rsv-dioc was titered in hep- cells as described above. for the rsv-dioc endocytosis assay, cells were detached with versene solution ( . mm edta, ph . ), washed with pbs and chilled on ice. purified rsv-dioc, diluted in dmem-hepes was bound to the cells on ice for h. the inoculum was washed away with pbs; the cells were resuspended in complete medium and transferred to uc. after desired times, the cells were fixed in % formaldehyde, washed, and resuspended in facs buffer (pbs, . % fcs, mm edta). for the facs data acquisition (bd biosciences, canto ii) samples were divided into two. one sample was acquired directly and the other after addition of trypan blue to a final concentration of . % (invitrogen). alternatively, after internalization cells were treated with . % trypsin for min on ice. than washed with pbs, fixed in % formaldehyde, permeabilized with . % triton x- , stained with anti-f or anti-n antibody at uc over night followed by the af- secondary antibody staining. mean fluorescence intensity measured by flow cytometry (bd biosciences, canto ii) was normalized to the mock control without bound virus. purified rsv ( mg) was resuspended in . ml of hbss-hepes buffer before adding of dioc ( . mm) and r ( . mm) mixture. labeling was performed for h at room temperature while gently shaking. to remove unincorporated dye, virus was filtered through . mm syringe filter (millipore) and used freshly for the fusion assay. for the rsv-r /dioc fusion assay, cells were detached with versene solution, washed with pbs and chilled on ice. purified rsv-r /dioc, diluted in dmem-hepes was bound to the cells on ice for h. the inoculum was washed away with pbs; the cells were resuspended in complete medium and transferred to uc. after desired times, the cells were fixed in % formaldehyde, washed, and resuspended in facs buffer. for the facs data acquisition (bd biosciences, canto ii) samples were divided into two. one sample was analyzed directly and the other after addition of trypan blue to a final concentration of . % (invitrogen). rsv (moi , ) was bound to subconfluent cells for h at uc. than cells were washed with pbs, supplemented with medium containing mm cycloheximide and incubated for h at uc. cells were moved on ice and treated with ice cold . % trypsin for - min, washed with pbs, fixed in % formaldehyde, permeabilized with . % triton x- and stained with anti-n antibody over night at uc followed by the goat anti-mouse af staining. the mfi (mean fluorescence intensity) of af staining determined by facs (bd bioscience, canto ii) and analyzed by for the trf-af ( mg/ml) internalization was performed in parallel in identical conditions with the exception that uptake was carried for min. adherent cells were detached with versene solution, washed with pbs, chilled on ice, and pelleted. cell pellets were resuspended in dmem-hepes, containing the virus inoculum, and incubated h at uc. when the effect of inhibitors was tested, cells were preincubated with media containing inhibitor for min, and inhibitor was continuously present at each step of the experiments. cells were washed with pbs to remove unbound virus, fixed with % formaldehyde, permeabilized with . % triton x- , and stained with rsv anti-f over night at uc, followed by the secondary antibody labeling. samples were reconstituted in facs buffer and analyzed by flow cytometry (bd biosciences, canto ii). mean fluorescence intensity values were normalized to the mock control without bound virus. cells were detached with versene solution, washed with pbs, chilled on ice, and pelleted. virus input (purified virus stock (moi , ) diluted in dmem-hepes) was divided in two portions. one was mixed with the cells and the other left for further analysis as virus input. virus was incubated with the cells for h at uc. the cells were pelleted by centrifugation and the supernatant collected as an unbound viruses sample. the cell pellets were washed in pbs and resuspended in dmem-hepes of equal volume to the collected supernatant. the sds-page loading buffer was added to all samples, followed by denaturation for min at uc. an equal volume of each sample was separated by the sds-page and subjected to western blot with anti-p or anti-n antibody. western blots were quantified by densitometry with quantityone software (bio-rad). subconfluent cells seeded in well plates were starved over night with serum free dmem-hepes medium. purified rsv was bound to pre-chilled cells for h at uc. virus inoculum was washed and cells were pulsed with the serum free dmem-hepes medium containing kda dextran-af ( . mg/ml) for or min at uc. the cells were washed with mm naoac, mm nacl ph . followed by pbs wash. the cells were trypsinized, fixed in % formaldehyde and analyzed by flow cytometry (bd biosciences, canto ii). for confocal microscopy, cells were fixed in % formaldehyde, permeabilized with . % triton x- , and stained with rsv specific antibodies. images were acquired with zeiss lsm laser scanning confocal microscope. for transfection, cells were trypsinized, pelleted, and electroporated with mg of plasmid dna using amaxa (nucleofactor solution r, program i- ). cells were seeded on mm cover glass in well plates for imaging or in the well plates for facs analysis. experiments were performed after h of transient expression. the experiment was performed according to the protocol provided by the f/g actin in vitro assay kit producer (cytoskeleton inc. cat # bk ). in brief, subconfluent cells in . cm dishes were inoculated with purified rgrsv (moi ) for or min at uc. cells were washed, lysed, and clarified by centrifugation. supernatants were centrifuged ( rpm, h, uc, tla . rotor, beckman optima tlx ultracentrifuge), the resulting supernatants were collected (g-actin), and the pellets (f-actin) were resuspended in equal to supernatant volume of water containing actin depolymerizing reagent provided in the kit. equal volume of each sample was resolved by sds-page and western blots were developed with an anti-actin antibody. to measure ratio of the g and f actin western blots were analyzed by densitometry with quantityone software (bio-rad). for the infection assays cells were plated in optical bottom well plates. after infection with rgrsv for - hours, the cells were fixed with % formaldehyde and counterstained with dapi. nine images per well were acquired with automated md microscope that autofocuses at each image acquisition ( objective). the total cells number and the number of infected cells expressing gfp were scored by the matlab-based infection counter software described previously [ ] . human phospho-rtk array kit was obtained from r&d systems (cat # ary ) and experimental procedure was followed according the manufacturers guidelines. in brief, serum-starved subconfluent cells in . cm dish were inoculated with purified rsv (moi ) or null virus prep extract for min at uc. then, cells were lysed and pre-clarified by centrifugation. the supernatant samples were incubated with the antibody array, and developed with provided reagents. developed arrays were analyzed by densitometry with quantityone software (bio-rad). subconfluent cells in well plates were chilled on ice for min. rsv (moi ) in dmem-hepes was bound to cells for h at uc. post binding samples were washed and lysed in ripa buffer ( mm tris, mm nacl, mm edta, % np- , . % sds, ph . ). remaining samples were washed with pbs supplemented with complete medium and transferred to uc for h; following cells were trypsinized, washed and lysed with ripa buffer. all samples were separated on the % bis-tris gels (invitrogen). western blots were probed with anti-f antibody ( : ), hrp conjugated goat ant-mouse ( : ) and developed with super signal west pico (pierce). immunofluorescence staining and analysis . cells were seeded on mm coverslips in the well plate h prior experiment. plates were chilled on ice and then rsv was bound to cells in dmem-hepes for h at uc. viruses were washed away and cells were supplemented with complete medium and transferred to uc. at desired times cells were fixed in % formaldehyde, permeabilized with . % triton x- and incubated with % goat serum for min. cells were then stained with appropriate primary and secondary antibody. coverslips were mounted to the glass slides with prolong gold anti-fade reagent (invitrogen). immunofluorescence images were captured with lsm zeiss microscope with the confocal laser scanning set up (objectives or ). per experiment, - images were captured and processed by imagej. for the virus particles detection imaris software was used set up to detect particles larger than . mm and quality greater than . live cell imaging was performed with olympus cellr, objective with dic setting, equipped with uc incubator. hep cells or gradient purified rsv particles were lysed in denaturing buffer containing m urea and mm nh hco . lysates were briefly sonicated and proteins were reduced with mm dtt for min at uc and alkylated with mm iodoacetamide for min at uc. the samples were diluted : with mm nh hco and digested with sequencing-grade porcine trypsin (promega) at an enzyme/substrate ratio of : . the digestion was performed overnight and stopped with formic acid at final concentration of %. the peptide mixtures were desalted on sep-pak c cartridges (waters), eluted with % acetonitrile, vacuum centrifuged until dryness and resuspended in . % formic acid. for each peptide, q and q masses, as well as collision energies (ce) for peptide fragmentation were calculated using skyline (v . , maccoss lab software). double and triple charged product ions from the y-and b-series and transitions in a mass range of - da were considered (see full list of srm transitions in the supporting information table s ). the peptide samples were measured in srm mode on a triple-quadrupole/ion trap mass spectrometer ( qtrap, absciex) equipped with a nano-electrospray ion source. for the chromatographic separation of the peptides, the instrument was coupled with an eksigent nano lc system (absciex) connected to a -cm fused silica column, mm inner diameter (bgb analytik), packed in-house with magic c aq, mm beads (michrom bioresources). the peptide mixtures were loaded from an autosampler cooled to uc (absciex) and separated with a linear gradient of acetonitrile/ water containing . % formic acid from to % acetonitrile in min, with a flow rate of nl/min. srm analysis was conducted with q and q operated at unit resolution ( . m/z half maximum peak width) with up to transitions per run (dwell time, ms; cycle time, . s). data were analyzed with the software skyline. peak area of the srm peaks was used for quantitation, after confirming co-elution and shape similarity of the transitions monitored for each peptide. outlier transitions (e.g., shouldered or noisy transition traces) were not considered in the calculations. results are presented as the sum of the areas of all srm peaks for a given peptide. all experiments were performed at least in triplicate, and presented as normalized values with +/ standard deviation (sd). . rsv (moi , . ) was bound to a cells at uc followed by min at uc. cells were by iif with anti-f-af (green), anti-n-af (red), and phalloidin-af (pseudocolored white) for confocal microscopy as, and z-stack image series acquired. the orthogonal views of image z-stacks (pseudo-colored white) were generated with imagej. (b). rsv (moi , . ) was bound to a cells at uc, virus inoculum was washed, cells warmed to uc, fixed at indicated times, and stained with phalloidin-af (pseudo colored white) and anti-f-af (red) antibody. images represent z-stack projections acquired with a confocal microscope. movie s rsv induces transient blebbing of hela cells. hela cells were inoculated with a purified rsv (moi ) and immediately imaged with olympus cellr microscope with dic settings with the objective, frame per sec speed at c. (avi) table s srm assays used to study f (uniprot accession number p , fus_hrsva). (docx) a review of palivizumab and emerging therapies for respiratory syncytial virus morphogenesis and ultrastructure of respiratory syncytial virus viral and host factors in human respiratory syncytial virus pathogenesis iduronic acidcontaining glycosaminoglycans on target cells are required for efficient respiratory syncytial virus infection functional analysis of recombinant respiratory syncytial virus deletion mutants lacking the small hydrophobic and/or attachment glycoprotein gene in vitro modeling of respiratory syncytial virus infection of pediatric bronchial epithelium, the primary target of infection in vivo characteristics of fusion of respiratory syncytial virus with hep- cells as measured by r fluorescence dequenching assay recombinant vesicular stomatitis virus expressing respiratory syncytial virus (rsv) glycoproteins: rsv fusion protein can mediate infection and cell fusion small interfering rna profiling reveals key role of clathrin-mediated endocytosis and early endosome formation for infection by respiratory syncytial virus cholesterol-rich microdomains as docking platforms for respiratory syncytial virus in normal human bronchial epithelial cells respiratory syncytial virus engineered to express the cystic fibrosis transmembrane conductance regulator corrects the bioelectric phenotype of human cystic fibrosis airway epithelium in vitro protein analysis of purified respiratory syncytial virus particles reveals an important role for heat shock protein in virus particle assembly a single-step, sensitive flow cytofluorometric assay for the simultaneous assessment of membrane-bound and ingested candida albicans in phagocytosing neutrophils dual wavelength imaging allows analysis of membrane fusion of influenza virus inside cells pharmacological inhibition of endocytic pathways: is it specific enough to be useful? role of the clathrin terminal domain in regulating coated pit dynamics revealed by small molecule inhibition inhibition of dynamin by dynole - induces cell death following cytokinesis failure in cancer cells penetration of semliki forest virus from acidic prelysosomal vacuoles inhibition of semliki forest virus penetration by lysosomotropic weak bases virus entry by macropinocytosis gulping rather than sipping: macropinocytosis as a way of virus entry drinking a lot is good for dendritic cells amiloride inhibits macropinocytosis by lowering submembranous ph and preventing rac and cdc signaling shaping cups into phagosomes and macropinosomes myosin iib isoform plays an essential role in the formation of two distinct types of macropinosomes sequential activities of phosphoinositide -kinase, pkb/aakt, and rab during macropinosome formation in dictyostelium inhibition of rab gtpase activity stimulates membrane fusion in endocytosis visualisation of macropinosome maturation by the recruitment of sorting nexins cleavage of the human respiratory syncytial virus fusion protein at two distinct sites is required for activation of membrane fusion proteolytic activation of respiratory syncytial virus fusion protein. cleavage at two furin consensus sequences chemotactic antiviral cytokines promote infectious apical entry of human adenovirus into polarized epithelial cells paramyxovirus membrane fusion: lessons from the f and hn atomic structures paramyxovirus fusion and entry: multiple paths to a common end fusion of sendai virus and individual host cells and inhibition of fusion by lipophosphoglycan measured with image correlation spectroscopy role of endocytosis and cathepsinmediated activation in nipah virus entry newcastle disease virus may enter cells by caveolae-mediated endocytosis measles virus glycoprotein-pseudotyped lentiviral vector-mediated gene transfer into quiescent lymphocytes requires binding to both slam and cd entry receptors nipah virus entry can occur by macropinocytosis distinct endocytotic pathways in epidermal growth factor-stimulated human carcinoma a cells respiratory syncytial virus inhibits apoptosis and induces nf-kappa b activity through a phosphatidylinositol -kinase-dependent pathway protein kinase c-alpha activity is required for respiratory syncytial virus fusion to human bronchial epithelial cells macropinosome maturation and fusion with tubular lysosomes in macrophages virus membrane-fusion proteins: more than one way to make a hairpin endosomal proteolysis of the ebola virus glycoprotein is necessary for infection characterization of severe acute respiratory syndrome-associated coronavirus (sars-cov) spike glycoprotein-mediated viral entry activation of the nipah virus fusion protein in mdck cells is mediated by cathepsin b within the endosome-recycling compartment a mature and fusogenic form of the nipah virus fusion protein requires proteolytic processing by cathepsin l long-term culture of normal and cystic fibrosis epithelial cells grown under serum-free conditions phosphatidylinositol -kinase-, actin-, and microtubule-dependent transport of semliki forest virus replication complexes from the plasma membrane to modified lysosomes differential response of dendritic cells to human metapneumovirus and respiratory syncytial virus respiratory syncytial virus. i. concentration and purification of the infectious virus role of endosomes in simian virus entry and infection we are grateful to drs. p. collins and m. peeples who provided us with recombinant rsv strains and dr. g. balistreri for the stocks of zsgreen-sfv and valuable advises. thanks to v-biosciences for anti-p serum. we also thank dr. t. hegar who made available the infectious counter software and m. bonvin for help with depletion experiments. finally, thanks to drs. p.y. lozach and j. mercer for helpful discussions. key: cord- - be gxe authors: poulain, florian; lejeune, noémie; willemart, kévin; gillet, nicolas a. title: footprint of the host restriction factors apobec on the genome of human viruses date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: be gxe apobec enzymes are innate immune effectors that introduce mutations into viral genomes. these enzymes are cytidine deaminases which transform cytosine into uracil. they preferentially mutate cytidine preceded by thymidine making the ’tc motif their favored target. viruses have evolved different strategies to evade apobec restriction. certain viruses actively encode viral proteins antagonizing the apobec s, others passively face the apobec selection pressure thanks to a depleted genome for apobec -targeted motifs. hence, the apobec s left on the genome of certain viruses an evolutionary footprint. the aim of our study is the identification of these viruses having a genome shaped by the apobec s. we analyzed the genome of , human viruses for the depletion of apobec -favored motifs. we demonstrate that the apobec selection pressure impacts at least % of all currently annotated human viral species. the papillomaviridae and polyomaviridae are the most intensively footprinted families; evidencing a selection pressure acting genome-wide and on both strands. members of the parvoviridae family are differentially targeted in term of both magnitude and localization of the footprint. interestingly, a massive apobec footprint is present on both strands of the b erythroparvovirus; making this viral genome one of the most cleaned sequences for apobec -favored motifs. we also identified the endemic coronaviridae as significantly footprinted. interestingly, no such footprint has been detected on the zoonotic mers-cov, sars-cov- and sars-cov- coronaviruses. in addition to viruses that are footprinted genome-wide, certain viruses are footprinted only on very short sections of their genome. that is the case for the gamma-herpesviridae and adenoviridae where the footprint is localized on the lytic origins of replication. a mild footprint can also be detected on the negative strand of the reverse transcribing hiv- , hiv- , htlv- and hbv viruses. together, our data illustrate the extent of the apobec selection pressure on the human viruses and identify new putatively apobec -targeted viruses. a a a a a the apobec s (apolipoprotein b mrna-editing enzyme, catalytic subunit or a s) are innate immune effectors restricting many exogenous viruses and endogenous retroelements [ ] [ ] [ ] . the human genome encodes for seven a genes (namely a a, b, c, d, f, g and h), with several spliced transcripts and allelic variants for each. these seven genes originate from gene duplications and rearrangements that have occurred during mammalian evolution and represent a classic example of the virus-host arms race [ ] . the a s are cytidine deaminases that convert cytosine to uracil present in single stranded dna or rna. such editing on viral genomes generally results in c to t (or u) transition after replication of the genome. the a s preferably mutate cytosine in a 'tc dinucleotide context with the notable exception of a g that favors a c before the mutated c [ ] . the antiviral activity of the a s has been first reported for the reverse transcribing viruses hiv- (human immunodeficiency virus- ), htlv- (human t-lymphotropic virus- ) and hbv (hepatitis b virus) [ ] [ ] [ ] . editing occurs during reverse transcription on the negative strand leading to g to a mutations on the positive strand [ ] [ ] [ ] [ ] [ ] [ ] . subsequently, a -introduced mutations have been reported on various double-stranded dna (ebv for epstein-barr virus, hsv- for herpes simplex virus- , α-hpvs for alpha human papillomaviruses, bk pyv for bk polyomavirus), single-stranded dna (tt virus) and single-stranded rna (hcov-nl for human coronavirus nl ) viruses [ ] [ ] [ ] [ ] [ ] . it is important to note that the antiviral action of the a proteins is not based solely on their deaminase activity. deaminase-independent restriction has been demonstrated against endogenous retroelements, reverse transcribing viruses, adeno-associated viruses and many rna viruses (hcv for hepatitis c virus, rsv for respiratory syncytial virus, hcov-nl , mumps virus and measles virus) [ ] [ ] [ ] [ ] [ ] [ ] . the co-evolution between virus and host leads to the selection of viral proteins capable of countering the restriction effect of a s. hiv- encodes for the vif protein which promotes a g degradation [ ] . htlv- evades a g restriction by excluding a g from virions [ ] . borf protein from ebv inhibits a b deaminase activity and re-locates it far from viral replication centers [ ] . besides these active viral mechanisms targeting a proteins, some viruses have evolved passive strategies to limit a restriction. one such strategy is the depletion of a -favored motifs from the viral genome. by repetitive exposure to a activity, non-lethal mutations can accumulate in the genomic sequence leading to the under-representation of the motifs favored by a s. this under-representation of a -favored motifs is called a evolutionary footprint. thus, the 'tc dinucleotide motif is under-represented in the genome of α-hpvs and bk polyomavirus [ , ] . similarly, 'tc and 'cc (favored by a g) motifs are under-represented in the negative strand of ltr (long terminal repeat) and non-ltr endogenous retroelements [ ] . conflicting data have been reported regarding evidence of an a footprint on the hiv- genome [ , ] . recently, an under-representation of certain a motifs has been shown in the genome of the γ-herpesviruses ebv and kshv (kaposi sarcoma herpes virus) [ ] . finally, codon usage in coronaviruses suggests that cytosine deamination is an important biochemical force which shapes the evolution of these viruses [ ] . different bioinformatics approaches have been used to search for evidence of an a evolutionary footprint in viral genomes [ , [ ] [ ] [ ] [ ] [ ] , ] . in this study, we adapted and extended the warren et al. approach [ ] to carry out a general screening for the a footprint of the genomes of all currently annotated human viruses. we first demonstrate the sensitivity and specificity of our approach: i. an a footprint is detected in viruses which have already been shown to be depleted for a -targeted motifs; ii. no a footprint has been reported in viruses from animals lacking a genes. we showed that as much as % of currently annotated human viral species are shaped by the a selection pressure. we confirmed previous reports showing that papillomaviruses and polyomaviruses are generally strongly footprinted by the a s. among the most a -footprinted viruses, we identified autonomous parvoviruses and in particular the b erythroparvovirus as deeply cleansed for a -favored motifs. importantly, we showed that the a footprint observed in coronaviruses is limited to the endemic viruses and absent from the zoonotic mers-cov (middle east respiratory syndrome coronavirus), sars-cov- (severe acute respiratory syndrome coronavirus) and sars-cov- viruses. we also carried out a gene-specific a footprint search and identified local footprint on ebv and adenoviruses consistent with genome targeting during the initiation of replication. the a footprint is defined as the under-representation of a -targeted motifs. different approaches have been devised to estimate the over/under-representation of a given motif [ , [ ] [ ] [ ] [ ] [ ] , ] . firstly, and because most of the a proteins (a a, a b, a c, a f and a h) favors deamination of cytosine to uracil in a 'tc dinucleotide context, we have chosen to look for the under-representation of the 'tc motif. moreover, as originally developed by warren et al., we refined our analysis by distinguishing the position of the 'tc motif relative to the coding sequence. namely, we differentiate three k-mers containing the tc motif; one k-mer having the c in the first position of the codon (nntcnn), one k-mer having the c in the second position of the codon (tcn) and one k-mer having the c in the third position of the codon (ntc). a -introduced deamination of cytosine in viral genome produces an uracil that can be fixed in the form of thymidine after genome replication. this transition will have different impacts depending on the position of the mutated c. the c to t mutation will be non-synonymous if the c is at the first or second position of the codon (fig a, nntcnn and tcn kmers). however, if the mutated c occupies the third position of the codon, the c to t mutation will always be synonymous (fig a, ntc k-mer). therefore, a -driven natural selection should deplete more intensively ntc codons than tcn or nntcnn motifs (as in those cases the c to u mutation will impact the encoded amino acid). obviously, a editing can also target the template strand where a c to t mutation will translate into g to a transition in the coding strand. again, this transition will have different impacts depending on the position of the mutated g. the g to a mutation will be non-synonymous if the g is at the first or second position of the codon (fig a, gan and nga k-mers). however, if the mutated g occupies the third position of the codon the mutation will be most of the time synonymous (fig a, nngann k-mer). because synonymous mutations are presumably more likely to be retained than non-synonymous, we define the a footprint (with the exception of a g-induced footprint) as the depletion of ntc or nngann k-mers. calculation of observed vs expected kmer ratio has been adapted from warren et al. and detailed in the material and methods section. briefly, a synthetic coding genome was generated by concatenating the different coding sequences allowing the counting of the occurrence of a given k-mer (fig b, n obs (k-mer) ). each synthetic coding genome has been randomly shuffled a thousand times. the expected count is calculated as the average of the occurrences of this k-mer over the thousand iterations ( fig b, n exp (k-mer)). a negative k-mer ratio indicates depletion of that k-mer. the observed vs expected ratio of the ntc k-mer will be compared to those of the nntcnn and tcn k-mers. similarly, the observed vs expected ratio of the nngann k-mer will be compared to those of the gan and nga k-mers. of note, for the sake of clarity and simplicity, we have chosen to stick with a dna genetic code throughout the manuscript. the reader will read a t as a u in the context of rna viruses. secondly, and because a g favors deamination of cytidine when preceded by another cytidine, we have chosen to look for the under-representation of the 'cc motif. following the same rationale, a g-footprinted viruses should display to a stronger depletion of ncc codons compared to ccn or nnccnn motifs (or a depletion of nnggnn motifs versus the gnn and ngg motifs) (s a fig). the ncc ratio will be compared to those of the nnccnn and ccn k-mers. similarly, the nnggnn ratio will be compared to those of the ggn and ngg k-mers. given that the bk polyomavirus has recently been demonstrated to induce a b expression and that it was depleted in 'tc motifs [ ] , we considered this virus as a positive control to validate our approach. fig a shows a strong depletion of the ntc motif. on the contrary, the dinucleotide 'tc in the context of the nntcnn and tcn k-mers are neither over-nor under-represented. the significant differences between the ntc ratio and the tcn (or nntcnn) ratios reveal that the frequency of the tc motif is dependent on its position within a codon. it suggests that ntc depletion can be tolerated because of the degeneration of the genetic code. importantly, the absence of nntcnn and tcn depletion infers that this virus is still vulnerable to a restriction because deamination of those cytidines would lead to changes in the amino acid sequence. fig b highlights the fact that ntc depletion is genomewide and can be observed in each gene. similarly, the 'ga motif is significantly less abundant in the nngann context than in the gan or nga codons. the 'ga depletion is also genome-wide ( fig b) . we read these observations as the consequence of an a activity acting on both coding and template strands. extending our analysis to other polyomaviruses shows that both jc polyomavirus and merkel cell polyomavirus bear an a footprint; footprint that is also genome-wide and present on both strands (fig c- f ). the magnitude of the a footprint appears lighter on the merkel a. a -induced cytidine deamination followed by viral replication leads to c to t mutations (in red). most of the a enzymes favor deamination in a 'tc context. the tc dinucleotide motif is depicted in three possible codon contexts on both coding and template strand. depending on the position of the mutated c, the c to t transition can be synonymous (s) or non-synonymous (ns). proportion of s and ns mutations is reported when the two types of mutation can be produced. because synonymous mutations are more likely to be retained, the a footprint can be defined as the depletion of the ntc and/or nngann codons. b. depletion or enrichment of a given kmer (e.g. ntc) is calculated as the log ratio of the observed occurrence of that k-mer (n obs) divided by its expected occurrence (n exp). for each human virus, its coding sequences (colored arrows) are concatenated to generate a synthetic coding genome from which we obtain the n obs of a given k-mer. the synthetic coding genome is then shuffled a thousand times and the n exp is calculated as the average count for that k-mer. we showed that our approach sensitively detects an ntc depletion in viruses known to promote a expression. we then wondered to what extent this depletion is widespread among viruses. we therefore downloaded genomic sequences of , human, , non-human primate, , avian, and fish viruses and calculated nntcnn, tcn and ntc ratios ( fig b) . with nntcnn, tcn and ntc median ratios close to zero, most of the sequences are not a -footprinted (fig b, box plots) . however, the distribution of the ntc ratios is bimodal in human and non-human primate viruses with a subpopulation of sequences strongly depleted for the ntc codon ( fig b, arrows and a red part of the distribution plot). crucially, such footprinted subpopulation is not detected in avian or fish viruses ( fig b) . hence, the absence of a detectable a footprint in avian and fish viral sequences is consistent with the restriction of the a genes family to the mammalian class [ ] . it is worth mentioning that each baltimore's group is represented in the human, non-human primate, avian and fish viral sequence data sets, albeit in different proportions ( fig a) . due to the redundancy of the genetic code, different codons can encode for the same amino acid. the third position of the codons is highly reiterated (redundant) and allows synonymous substitutions. this is notably the case for the ntc and ntt codons where the c or t at the third position are perfectly interchangeable (fig c, colored in red) . while we observe a subpopulation of sequences depleted for ntc, such depletion is not observed for ntt. hence, the ntc depletion cannot simply be explained by the under-representation of an amino acid. the pair ntc/ntt is not the only interchangeable pair. the naa/nag, nac/ nat, ngt/ngc duos and the ncc/nct/ncg/nca quartet are also interchangeable. the distribution of ntc ratios remains the sole being bimodal with a subpopulation of strongly depleted sequences. the general ncg depletion (monomodal distribution with a median significantly less than zero) is the result of the well characterized cg dinucleotide under-representation in viral genomes [ ] . this depletion is shared in all viral datasets while the ntc depletion is specific to a subpopulation of human and non-human primate viral sequences. by breaking down the human viruses into their respective baltimore's group (s fig), we observed that ntc depletion is not present in reverse transcribing nor in negative sense single strand rna viruses. a mild general depletion is present in double strand rna viruses. importantly, a strong general depletion can be observed in double strand dna viruses. finally, in single strand dna and positive sense single strand rna viruses, certain specific viral sequences appear also significantly depleted. we also observed a mild general ncc depletion in single strand dna and double strand rna viruses, justifying further investigation for a possible a g-induced footprint (s fig). no ncc depletion is observed in double strand dna, single strand rna nor in reverse transcribing viruses. in order to identify a -footprinted viruses, we detailed the ntc and nngann ratios for human viral species (fig a) . we observed that the ntc and nngann distributions are bimodal with a subpopulation of depleted sequences in each case. we consider a viral species as footprinted by a s when its ntc or nngann median ratio is inferior to the population median by at least two times the standard deviation. hence, about % of the viral species are depleted for ntc ( species over ) and about % are depleted for nngann motifs ( species over ). in total, species ( %) present an a footprint on either one or both strands. this subgroup is essentially composed by double-stranded dna viruses with numerous alpha-, beta-and gamma-papillomaviridae (αpv, βpv and γpv) but also beta-polyomaviridae (bkpyv, jcpyv, kipyv, wupyv and hpyv ) and the delta-polyomavirus mwpyv ( fig b) . these viruses show a strong depletion for both the ntc and nngann motifs by comparison to nntcnn/tcn and gan/nga ( fig b) . of note, ntc depletion generally goes with a mild to a significative ntt enrichment (s fig). in the strongly ntc-depleted viruses hpv , hpv and hpv , a tc depletion is also observed in the non-coding region of the genome regardless of the analyzed motif (s fig). to recapitulate, the a footprint on the papillomaviridae and polyomaviridae is genome-wide and on both strands (fig , fig b and s fig) . importantly, we also identified the single-stranded dna virus erythroparvovirus b and the singlestranded rna virus hku beta-coronavirus as strongly footprinted by a s (fig b, highlighted) . we will further detail the a footprint of these viruses in the following sections. in order to specifically look for a g-footprinted viruses, we calculated the ncc and nnggnn ratios for human viral species (s b fig). we observed that the ncc and nnggnn ratios are mostly narrowly distributed around the zero value. viruses depleted for ncc are generally single stranded dna viruses (s b fig). however, in many of them we observed a concomitant depletion of the nnccnn motif casting doubts on the causal link between the observed ncc depletion and a g editing (s c fig). one of the most a -footprinted virus is the b erythroparvovirus. among the parvoviridae family, the tc ratio analysis showed a strong ntc depletion for erythroparvovirus b and to a lesser extent for parvovirus and bocavirus ( fig a) . we also observed a significant depletion of the nngann k-mer for each autonomous parvovirus (erythroparvovirus, parvovirus and bocaparvovirus , , and ) ( fig a) . thus, autonomous parvoviruses appear to be footprinted either on both strands as for the erythroparvovirus, the parvovirus and bocaparvovirus or only on the template strand for the bocaparvovirus , and . it is interesting to note that the aav- (adeno-associated dependoparvovirus ) shows a totally different pattern with even a slight enrichment in ntc codons. this dependoparvovirus is not footprinted by the a s. detailed analysis of the erythroparvovirus b sequences shows a nearly complete ntc cleansing along the whole genome ( fig b, red marks) . on the contrary, ntt codons are distributed all along ( fig b, green marks) . some ntc codons remain present in a short, discrete section of the ns gene. this region also encodes for the . k protein in another coding frame. hence, the remaining tc motifs in the ns gene are tcn codon context in the . k protein a. the ntc and nngann observed/expected ratios for , human viruses' genomes (from unique species) were calculated, grouped by species and colored according to the baltimore classification. each point represents a unique viral genome. abundance distribution is depicted by a histogram on the right-hand side of the panel. viral species with an ntc or nngann ratio below two times the standard deviation (dotted grey line) from the population median (red line) are the putative a -footprinted viruses. b. the observed/expected ratios of tc dinucleotide at various codon positions and on both strands (i.e. nntcnn, tcn, ntc, gan, nga and nngann) were calculated for the putative a -footprinted viral species and depicted by a heatmap. a colored scale with increasing shades of blue indicating depletion and increasing shades of red indicating enrichment. p-values were calculated by student's unpaired, two-tailed t-test (ns for not significant, � p< . , �� p< . , ��� p< . ). (pv stands for papillomavirus, pyv for polyomavirus). https://doi.org/ . /journal.ppat. .g a. the observed/expected ratios of tc dinucleotide at various codon positions for the b erythroparvovirus were compared to those of the other human members of the parvoviridae family and depicted by a heatmap. a colored scale with increasing shades of blue indicating depletion and increasing shades of red indicating enrichment. p-values were calculated by student's unpaired, two-tailed t-test (ns for not significant, � p< . , �� p< . , ��� p< . ). b. coding sequences (ns , . k, vp , x, vp and k) from full-length b erythroparvirus were depicted by grey lines overlaid by red marks to symbolize ntc and green marks to position ntt codons. zoom-in detailed a bp-long sequence from the ns and . k genes (from nucleotide to ). a second zoom-in detailed a bp-long sequence from the vp -vp genes (from nucleotide to ). gene. the mutation of those tcs would introduce non-synonymous mutation in the . k protein. this probably explains the conservation of those tc motifs. moreover, among the sequences illustrated in fig b, some locations harbor a mix of ntc and ntt codons, suggesting that c to t transition is still an active process (zoom for vp -vp sequence). an a footprint can also be observed in the template strand as shown by the nngann depletion (fig a and s fig) . these observations show that the b erythroparvovirus is submitted to an ongoing and strong a selection pressure acting on both strands of the virus. by comparing the ntc ratio to the nntcnn and tcn ratios, we observed a common ntc depletion in the nl , e, hku and oc coronaviruses; the hku -cov being the most strongly deleted in ntc codons (fig ) . in coronaviruses, all viral genes are encoded by the positive strand. in other words, the coding strand of each gene is on the positive strand. therefore, the depletion of ntc codons is indicative of an a activity on the positive strand. these observations corroborate the in vitro detection of a soft rate of a c, a f and a h editing on the nl genome and the nnu/nnc codon bias previously reported for the hku coronavirus [ , ] . next, we investigated the presence of an a footprint on the template strand (corresponding to the negative strand in coronaviruses) by comparing the 'ga ratios in different codon contexts. however, we did not observe a progressive depletion of the ga motif (i.e. nngann ratio < nga ratio � gan ratio) which would be expected in the presence of a ga to aa mutational pressure. for that reason, we cannot rule on the presence of an a footprint on the negative strand (fig ) . finally, unlike endemic viruses, the zoonotic viruses mers-cov, sars-cov- and sars-cov- and their animal ancestors camel-mers, bat-mers and bat-sars are not depleted for ntc codons (fig ) . since a non-random distribution for a mutations has been reported for some viruses, we then looked for spatially circumscribed a footprint; i.e. a footprint limited to certain viral genes to limit our screening on genes which are depleted compared to the whole genome, we subtracted to the genic k-mer ratio, the corresponding genomic k-mer ratio to define the differential ratio for ntc and nngann k-mers ( fig a) . in other words, we looked for viruses harboring local a footprint amongst an otherwise non-footprinted genome. thus, differences between genic and genomic k-mer ratios were calculated for , viral genes. fig b shows the viral genes having an ntc (or nngann) differential ratio inferior to the median by at least two times the standard deviation. thus, we identified many genes being footprinted by a among otherwise unaffected viral genomes. most of these genes belongs to two families of double-stranded dna viruses; i.e. herpesviridae (hhv- , , , , and ) and adenoviridae (adv a, b, c, d, e and f). we also observed a -footprinted genes in the reverse transcribing hbv, hiv- and htlv- . in order to better shed light on the possible mechanisms responsible for such editing, we position the identified genes along the corresponding viral genomes and detailed these analyses in the following sections. an equivalent analysis was performed to report a local a g footprint amongst an otherwise non-footprinted genome. ncc and nnggnn-depleted genes are listed in s d fig. similarly to what has been observed at the genome level, many of the ncc-depleted genes are also depleted for the nnccnn motif making difficult to ascribe the observed ncc depletion to the sole a g editing activity. adenoviruses a and b present a strong ntc depletion for the e a and e genes ( fig a) ; genes localized at both ends of the linear genome (s fig). the same trend can be observed in adenovirus c, d and e, although to a lesser extent (s fig) . importantly, these e a and e genes are being strongly depleted for ntc but not for the nngann motif ( fig b) . in other apobec footprint on human viruses words, these two genes are a -footprinted on their coding strand only. due to the relative position of those genes and the strand-displacement strategy used for genome replication, we propose a model where a editing would occur specifically during the initiation of genome replication on the displaced strands ( fig c) . indeed, at the beginning of dna replication, the displaced strand corresponds to the coding strand of e a at one end of the linear genome and to the coding strand of e at the other extremity. one might also notice an nngann depletion for most of the l genes (fig b and s fig). considering the position and orientation of that gene, such footprint might also reflect an a activity on the displaced strand (fig c) . among the ebv genes, only are significantly depleted for ntc (fig b) . interestingly, the five most depleted are localized around the lytic origins of replication. bhlf and bhrf are localized on both sides of the first lytic origin of replication and lf , rpms and search for an a footprint at the gene level. a. alongside the observed/expected k-mer ratios calculated from the synthetic coding genomes (named genomic kmer ratios), k-mer ratios were also computed for each viral coding sequence individually (named genic k-mer ratios). differential ratio is defined as the subtraction of genic k-mer ratio to the corresponding genomic k-mer ratio. b. list of the putative a -footprinted viral genes and belonging to an otherwise non-depleted viral genome (having at least five reported sequences). https://doi.org/ . /journal.ppat. .g apobec footprint on human viruses a are on both sides of the second lytic origin of replication (fig b) . similar to the adenoviruses, this local a footprint is very much strand-specific and present on the lagging strands of the replication forks surrounding the lytic origins ( fig b) . thus, the ebv specific footprint is pointing toward a editing during the beginning of replication at the lytic origins. we summarize these observations by a scheme in fig c. we observed that the hbz gene of the htlv- virus is depleted for ntc codons (fig b) . because hbz is an antisense transcript, its coding strand corresponds to the genomic negative strand. hence, the ntc depletion of the hbz gene is indicative of an a editing activity on the negative strand (fig a and b) . we then wondered whether such a footprint was restricted to the hbz coding region or rather extend further. we observed that the coding sequences of the sense transcripts gag, pro, pol and tax are depleted for the nngann motif (fig a and b) . these observations suggest that a s left an evolutionary footprint on the htlv- virus through editing during reverse transcription. depletion for the nngann motif has been observed for the c, prec/hbeag coding sequences of hbv (fig b) . these observations support the involvement of an a editing activity on the dna negative strand during the reverse transcription process. however, it is interesting to report that nor the pol neither the s and x coding sequences are being footprinted (fig c and d ) conflicting data were reported concerning the presence of an a evolutionary footprint on the hiv- genome [ , ] . fig reports ratios of the tc and ga-containing k-mers for the hiv- , hiv- and siv genomes. the hiv- genomes were spread out into their respecting groups (m, n, o) and subtypes (group m subtypes a, b, c, d and e). no ntc depletion was observed on the hiv- , hiv- and siv genomes (fig ) . we concluded that a s did not leave a footprint on the plus strand. importantly, a mild but consistent nngann depletion was observed in hiv- , hiv- and siv genomes, depletion compatible with a -editing during reverse transcription. the apobec family of genes also counts the aid (or aicda), apobec , apobec and apobec genes. aid is critical for somatic hypermutation and class switch recombination by editing the immunoglobulin loci in b cells [ ] . apobec plays an important role in lipid metabolism by editing the apolipoprotein b pre-mrna [ , ] . importantly, apobec and aid appear also to participate to the restriction of viruses and retroelements [ , ] . evidence for aid and apobec evolutionary footprints were investigated by looking for the depletion of their favored motifs, respectively wrc for aid [ ] and wcw for apobec [ ] . the distributions of the wrc and nngywn ratios do not point towards viruses significantly footprinted by aid at the whole genome level (s b fig). nevertheless, putatively aidfootprinted genes were identified in several double strand dna viruses, notably the b-cellinfecting virus ebv (s c fig). the distributions of the nwcwnn and nwgwnn ratios show evidence of genome-wide footprinted viruses (s b fig) . however, it is not possible to disentangle the apobec footprint from the apobec footprint as the -mers nwcwnn contains the -mers ntc. the putatively apobec -footprinted viruses are those that also bear the putative apobec footprint (fig b and s c fig). of note, apobec and apobec -favored motifs have not been described so far. in this study, we investigated the distribution of the a footprint along a large set of , human virus complete genomes. we first observed that no less than % of all referenced apobec footprint on human viruses human viral species have a genome-wide a footprint. among these, we mainly identified viruses from the papillomaviridae, polyomaviridae, coronaviridae and autonomous parvoviridae families. in addition to this category of viruses targeted over their entire sequence, we have identified viruses which have an a footprint spatially limited to a short section of their genome. this is notably the case for certain herpesviridae and adenoviridae where the a footprint is localized on genomic sequences used to initiate replication of viral dna. our study is in line with previous publications reporting the presence of an a footprint on papillomaviridae [ ] and on the bk polyomavirus [ ] . above all and because we analyzed all currently annotated human viruses with the same approach, we can compare the magnitude of the a selection pressure between different viral families. thus, we show that the papillomaviridae and the polyomaviridae families are those whose footprint is most intensive (fig a) . those viruses have evolved to thrive under an ongoing and strong a selection pressure. the strong ntc depletion reduces exposure of the viral genome to the introduction of uracil and consequently to the base excision repair-mediated dna degradation. importantly, not only do these viruses tolerate such pressure, but they even actively promote the expression of certain a proteins. indeed, high risk α-hpvs have been shown to trigger and stabilize a a and a b via their oncoproteins e and e [ , [ ] [ ] [ ] . likewise, it has recently been shown that bk and jc β-pyv upregulate a b through their large t antigen [ , ] . in both the α-hpvs and β-pyvs, the induced a proteins are enzymatically active and therefore capable of deamination [ , ] . the selective advantage which would provide a sustained expression of a a and/or a b proteins is still debated. on the one hand, a a has been shown to restrict hpv in vitro [ ] . those viruses are still susceptible to a restriction. indeed, deamination of the remaining tc motifs are most of the time non-synonymous. on the other hand, the deaminase activity could positively impact viral fitness by participating to the genetic diversification of the virus or even by protecting the host cell against the reactivation of retroelements [ , ] . we speculate that the error rate of the host dna polymerase could be too low for viruses with such small dna genome, hence requiring the a editing activity to drive their evolution. within the polyomaviridae family, the magnitude of the a footprint differs significantly between species; species of the betapolyomavirus genus appearing to be the most strongly footprinted (s fig). such differences could find their origin in the capacity of the large t ag at inducing the a proteins. to draw a parallel with the alpha-papillomaviridae, e from high-risk α-hpvs were found to be more potent at inducing a b than those of low-risk strains [ ] . besides, the cell type hosting the virus can also influence the level of a expression. the difference in tissue tropism between the alpha-and beta-papillomaviridae has been proposed to explain the stronger footprint on the former [ ] . the full spectrum of tissue and cell tropism has not been clearly established for the polyomaviridae, making this type of correlative analysis tricky. our analysis also shows that the a footprint is present on both strands of the papillomaviridae and polyomaviriridae genomes. this is compatible with an editing activity during viral dna replication. among the parvoviridae family, the erythroparvovirus b exhibits an intensive footprint on both strands of its genome, the bocaparvoviruses being mainly footprinted on the negative strand and the dependoparvovirus adeno-associated virus- showing no evidence of a selection pressure. these dissimilarities might be explained by differences at the replicative and packaging levels. thus, the parvoviridae family consists of viruses that package a single copy of their short linear single-stranded dna genome into preformed capsids. the packaging takes place in the nucleus of the infected cell. while most can encapsidate dna strands of either polarity with equal efficiency, some family members, predominantly package negative strand genome. in the case of the erythroparvovirus, there is an equivalent amount of positive and negative genome that is produced during replication and subsequently encapsidated (reviewed in [ ] ). for bocaparvoviridae, the replication produces % of negative ssdna [ ] . such difference could explain the location of the a footprint in the negative strand of the bocaparvoviridae. thus, we propose that a editing activity takes place inside the nascent virions of the autonomous parvoviridae. our screening also reported the coronaviridae as a -footprinted. the canonical substrate for the a proteins is single stranded dna and until recently, the viruses identified as being restricted by the a deaminase activity were either dna viruses or viruses having a dna intermediate (i.e. reverse transcribing viruses). however, recent reports demonstrated that a a and a g can deaminate ribocytidine within a single stranded rna molecule [ ] [ ] [ ] . in vivo, a mutational signature has been reported in the positive single strand rna rubella virus [ ] . importantly, milewska and colleagues demonstrated that cytoplasmic a s can restrict the nl coronavirus in vitro [ ] . the a -mediated restriction of the hcov-nl appears to be both deaminase-dependent and independent. a restriction did not cause hypermutation on the viral genome, but c to t and g to a point mutations were observed in hcov-nl viruses passaged in a -expressing cells but not in wild-type cells. it is a matter of debate whether the hypermutated genomes could not be retrieved because of the high fitness cost of such mutations or because the a are less processive on coronaviral rna. additionally, a proteins have been shown to interact with the nucleoproteins of the hcov- e, hcov-nl and sars-cov- viruses [ , ] . finally, a recent report demonstrates the presence of apobec and adar editing on the sars-cov- transcriptome [ ] . thus, knowing that a s can bind the nucleoprotein and that a footprint is present on the positive strand of the viral genome, we suggest that a editing occurs on the packaged genome. two beta-coronaviridae are endemic to humans (hcov-oc and hcov-hku ), they are widespread, have been circulating in human for at least several decades and may cause to % of common colds (review in [ ] ). both have an a footprint on the positive strand. in comparison, no evidence of footprint was observed on the zoonotic beta-coronaviridae sars-cov- , mers-cov or sars-cov- . the absence of an evolutionary footprint on sars-cov- and mers-cov could find its explanation in the relative low number of infected individuals and the short duration of viral circulation. according to the world health organization, sars-cov- infected about . people over a period of few months and have been declared eradicated in may . the mers-cov infected so far less than . people by causing episodic outbreaks in the middle east. the figures for the sars-cov- are radically different with more than million confirmed cases as of may . in that respect, it will be interesting to track the evolution of the pandemic sars-cov- regarding a possible introduction of an a footprint through its interhuman transmissions. it is worth reiterating that no footprint was detected on the sars bat isolates, although the bat a locus is the largest and most diverse known repertoire of a genes in mammals [ ] . perhaps sars-like viruses possess a yet unknown and unique a inhibiting mechanism. interestingly, the sars-cov- genome contains a novel orf, called orf . orf encodes a protein that has been demonstrated to interact with the cul complex [ ] . this interaction is reminiscent of the interaction between the vif of biv (bovine immunodeficiency virus) and cul [ ] . one could speculate that orf could play a vif-like role in bat and also in human. also, a shorter version of sars-cov- orf is present in all sars-like viruses [ ] . this could explain why the sars-like viruses are not a -footprinted. in addition to this category of viruses that are footprinted on their entire sequence, we identified viruses that show an a footprint only on a very limited section of their genome. this is notably the case for the gamma-herpesviridae ebv and adenoviridae. the a footprint on ebv is spatially limited to the lagging strands around the lytic replication origins. interestingly, both ebv and kshv were recently demonstrated to encode viral proteins capable of inhibiting a b activity [ , ] . those viral proteins (ebv borf and kshv orf ) are both the large subunit of the ribonucleotide reductase, expressed during lytic replication and providing the precursors necessary for viral dna synthesis. we speculate that their expression could avoid the extension of the a -editing further along the viral genome. of course, these viral proteins inhibiting a b may not be the sole actors protecting the viral genome. the coating of the viral dna by the major dna binding protein (dbp), the compartmentalization of the viral dna replication [ ] and the switch from theta to the rolling circle replication might also limit access to the viral single-stranded dna. the fact that no footprint was detected around the latency origin of replication, ori-p, points toward an a -editing acting during lytic replication. the strategy deployed by ebv and kshv to cope with a restriction is somehow opposite to the one used by the papilloma and polyomaviruses. indeed, ebv and kshv actively protect their genome from the a s as opposed to the papilloma and polyomaviruses which simply cope with a high mutational rate. we speculate here that the large size of the herpesvirus genome would not tolerate an unrestrained a activity. finally, we identified several aid-footprinted genes in the ebv genome which supports recent observations made by martinez et al. [ ] . those genes are not spatially clustered and further investigation would be necessary to link the detected footprint to aid activity. similarly, we identified an a footprint on adenoviridae localized at the ends of their linear genome where the origins of replication are located. the presence of an a footprint on the lagging strand of the origins of replication and its absence on the leading strand is striking. it parallels the footprint on the lytic origins of replication in ebv and it is also reminiscent of the a activity in cancer genomes. thus, a -related mutations in cancer genomes are strongly enriched on the lagging strand and early-replicating euchromatic regions [ ] [ ] [ ] [ ] . the leading strand, being protected by the nascent complementary dna, is less or even not accessible for deamination. another circumstance where the viral dna is transiently single-stranded is during transcription. indeed, the coding strand within the transcriptional bubble is temporally single stranded. in cancer genomes, the observed distribution of apobec -signature mutations is transcription independent [ ] . again, it is very similar to our observations on viral genomes where no evidence points toward a -editing during viral transcription. overall, the similarities between adenoviridae and herpesviridae regarding their relationship to a s appear substantial. both are large double-stranded dna viruses replicating their genome in the nucleus and capable of lytic and latent/persistent phases. it would not be surprising to find that adenoviridae are also able to inhibit a activity. along with these speculations, we found important to underline that the dependoparvovirus aav- is the only member of the parvoviridae family which does not have an a footprint. it would be interesting to test whether it is an intrinsic characteristic of the satellite virus or whether it is mediated by the helper virus (usually an adenovirus or a herpes virus). the detection of an a footprint on the negative strand of the c (core) and prec/hbeag coding sequences of hbv is compatible with a -editing on the dna negative strand during reverse transcription. a -related mutations have been detected on the negative strand of the c and prec region and it has been proposed that these mutations could be beneficial for the virus [ ] . indeed, during the natural course of hbv infection, the hbeag expression is being lost after production of antibodies against it. hbeag is an accessory non-particulate protein encoded by the prec mrna and displaying immunomodulatory properties. hbeag is described as a tolerogen that allows the virus to establish infection. seroconversion against the hbeag leads to the selection of hbeag-negative mutants. the ability to develop mutations, altering hbeag expression, can influence the length of the hbeag-positive phase, which is important for determining the clinical course (reviewed in [ ] ). our observation of an a footprint in the prec and c region further supports the idea that a can positively participate to the immune escape. that would not be the first example of the hijacking an antiviral weapon for the benefit to the pathogen. hepatitis d virus is a circular complementary single-stranded rna virus that requires editing of its genome by a cellular adenosine deaminase (adar- ) to complete its life cycle (reviewed in [ ] ). likewise, it is striking to note that only the c and prec/hbeag coding regions are being footprinted and not the others coding sequences downstream. in fact, the mutational load introduced by the a s is much stronger on the ' end of the negative strand (corresponding to the pol, s and x regions) because the newly synthesized double strand dna eventually displaces the single strand dna from the capsid walls, making it accessible to deaminase activity [ ] . thus, the ' end of the negative strand is found to be frequently hypermutated; the term hypermutation referring to as mutations clustered on a short sequence. it is essential to underline that our observations reflect the a -induced mutations which were conserved and not those which put an end to the viral cycle. consequently, the phenomenon of hypermutation will not leave an evolutionary footprint. in this respect, the hbv mutation spectrum from in vivo cirrhotic samples shows that even though the majority of hbv genomes are strongly mutated by a s and these virions are probably defective, a small fraction is slightly modified and may therefore still be infectious [ ] . finally, the pol, s and x genes overlap on different reading frames, which implies that these coding regions are less permissive to mutations (a silent mutation in one frame may not be in the other frame). we paid particular attention to hiv in our analysis as the apobec antiviral activity has been historically discovered in that field of research [ ] . we observed a weak a evolutionary footprint on the minus strand of the hiv genomes in support of the observations made by jern et al. [ ] . the weakness of the footprint can be explained at least in part by the efficiency of the a -inhibiting protein vif. also, the error-prone rt could be responsible for the reversion of some a -induced mutations providing that the virus can still complete its life cycle. finally, a s are well known to also restrict hiv through a deaminase independent activity, therefore without leaving any footprint. the intensity of the a footprint is very strong in many papillomaviridae and polyomaviridae. several viruses of these families are well-known tumor viruses (hpv- , hpv- , merkel cell polyomavirus, etc.) and a mechanistic link between a expression and the development of cancer has been established in hpv positive cervical and oesopharyngeal cancers [ , ] . s fig shows oncogenic viruses (confirmed or suspected) and their respective a footprint. it illustrates that an a footprint is not present in every tumor virus. hcv shows no a -footprint, is still definitely a tumor virus. nevertheless, we wonder whether the presence of a strong footprint may suggest involvement in cancer. the bk and jc polyomaviruses have a footprint as intensive as hr-hpvs (s fig). bk pyv infects the kidneys and the urinary tract and is suspected of playing a role in certain bladder cancer where viral expression and integration have been reported [ , ] . bk pyv triggers a expression in vitro and the a -related mutations found in bladder tumors account for two-thirds of the total mutational load [ , ] . similarly, along with the alpha, the beta-papillomaviridae show a similar a footprint (fig b) . some members of the beta-papillomavirus genus are suspected to play a role in non-melanoma skin cancer (cutaneous squamous-cell carcinoma) [ , ] . even so they do not seem to insert into the cell genome, they might promote carcinogenesis initiation. some β-hpvs were demonstrated to potentiate the deleterious effect of uv radiations and to drive skin carcinogenesis in mice with a hit-and-run mechanism [ ] . finally, the erythroparvovirus b is one of the most footprinted virus. while its replication occurs primarily in erythroid tissues, the erythroparvovirus b commonly persists in a wide range of tissues [ ] . a link between the erythroparvovirus b and thyroid cancer has been proposed but evidence is scarce to date ( ) ( ) ( ) . of note, the a -related mutations in thyroid tumors make about % of the total mutational load [ ] . we think that further research should be carried out to rule in or out the involvement of these later viruses in cancer. in conclusion, the present study represents the first global screening for the a selection pressure on all currently annotated human viruses. we demonstrate that many papillomaviridae, polyomaviridae, autonomous parvoviridae and coronaviridae can thrive despite being under the selective pressure of the a proteins. those viruses cope with a editing activity thanks to a deep cleansing of a -favored motifs in their genome. herpesviridae and adenoviridae display a subtler a footprint limited to the lytic origins of replication, probably thanks to active mechanisms of a inhibition. a deamination appears to occur during replication of viral dna (sometimes limited to the lagging strand) for the double-stranded dna viruses and/or inside the capsid for the single-stranded dna and rna viruses. the causal link established between hpv infection and the a mutational signature in human cancer also lead us to propose to consider the beta-papillomaviridae and the erythroparvovirus b as potentially promoting a expression and therefore exposing the cell genome to a mutagenic activity. we downloaded complete viral genomes from the "ncbi virus" database (https://www.ncbi. nlm.nih.gov/labs/virus/vssi/#/) as released in april . we retrieved only full-length genomes by selecting "complete" for the criterion "nucleotide completeness". we retrieved human viruses by selecting "humans" for the criterion "host". we retrieved non-human primate viruses by selecting "primate" for the criterion "host" and by deducting the human viruses from this data set. we retrieved avian viruses by selecting "aves (birds)" for the criterion "host". we retrieved fish viruses by selecting "actinopterygii (ray-finned fish)" for the criterion "host". we also retrieved camel mers viruses by selecting "camelus dromedaries (arabian camel)" for the criterion "host" and "mers-cov" for criterion "virus". we retrieved bat-mers viruses by selecting "chiroptera (bats)" for the criterion "host" and "mers-cov" for the criterion "virus". we retrieved bat-sars viruses by selecting "chiroptera (bats)" for the criterion "host" and "severe acute respiratory syndrome-related coronavirus" for criterion "virus". the dataset of human viruses was supplemented by manually curated human virus complete genome sequences from the "ncbi nucleotide" database. using these criteria, , human, , non-human primate, , avian, fish, camel mers, bat mers and bat sars full-length viral genomes were collected. genbank accession id's are treated as unique and listed in the s , s and s tables. a k-mer encompasses a collection of sequences with a common motif. for instance, the ntc k-mer includes the atc, ctc, gtc and ttc sequences. in addition, as we limit our analysis to coding sequences, we force our k-mers to be in the reading frame and therefore to correspond to codons. for example, the ntc k-mer actually includes the atc, ctc, gtc and ttc codons. following the same logic, the nntcnn k-mer comprises the pair of codons having a t at the end of the first codon and a c to start the second codon. we calculated the observed vs. expected k-mer representation ratio as described by warren et al. [ ] . briefly, each coding sequence has been randomly shuffled a thousand times, retaining only the nucleotide composition. the expected count of a given k-mer is calculated as the average of the occurrences of this k-mer over the thousand iterations. the k-mer ratio is given as the log ratio of the observed occurrence of this k-mer to the expected occurrence. to calculate the ratio of a given k-mer for an entire viral genome, a "synthetic coding genome" was generated by concatenating the different coding sequences (fig b) . the synthetic coding sequence is then randomly shuffled a thousand times and k-mer ratio calculated as above. a k-mer ratio << indicates k-mer under representation and a k-mer ratio equal to zero means that no representation bias is observed. unpaired student's t test has been used where appropriate. the results were considered statistically significant at a p-value of < . . all boxplot, heatmap and map representations have been generated using ggplot r package. supporting information s fig. search for a g-footprinted human viruses. a. a g favors deamination of cytidine when preceded by another cytidine. the 'cc dinucleotide motif is depicted in three possible codon contexts on both coding and template strand. depending on the position of the mutated c, the c to t transition can be synonymous (s) or non-synonymous (ns). proportion of s and ns mutations is reported when the two types of mutation can be produced. because synonymous mutations are more likely to be retained, a g-footprinted viruses should display to a stronger depletion of ncc codons compared to ccn or nnccnn motifs (and/or a depletion of nnggnn motifs versus the gnn and ngg motifs). b. the ncc and nnggnn observed/expected ratios for , human viruses' genomes (from unique species) were calculated, grouped by species and colored according to the baltimore classification. each point represents a unique viral genome. viral species with an ncc or nnggnn ratio below two times the standard deviation (dotted grey line) from the population median (red line) are retained for further analysis in panel c. c. the observed/expected ratios of 'cc dinucleotide at various codon positions and on both strands (i.e. nnccnn, ccn, ncc, ggn, ngg and nnggnn) were calculated for the ncc and/or nnggnn depleted viral species and depicted by a heatmap. a colored scale with increasing shades of blue indicating depletion and increasing shades of red indicating enrichment. p-values were calculated by student's unpaired, twotailed t-test (ns for not significant, � p< . , �� p< . , ��� p< . ). d. list of the viral genes displaying ncc or nnggnn depletion and belonging to an otherwise non-depleted viral genome. (pdf) fig. a footprint on hpv , hpv and hpv . ntc and nngann observed/ expected ratios were calculated for the different genes of the hpv , hpv and hpv and were reported on their genomic maps using a colored scale with increasing shades of blue indicating ntc depletion and increasing shades of red indicating ntc enrichment. replication origin is illustrated by a black dot and gene transcriptional orientation is symbolized by black arrow. (pdf) a. aid favors cytidine deamination in a ' wrc context. the wrc trinucleotide motif is depicted in three possible codon contexts on both coding and template strand. depending on the position of the mutated c, the c to t transition can be synonymous (s) or non-synonymous (ns). proportion of s and ns mutations is reported when the two types of mutation can be produced. b. the wrc and nngywn observed/expected ratios for , human viruses' genomes (from unique species) were calculated, grouped by species and colored according to the baltimore classification. each point represents a unique viral genome. c. list of the putative aid-footprinted viral genes (displaying wrc or nngywn depletion) and belonging to an otherwise non-depleted viral genome. (pdf) s fig. search for apobec -footprinted viruses. a. apobec favors cytidine deamination in a ' wcw context. the wcw trinucleotide motif is depicted in three possible codon contexts on both coding and template strand. depending on the position of the mutated c, the c to t transition can be synonymous (s) or non-synonymous (ns). proportion of s and ns mutations is reported when the two types of mutation can be produced. b. the nwcwnn and nwgwnn observed/expected ratios for , human viruses' genomes (from unique species) were calculated, grouped by species and colored according to the baltimore apobec footprint on human viruses classification. each point represents a unique viral genome. c. the observed/expected ratios of wcw trinucleotide at various codon positions and on both strands (i.e. nwcwnn, wcw, nnwcwn, nwgwnn, wgw and nnwgwn) were calculated for the nwcwnn and/or nwgwnn depleted viral species and depicted by a heatmap. a colored scale with increasing shades of blue indicating depletion and increasing shades of red indicating enrichment. p-values were calculated by student's unpaired, two-tailed t-test (ns for not significant, � p< . , �� p< . , ��� p< . ). d. list of the putative apobec -footprinted viral genes (displaying nwcwnn or nwgwnn depletion) and belonging to an otherwise non-depleted viral genome. (pdf) s fig. a footprint in human oncogenic viruses. ntc and nngann observed/expected ratios were calculated for each available coding sequence of eleven well-known cancer-related viruses. each point represents a unique viral coding sequence. the coding sequences are grouped and colored according to gene name. (pdf) s apobecs and virus restriction apobec interference during replication of viral genomes the apobec protein family: united by structure, divergent in function an ancient history of gene duplications, fusions and losses in the evolution of apobec mutators in mammals dna deaminases induce break-associated mutation showers with implication of apobec b and a in breast cancer kataegis isolation of a human gene that inhibits hiv- infection and is suppressed by the viral vif protein apobec g targets human t-cell leukemia virus type inhibition of hepatitis b virus replication by apo-bec g hypermutation of hiv- dna in the absence of the vif protein broad antiretroviral defence by human apobec g through lethal editing of nascent reverse transcripts extensive editing of a small fraction of human t-cell leukemia virus type genomes by four apobec cytidine deaminases apobec g generates nonsense mutations in human t-cell leukemia virus type proviral genomes in vivo extensive editing of both hepatitis b virus dna strands by apobec cytidine deaminases in vitro and in vivo genetic editing of hbv dna by monodomain human apobec cytidine deaminases and the recombinant nature of apo-bec g genetic editing of herpes simplex virus and epstein-barr herpesvirus genomes by human apobec cytidine deaminases in culture and in vivo evidence for editing of human papillomavirus dna by apobec in benign and precancerous lesions functional upregulation of the dna cytosine deaminase apobec b by polyomaviruses resistance of human t cell leukemia virus type to apobec g restriction is mediated by elements in nucleocapsid epstein-barr virus borf inhibits cellular apobec b to preserve viral genome integrity role of the host restriction factor apo-bec on papillomavirus evolution footprint of apobec on the genome of human retroelements likely role of apobec g-mediated g-to-a mutations in hiv- evolution and drug resistance evolutionary effects of the aid/apobec family of mutagenic enzymes on human gamma-herpesviruses cytosine deamination and selection of cpg suppressed clones are the two major independent biological forces that shape codon usage bias in coronaviruses the preferred nucleotide contexts of the aid/apobec cytidine deaminases have differential effects when mutating retrotransposon and virus sequences compared to host genes the cytidine deaminase under-representation reporter (cdur) as a tool to study evolution of sequences under deaminase mutational pressure the artiodactyl apo-bec innate immune repertoire shows evidence for a multi-functional domain organization that existed in the ancestor of placental mammals why is cpg suppressed in the genomes of virtually all small eukaryotic viruses but not in those of large eukaryotic viruses? class switch recombination and hypermutation require activation-induced cytidine deaminase (aid), a potential rna editing enzyme a novel form of tissue-specific rna processing produces apolipoprotein-b in intestine proposed nomenclature for the catalytic subunit of the mammalian apolipoprotein b mrna editing enzyme: apobec- aid and apobecs span the gap between innate and adaptive immunity the mutation spectrum of purified aid is similar to the mutability index in ramos cells and in ung(-/-)msh (-/-) mice transcriptome-wide sequencing reveals numerous apobec mrna-editing targets in transcript ' utrs apobec a functions as a restriction factor of human papillomavirus human papillomavirus e upregulates apobec b via the tead transcription factor human papillomavirus e stabilizes apobec a protein by inhibiting cullin -dependent protein degradation polyomavirus t antigen induces apobec b expression using an lxcxe-dependent and tp -independent mechanism human papillomavirus e triggers upregulation of the antiviral and cancer genomic dna deaminase apobec b. mbio roles of apobec a and apobec b in human papillomavirus infection and disease progression the curious case of apobec activation by cancer-associated human papillomaviruses human parvoviruses analysis of the termini of the dna of bovine parvovirus: demonstration of sequence inversion at the left terminus and its implication for the replication model apobec a cytidine deaminase induces rna editing in monocytes and macrophages transient overexpression of exogenous apobec a causes c-to-u rna editing of thousands of genes mitochondrial hypoxic stress induces widespread rna editing by apobec g in natural killer cells infectious vaccine-derived rubella viruses emerge, persist, and evolve in cutaneous granulomas of children with primary immunodeficiencies apobec g cytidine deaminase association with coronavirus nucleocapsid protein evidence for host-dependent rna editing in the transcriptome of sars-cov- human coronaviruses: what do they cause? antivir ther differential evolution of antiretroviral restriction factors in pteropid bats as revealed by apobec gene complexity a sars-cov- protein interaction map reveals targets for drug repurposing cellular requirements for biv vif-mediated inactivation of bovine apobec proteins coding potential and sequence conservation of sars-cov- and related animal viruses a conserved mechanism of apobec relocalization by herpesviral ribonucleotide reductase large subunits. biorxiv four-dimensional analyses show that replication compartments are clonal factories in which epstein-barr viral dna amplification is coordinated apobec a and apobec b preferentially deaminate the lagging strand template during dna replication apobecinduced mutations in human cancers are strongly enriched on the lagging dna strand during replication mutational strand asymmetries in cancer genomes reveal mechanisms of dna damage and repair apo-bec-induced cancer mutations are uniquely enriched in early-replicating, gene-dense, and active chromatin regions massive apobec editing of hepatitis b viral dna in cirrhosis immunomodulatory function of hbeag related to short-sighted evolution, transmissibility, and clinical manifestation of hepatitis b virus. front microbiol adenosine deaminases acting on rna (adars) and a-to-i editing asymmetric modification of hepatitis b virus (hbv) genomes by an endogenous cytidine deaminase inside hbv cores informs a model of reverse transcription apobec-mediated cytosine deamination links pik ca helical domain mutations to human papillomavirus-driven tumor development the apobec genes and their role in cancer: insights from human papillomavirus the landscape of viral expression and host gene fusion and adaptation in human cancer the case for bk polyomavirus as a cause of bladder cancer signatures of mutational processes in human cancer beta genus papillomaviruses and skin cancer the biology of beta human papillomaviruses beta hpv oncoproteins act with a hit-and-run mechanism in ultraviolet radiation-induced skin carcinogenesis in mice persistent parvovirus b infection in non-erythroid tissues: possible role in the inflammatory and disease process we thank members of the urvi lab for valuable discussions.validation: florian poulain. writing -review & editing: nicolas a. gillet. key: cord- -t l i f authors: kovalev, nikolay; nagy, peter d. title: the expanding functions of cellular helicases: the tombusvirus rna replication enhancer co-opts the plant eif aiii-like atrh and the ddx -like atrh dead-box rna helicases to promote viral asymmetric rna replication date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: t l i f replication of plus-strand rna viruses depends on recruited host factors that aid several critical steps during replication. several of the co-opted host factors bind to the viral rna, which plays multiple roles, including mrna function, as an assembly platform for the viral replicase (vrc), template for rna synthesis, and encapsidation during infection. it is likely that remodeling of the viral rnas and rna-protein complexes during the switch from one step to another requires rna helicases. in this paper, we have discovered a second group of cellular rna helicases, including the eif aiii-like yeast fal p and the ddx -like dbp p and the orthologous plant atrh and atrh dead box helicases, which are co-opted by tombusviruses. unlike the previously characterized ddx -like atrh /ded p helicases that bind to the ′ terminal promoter region in the viral minus-strand (−)rna, the other class of eif aiii-like rna helicases bind to a different cis-acting element, namely the ′ proximal riii(−) replication enhancer (ren) element in the tbsv (−)rna. we show that the binding of atrh and atrh helicases to the tbsv (−)rna could unwind the dsrna structure within the riii(−) ren. this unique characteristic allows the eif aiii-like helicases to perform novel pro-viral functions involving the riii(−) ren in stimulation of plus-strand (+)rna synthesis. we also show that atrh and atrh helicases are components of the tombusvirus vrcs based on co-purification experiments. we propose that eif aiii-like helicases destabilize dsrna replication intermediate within the riii(−) ren that promotes bringing the ′ and ′ terminal (−)rna sequences in close vicinity via long-range rna-rna base pairing. this newly formed rna structure promoted by eif aiii helicase together with atrh helicase might facilitate the recycling of the viral replicases for multiple rounds of (+)-strand synthesis, thus resulting in asymmetrical viral replication. host factors co-opted for replication of plus-stranded (+)rna viruses are critical in each step of the well-orchestrated infection process. after translation of the viral mrna-sense genomic rna(s), the viral (+)rna and the viral replication proteins together with host rna-binding proteins (rbps) are recruited to the site of viral replication in membranous cellular compartments. ultimately, the process leads to the assembly of the membranebound viral replicase complexes (vrcs), followed by the activation of the polymerase function of the viral rna-dependent rna polymerase (rdrp), and initiation of complementary rna synthesis on the viral (+)rna template [ ] [ ] [ ] [ ] . subsequent (+)strand synthesis in the vrcs takes place in an asymmetric manner, producing excess amounts of (+)-strand progeny, which is released from replication to participate in encapsidation, cell-tocell movement and other viral processes. although the roles of host factors in facilitating the replication process of (+)rna viruses have been extensively characterized in recent years [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , our current understanding of the role of cellular rbps, which constitute one of the largest groups of host factors identified is incomplete [ , , ] . the co-opted rbps likely affect several steps in viral rna replication, including viral (+)rna recruitment, stabilization of the viral rna, vrc assembly and viral rna synthesis. tomato bushy stunt virus (tbsv) is a plant rna virus with a single , , nt genomic rna and has two essential replication proteins, p and p pol , required for tbsv replicon (rep)rna replication in yeast (saccharomyces cerevisiae) model host [ , ] . the membrane-bound tombusvirus vrc contains p and p pol , and the tombusviral (+)reprna, which serves both as a template and as a platform during vrc assembly and activation [ ] [ ] [ ] [ ] [ ] . interestingly, the tombusvirus vrc contains at least seven host proteins as resident members, including glyceraldehyde- -phosphate dehydrogenase (gapdh, encoded by tdh and tdh in yeast) [ ] , the heat shock protein chaperones (hsp , ssa / p in yeast) [ ] [ ] [ ] [ ] , pyruvate decarboxylase (pdc p) [ ] , cdc p e ubiquitin conjugating enzyme [ ] , eukaryotic translation elongation factor a (eef a) [ , ] , eef bc [ ] , and ded p dead-box helicase [ ] . the vrc also contains two temporary resident proteins, pex p shuttle protein [ ] and the vps p adaptor escrt protein [ , , ] . detailed mechanistic studies revealed that the cellular hsp , eef a and vps p are involved in the assembly of the vrc, while the functions of host rbps, such as eef a, eef bc, gapdh and ded p, are to regulate viral rna synthesis by the vrc [ , [ ] [ ] [ ] [ ] , , ] . in spite of our growing understanding of tbsv replication and tbsv-host interaction, many questions remain. indeed, multiple genome-wide screens and global proteomics approaches with tbsv using yeast as host identified , host factors, which interact with viral replication proteins or affect tbsv replication [ , , , [ ] [ ] [ ] [ ] [ ] . among the host proteins identified are host atp-dependent rna helicases out of known yeast helicases that could be involved in tbsv replication. dead-box proteins constitute the largest family of rna helicases, which perform atp-dependent rna duplex unwinding, rna folding, remodeling of rna-protein complexes, and rna clamping in cells. dead-box helicases are involved in all aspects of cellular metabolism [ ] [ ] [ ] and affect replication of many viruses [ ] , including plant rna viruses [ ] . plant rna helicases are also implicated in plant responses to abiotic stress and pathogen infections [ ] [ ] [ ] . the many cellular rna helicases identified in tbsv screens are intriguing because, tombusviruses and other small rna viruses do not code for their own helicases [ , ] . these viruses likely recruit host helicases in order to facilitate viral replication. the best-characterized member of the helicase family involved in tombusvirus replication is the yeast ded p (the human ddx like) and the similar plant atrh dead-box helicases, both of which promote (+)-strand synthesis [ ] . ded p and atrh bind to the -end of the tbsv minus-strand rna, making the promoter sequence accessible to p pol for initiation of (+)-strand rna synthesis. additional characterization of ded p and the similar yeast dbp p (similar to human p ) dead-box helicases revealed that these helicases play major and overlapping roles in (+ )-strand synthesis [ , ] . altogether, the identification of yeast rna helicases involved in tombusvirus replication suggests that tombusviruses likely co-opt a number of host rna helicases and these helicases might have a number of unique functions in viral replication. in this paper, we characterized the novel pro-viral functions of two yeast rna helicases, which were among those identified in previous screens, and their plant orthologs in tbsv replication. we found that the yeast dbp p (dead box protein , human ddx -like) and fal p (eukaryotic translation initiation factor aiii-like) dead box helicases, which are involved in ribosome biogenesis in yeast [ ] [ ] [ ] , and the orthologous arabidopsis rh and rh helicases bind to a critical replication enhancer element (ren) present in a proximal region of the tbsv minus-strand rna. we show that these cellular helicases can locally unwind the double-stranded (ds) structure within the ren of the replication intermediate in vitro. these activities by the host helicases enhance in vitro replication and plus-strand rna synthesis, and the accumulation of tbsv rna in yeast and plants. we also demonstrate that atrh and atrh helicases work synergistically with the ddx -like atrh helicase (that binds to the promoter sequence) to facilitate plus-strand synthesis in an asymmetric manner. altogether, these co-opted host dead box helicases greatly enhance tbsv replication by interacting with the viral ( )rna and the replication proteins within the vrcs. overexpression of yeast eif aiii-like fal p or dbp p dead-box rna helicases and the orthologous plant atrh and ddx -like atrh helicases enhances tbsv rna replication in yeast and plants to characterize the functions of host rna helicases in tbsv replication, first we overexpressed yeast rna helicases in yeast and tested their effects on tbsv replicon (rep)rna accumulation via northern blotting (fig. s ). these helicases were chosen from the previously identified yeast helicases from several complementary high throughput screens using yeast and tombusviruses [ , , , , , , ] . overexpression of all host rna helicases increased tbsv accumulation, with the yeast dbp p showing the highest (over -fold increase) stimulation ( fig. s a-b) . the overexpression of these host helicases did not affect the accumulation of p replication protein ( fig. s a-b) , suggesting that the effects of this group of rna helicases are not through increased translation of viral replication proteins. altogether, the observed -to- % increase in tbsv rna accumulation due to overexpression of these yeast helicases is significant since overexpression of most yeast proteins nonspecifically reduces tbsv accumulation by - % as demonstrated before based on individual overexpression of , yeast proteins [ , ] . thus, these helicases likely play stimulatory roles in tbsv replication. for additional in-depth studies, we have selected the yeast dbp p and the highly similar fal p (human eif aiii-like) together with the orthologous arabidopsis atrh (fal p ortholog and human eif aiii-like) and atrh (dbp p ortholog, human ddx -like) helicases [ ] ]. overexpression of atrh and atrh stimulated (up to -fold increase) tbsv reprna accumulation in yeast (fig. a) . the stimulation of tombusvirus accumulation (we used cucumber necrosis virus, cnv, which is very closely related to tbsv) was also robust in nicotiana benthamiana host plants when atrh and atrh rna helicases were overexpressed (up to , - genome-wide screens for host factors affecting tombusvirus replication in yeast indicated that subverted cellular rna helicases likely play major roles in virus replication. tombusviruses do not code for their own helicases and they might recruit host rna helicases to aid their replication in infected cells. accordingly, in this paper, the authors show that the yeast eif aiii-like fal p and dbp p and the orthologous plant atrh and atrh dead-box helicases are co-opted by tomato bushy stunt virus (tbsv) to aid viral replication. the authors find that eif aiii-like helicases bind to the replication enhancer element (ren) in the viral ( )rna and they promote (+)strand tbsv rna synthesis in vitro. data show that eif aiiilike helicases are present in the viral replicase complex and they bind to the replication proteins. in addition, the authors show synergistic effect between eif aiii-like helicases and the previously identified ddx -like ded p/ atrh dead box helicases, which bind to a different cisacting region in the viral ( )rna, on stimulation of plusstrand synthesis. in summary, the authors find that two different groups of cellular helicases promote tbsv replication via selectively enhancing (+)-strand synthesis through different mechanisms. fold increase in tombusvirus genomic rna accumulation, and , -fold increase in subgenomic rna accumulation, fig. b , lanes - and - versus - ), suggesting that these cellular helicases are important host factors. while overexpression of atrh and atrh did not affect the phenotype of uninoculated n. benthamiana plants, the tombusvirus-induced symptoms were intensified (fig. ) and the symptoms appeared faster (not shown), when compared with the control host plants not overexpressing the atrh and atrh helicases. simultaneous co-overexpression of atrh and atrh increased tombusvirus replication by up to -fold ( fig. b, lanes - ) , similar to the level obtained with individual overexpression of atrh and atrh . also, the symptoms induced by tombusvirus infection in plants with cooverexpression of atrh and atrh were comparable to those induced by the individual overexpression of atrh and atrh (fig. ) . therefore, it is likely that atrh and atrh play comparable and overlapping functions in tombusvirus replication. overall, the host helicase overexpression studies in yeast and plant established that atrh and atrh helicases and the orthologous yeast dbp p and fal p helicases could support increased level of tombusvirus rna replication in host cells. novel pro-viral function of atrh , atrh and the yeast dbp p and fal p is to bind to the viral replication enhancer present in the tombusvirus minus-strand rna to test if atrh , atrh and the yeast dbp p and fal p play a comparable role with the previously analyzed yeast ded p (human ddx -like) and dbp p (human p -like) and the ortologous atrh dead-box helicases [ , ] , first we performed in vitro rna binding experiments with affinity-purified recombinant helicase proteins. using four different cis-acting regions present in the tbsv ( )reprna (fig. a) , we found that atrh and atrh bind to a unique cis-acting sequence in a proximal region of the minus-strand rna, called riii( ), which carries a well-defined rna replication enhancer (ren) element (fig. b , lanes - ; and s a, lanes - ) [ , ] . the recombinant yeast dbp p and fal p rna helicases showed similar rna binding characteristics to the riii( ) ren in vitro ( fig. s b-c) . importantly, binding of atrh , atrh and the yeast dbp p and fal p to the tbsv riii( ) ren element is a novel feature for co-opted host helicases. indeed, the previously characterized atrh and the yeast ded p and dbp p dead-box helicases bound the most efficiently to ri( ) sequence carrying the plusstrand initiation promoter ( fig. a; fig. d versus c) [ , ] . this striking difference in recognition of two separate cis-acting elements [indeed riii( ) ren is located close to the end, while ri( ) promoter region is situated at the end of the viral ( )rna, fig. a ] by these host helicases indicate that their functions and the mechanism of stimulation of tbsv rna replication must be different. to confirm binding of atrh to the riii( ) ren, we also performed uv cross-linking experiments with p-labeled di- ( ) rna and cold competitors (fig. e ). the nt complete riii( ) ren was a better competitor than similar-sized rna lacking one of the stem-loop structure and a ''bridge'' sequence that can base-pair with ri( ) sequence via long-range interaction (fig. f , lanes - versus - ) [ ] . our data also support a role in vitro binding assay with purified atrh . the assay contained the p-labeled di- ( )reprna (, . pmol) plus increasing amount of unlabeled competitor rnas, each used in the same amounts, including ri( ) ( and pmol), rii( ) ( and pmol), riii( ) ( and pmol) or riv( ) ( and pmol). the free or atrh -bound ssrna was separated on nondenaturing % acrylamide gels. (c) rna gel shift analysis shows that atrh binds the most efficiently to riii( ). p-labeled di- ( )reprna template (, . pmol) from fhv and unlabeled competitor rnas ( and pmol) representing one of the four regions of tbsv di- rna from both rna strands (see panel a) were used in the competition assay. the atrh - p-labeled ssrna complex was visualized on nondenaturing % acrylamide gels. each experiment was repeated at least three times. note that we used the heterologous fhv di- ( )rna in the binding assay to allow comparison of (+) versus ( )rna regions of tbsv rna. the template competition assay showed efficient binding/competition by the riii( ) and ri(+) sequences for atrh . (d) comparable viral rna binding assay reveals different binding specificity for ddx -like atrh and the ddx -like atrh dead-box helicases. see additional details in panel c. the template competition assay showed efficient binding/competition by the ri( ), rii( ), riv( ) and ri(+) sequences for atrh . (e) schematic representation of the long-range rna-rna interaction between the ''base'' sequence in the cpr promoter in ri( ) and the complementary ''bridge'' sequence in riii( ) ren [ ] . the competitor rnas used in panel f are shown schematically. (f) the bridge sequence contributes to binding of riii ( ) to atrh in vitro. top image: rna-binding analysis of atrh -di- ( )reprna interaction after uv cross-linking. for the bridge sequence in binding to atrh (compare lanes - with - , fig. f ). the efficient binding of atrh , atrh and the yeast dbp p and fal p to the riii( ) ren region indicates that these helicases might facilitate the unwinding of rna structures within the riii( ) ren during replication. therefore, we tested if recombinant atrh and atrh could unwind partial rna duplexes, which are known to hinder rdrp-driven rna synthesis [ , ] . we chose partial duplex for this assay, because dead-box helicases are not processive enzymes and can only unwind short duplexes [ ] . interestingly, addition of purified atrh and atrh unwound the partial rna duplex (fig. b , lanes and ) and the yeast dbp p and fal p showed similar activities (lanes - ). in contrast, ded p and atrh helicases did not efficiently unwind the rna duplex formed only within the riii( ) ren ( fig. b , lanes and ). the failure to unwind this partial duplex by ded p and atrh is likely due to the preference of these helicases to bind the ri( ) sequence of the tombusvirus rna ( fig. ) [ , ] . overall, the unwinding assay further supported that atrh , atrh and the yeast dbp p and fal p have a novel pro-viral function during tbsv replication that is based on interaction with the riii( ) ren element. to test the direct effect of atrh and atrh helicases on tbsv rna synthesis, we utilized detergent-solubilized and affinity-purified tombusvirus replicase from yeast with down regulated fal p (fig. a ). the requirement for fal p helicase on rna synthesis was supported by the observed , % decrease of the in vitro activity of the purified replicase obtained from yeast with depleted fal p when compared with the replicase from yeast expressing fal p at high level (fig. b ). this purified replicase can only synthesize complementary rna products on added tbsv templates allowing for the measurement of the level of rna synthesis [ , ] . we found that addition of purified recombinant atrh and atrh helicases to the purified tombusvirus replicase (obtained from fal p depleted yeast) programmed with the di- ( )reprna stimulated (+)-strand synthesis by up to -fold ( fig. c , lanes versus and versus ). interestingly, atrh and atrh helicases stimulated the production of both full-length (+)-strand reprna product (via de novo initiation) and the -terminal extension product ( tex; due to initiation of complementary rna synthesis by selfpriming from the end of the template, instead of de novo initiation [ ] [ ] [ ] ). this is in contrast with atrh and ded p helicases, which stimulated the production of mostly full-length (+)-strand reprna product (fig. c , lanes and ) [ , ] . time-course experiments with the purified tombusvirus replicase and a minimal template carrying ri( ) and riii( ) ren sequences confirmed that atrh and atrh helicases stimulated the production of both full-length (+)-strand rna and tex products by ,two-fold at both early and late time points (fig. d) . interestingly, the stimulation of rna synthesis by atrh and atrh helicases is lost when we used a ( )reprna lacking sequences including riii( ) ren region (fig. e , lanes - versus ). this is in contrast with atrh , which was able to stimulate the tombusvirus replicase activity on this template rna (fig. e , lane ). this observation was confirmed in in vitro timecourse experiments with the purified tombusvirus replicase and a template lacking riii( ) ren by showing the absence of stimulation of rna synthesis products by atrh and atrh helicases at both early and late time points (fig. f ). therefore, we suggest that atrh and atrh helicases depend on riii( ) ren to facilitate the overall efficiency of template use and rna synthesis on the ( )rna template by the tombusvirus replicase. to test if atrh and atrh helicases could also stimulate rna synthesis by the tombusvirus replicase on dsrna templates, which are formed during tbsv replication (kovalev et al, in press), we used partial dsrna duplexes (fig. a ). the tombusvirus replicase is inefficient utilizing these dsrna templates in vitro [ , , ] . the addition of recombinant atrh and atrh helicases stimulated rna synthesis by up to , . -fold in vitro on a partial dsrna template that had both ri( ) and riii( ) ren as part of the duplex (fig. a , construct drii and b, lanes and versus and ). this level of stimulation was comparable to that obtained with atrh that targets the ri( ) sequence ( fig. b , lanes and ). atrh and atrh helicases also stimulated rna synthesis on a complete dsrna template ( fig. s a and s b, lanes and versus ), producing mostly (+)rna products (fig. s c) . importantly, the stimulation of rna synthesis by atrh and atrh helicases was lost when a partial dsrna template lacking the riii( ) ren was used (fig. a , construct driii and b, lanes and versus and ). in contrast, atrh was still able to stimulate rna synthesis on this template (fig. b , lanes and ), as predicted based on the ability of atrh to bind to ri( ) sequence [ , ] . based on these data, we conclude that atrh to further study the roles of atrh and atrh helicases in tbsv replication, we used whole cell extracts (cfe) prepared from yeast containing temperature-sensitive (ts) fal p and lacking dbp p to support cell-free tbsv replication. tbsv (+)rna has been shown to perform one full cycle of replication, starting with vrc assembly, ( )rna synthesis and finally production of excess amount of (+)-strands, in the cfe-based replication assay when purified recombinant p and p pol replication proteins are included [ , ] . the cfe-based replication assay showed that (+ )rna synthesis decreased by , -fold when compared with the control cfe prepared from yeast with high level of dbp p and wt fal p (fig. s b , lane versus ). this is in contrast with ( )rna synthesis (represented by dsrna product), which was unchanged when the cfe was prepared from dbp d/ts-fal yeast versus wt yeast. however, addition of purified atrh or atrh to the cfe assay increased (+)rna production by , - %, while the ( )rna (in the form of dsrna) was unchanged (fig. s c-d) . thus, these data with cfe-based approaches confirm that atrh and atrh and the ortologous yeast helicases are important for (+ )rna synthesis during tbsv replication in vitro. to examine if atrh and atrh helicases are present within the tombusvirus replicase complex, we flag affinity-purified the tombusvirus replicase from yeast cells actively replicating tbsv reprna [ , ] . the yeast cells also expressed either his -tagged atrh or his -atrh helicases from plasmids. we found that the solubilized and affinity-purified tombusvirus replicase preparation, which is highly active on added templates in vitro (not shown), contained his -atrh (fig. a, lane ) , while his -atrh was undetectable in the control yeast sample obtained using the same affinity purification (fig. a, lane ) . formation of active replicase complex was not necessary for his -atrh to become co-opted since the inactive purified replicase [the tombusvirus replicase is inactive in the absence of the viral rna; [ , ] ] contained his -atrh when derived from yeast lacking the viral reprna (fig. a, lane ) . we found that his -atrh showed similar characteristics in these co-purification experiments (fig. s a) . to test if the tbsv p replication protein interacts directly with atrh and atrh , we performed membrane-based splitubiquitin yeast two-hybrid assay. this assay confirmed the interaction between p and atrh and atrh (fig. b) . the yeast dbp p and fal p dead-box helicases also interacted with p in this assay (fig. s b) . to test what region within the tbsv p replication protein is involved in the interaction with atrh and atrh , we performed pull-down experiments with mbp-tagged p derivatives from e. coli. these experiments revealed that the rpr-motif in p involved in viral rna-binding was responsible for interacting with both atrh and atrh (fig. s a-b) . interestingly, the interaction of p with atrh and atrh did not affect the ability of p to bind to the viral (+)reprna in vitro (fig. s ) . the interaction between p and (+)reprna is required for recruitment of the viral (+)rna into replication [ , ] . based on the above interaction data, we suggest that the viral p replication protein (and p replication protein, which within its n-terminal region contains the p sequence due to the expression strategy) co-opts atrh and atrh dead-box proteins from the host cells into the viral replicase complexes to aid the replication process. the difference in viral rna-binding by atrh /atrh versus atrh suggests that these groups of helicases could have synergistic effect on tombusvirus replication. this was tested by coexpressing atrh and atrh in n. benthamiana leaves replicat- ing the tombusvirus rna (fig. ) . interestingly, atrh and atrh host proteins, when co-expressed together, had the largest (up to , . -fold, fig. a, lanes - ) effect on viral genomic rna accumulation in comparison with the , -fold increase for separate expression of atrh (lanes - ) and atrh (lanes - ) . also, the symptom development of tombusvirus-infected plants was the most severe and the fastest when the two helicases were co-expressed (fig. c ). based on these data, we suggest that atrh and atrh have a synergistic effect on tombusvirus replication (see further explanation in discussion). replication of (+)rna viruses is performed by viral replicases (vrcs), which are membrane-bound ribonucleic acid-protein complexes (rnp) [ , , ] . the vrcs likely have to remodel viral rna structures, including the dsrna formed during replication. in addition, the viral rna plays multiple roles, such as template for translation and rna synthesis, and as a vrc assembly platform and the viral rna also becomes encapsidated during infection [ ] [ ] [ ] ] . it is likely that remodeling of the viral rnas and rnp complexes during the switch from one step to another requires rna helicases or rna chaperones. while the larger rna viruses over , nt genome-size all code for rna helicase-like proteins [ , ] , small rna viruses usually do not code for rna helicases. however, the small rna viruses likely coopt cellular rna helicases during infections as shown for tbsv [ , ] . the yeast ddx -like ded p and the orthologous plant atrh helicases are recruited for tbsv replication to promote (+)-strand rna synthesis by aiding initiation by the viral rdrp. however, genome-wide screens and global proteomics approaches with tbsv have identified host helicases, suggesting that several host helicases might be co-opted during tbsv infections [ , ] . in the current work, we have discovered another class of cellular dead box rna helicases, including the yeast eif aiii-like fal p and dbp p and the orthologous plant atrh and ddx -like atrh dead box helicases, which are co-opted by tombusviruses for distinct pro-viral functions. eif aiii-like helicases, which assist ribosome biogenesis, are likely involved in local remodeling of large ribosomal rnp structures [ , ] . although eif aiii helicase has striking homology with eif a, yet eif aiii is functionally distinct from eif a, having no known role in translation initiation, and no interaction with ribosome in vitro [ ] . the similar dbp p helicase, which is also involved in ribosome biogenesis, affects the endonuclease rnase mrp-driven cleavage of pre-ribosomal rna [ ] . it is currently not known how eif aiii or dbp p rna helicases are selected for their cellular functions. in spite of their different cellular functions, fal p and dbp p helicases play comparable roles during tbsv replication. we find that, unlike the previously characterized atrh /ded p helicases, the eif aiii-like rna helicases (i.e., atrh , atrh , fal p and dbp p) bind to a different cis-acting element, the riii( ) ren, which is present at the region of the tbsv ( )rna (fig. a) . also dissimilar with atrh /ded p helicases is the ability of atrh , atrh and the yeast dbp p and fal p to unwind the dsrna structure within the riii( ) ren (fig. ) . this unique characteristic allows these eif aiii-like rna helicases to perform unique functions that involves the riii( ) ren. the atrh and atrh helicases are components of the tombusvirus vrcs as demonstrated by co-purification experiments ( fig. and s ). in addition to binding to the viral rnas, these eif aiii-like rna helicases also bind to the p /p replication proteins, likely facilitating the recruitment of these cellular helicases into vrcs. it is possible that additional members of the large helicase family could perform a similar function to eif aiii-like rna helicases during tbsv replication. indeed, rna helicases are present in yeast and - rna helicases are described in plants [ , ] , indicating that additional members of this large protein family could also be involved in tbsv replication. one of the hallmark features of (+)rna virus replication is the asymmetric nature of rna synthesis [ , [ ] [ ] [ ] . the replication process leads to the production of abundant (+)rna progeny, while the ( )rna templates are likely sequestered in dsrna forms within the vrcs. the presented in vitro data based on the solubilized/purified tombusvirus replicase and the cfe assay containing the membrane-bound vrc indicate that the eif aiiilike rna helicases can mainly stimulate tbsv (+)-strand synthesis, while their effects on ( )rna synthesis have not been observed (not shown). the recombinant eif aiii-like rna helicases enhanced (+)strand synthesis by the purified recombinant tombusvirus replicase, it is possible that these helicases directly affect tbsv rna synthesis via affecting the structure of the rna templates, including the riii( )ren. however, we cannot fully exclude that atrh , atrh and the yeast fal p and dbp p helicases could also affect the activity of the vrc due to their interactions with p and p (fig. ) . overall, the recruitment of eif aiiilike dead-box helicases for replication of a small rna virus is remarkable, and we suggest that small (+)rna viruses likely co-opt two or more different host helicases that interact with different cisacting elements in the viral rna to aid viral replication. based on their rna binding features and their abilities to unwind dsrna regions only locally, we propose that the helicase functions of atrh , atrh and the yeast fal p and dbp p are likely important for unwinding of the riii( ) ren region in the dsrna structure formed within the vrcs during tbsv replication. why is local unwinding of dsrna within the riii( ) ren stimulatory for replication? we suggest that the locally opened riii in the dsrna form might allow the bridge sequence within the riii( ) ren to participate in a long-range basepairing with the end of the ( )rna, thus bringing the and terminal sequences of the ( )rna in close vicinity (fig. ). this could facilitate (+)-strand synthesis and the reutilization of the viral replicases (vrcs) for multiple rounds (as discussed below). however, the long-distance base pairing between the ''bridge'' in riii( ) ren and the cpr promoter in ri( ), both of which are buried in the dsrna structure, should also depend on opening the dsrna form within ri( ). this function is unlikely performed by eif aiii-like rna helicases. instead, we have previously demonstrated that the subverted ddx -like atrh /ded p helicases could open up the dsrna structure within the ri( ) sequence [ ] . in summary, based on this and previous publications [ , , ] , the emerging picture with tbsv is that this virus utilizes co-opted rna-binding host proteins to regulate asymmetric viral rna replication. the recruited host proteins are needed for specific interactions with various cis-acting sequences in the viral ( )rna because the viral p /p replication proteins bind to tbsv ( )rna nonspecifically [ ] . we propose that, first, the recruited eif aiii-like rna helicase proteins bind to riii( ) ren, while the ddx -like atrh /ded p helicases bind to ri( ) sequence. the interactions of two groups of helicases with the viral dsrna likely opens up the proximal riii( ) ren and the terminal promoter region from the dsrna structure present in the vrcs. then, long-distance rna-rna interaction between the bridge sequence in the riii( ) ren and the terminal sequence [ ] could ''circularize'' the ( )rna template and bring the p rdrp protein from the end back to the end for a new round of (+)strand synthesis (fig. ) . as proposed earlier [ , ] , an additional function of atrh /ded p is to further unwind local secondary structure within ri( ) to promote the association of the cellular gapdh with an au-rich internal site and proper positioning of the gapdh-p rdrp complex [ ] over the (+)-strand initiation promoter, leading to robust (+)rna synthesis. therefore, we propose that the synergistic effect between the two groups of subverted helicases, host gapdh and the viral p pol might promote efficient recycling of the viral rdrp, resulting in multiple rounds of (+)rna synthesis on the same dsrna template (fig. ) . this strategy could be beneficial for the virus by allowing asymmetric rna synthesis on dsrna templates, thus leading to excess amount of progeny (+)rnas. it is currently not known if other viruses might also use two different groups of cellular helicases to aid their replication. however, hiv retrovirus, which also lacks viral-coded helicases, has been shown to recruit several cellular helicases, including ddx , for various steps of its infection cycle [ ] [ ] [ ] . in addition, host dead-box helicases have been shown to affect virus infections, including translation of viral proteins [ ] [ ] [ ] ; viral rna replication [ , [ ] [ ] [ ] [ ] ; subgenomic rna synthesis [ ] ; reverse transcription [ ] ; virus assembly [ ] ; virus-mediated regulation of host gene transcription [ ] , and the activity of many anti-viral proteins [ ] [ ] [ ] . therefore, the emerging picture is that rna viruses subvert multiple members of the cellular rna helicase family during infections. saccharomyces cerevisiae strain by , ddbp (yko library) and tet::fal yeast strain (ythc library), were obtained from open biosystems (huntsville, al, usa). ts-fal yeast strain was from a yeast ts strain collection [ ] . yeast strain nmy was obtained from dualsystems. ddbp /ts-fal yeast strain was generated as follows: plasmid pym- (euroscarf) [ ] was used for pcr with primers # and # (table s ) to amplify the dbp deletion cassette. ts-fal yeast was transformed with the obtained figure . model on the roles of co-opted cellular helicases in asymmetrical replication of tombusviruses. cis-acting replication elements present at the and ends of the viral ( )rna are recognized by cellular helicases to locally open the viral dsrna replication form. namely, the eif aiii-like helicases unwind the dsrna structure within the riii( ) ren, while the ddx -like helicases open up the dsrna structure within ri( ) (as shown schematically). long-range rna-rna interaction between the ''bridge'' and the cpr region (promoter sequence at the end) in the ( )rna is proposed to lead to circularization of the viral ( )rna, which is still part of the dsrna. this rna structure is suggested to facilitate the transfer of the viral p pol from the end of the ( )rna [after completion of the (+)rna strand synthesis in the previous round] back to the cpr in the ( )rna for a new round of (+)rna strand synthesis. another co-opted cellular protein, gapdh (called tdh / in yeast) is likely involved in this process by facilitating the binding of p pol to the end of the ( )rna. altogether, the re-use of the viral p pol multiple times on the same ( ) rna template could result in production of multiple (+)rna progeny, resulting in the characteristic asymmetric viral rna replication. note that it is also possible that p pol is standing still, while the rna template is moved during rna synthesis within the membrane-bound vrcs. doi: . /journal.ppat. .g pcr product and the suitable yeast strain was selected on g containing plates. then, yeast strains were grown in liquid media and genomic dna was isolated. the correct deletion site was checked by pcr with primers # and # using genomic dna as a template. pcr products of yeast helicase genes were obtained as follows: yeast genomic dna was used as a template for amplification by pcr with primers # and # for dbp ; # and # for dbp ; # and # for dbp ; # and for tif ; and # and # for fal . the generated pcr products were digested with bamhi and xbai in the case of dbp and dbp and with bglii and xbai in the case of dbp . plasmids pyc-his (provided by dr. daniel barajas) and pmalc- x (new england biolabs) were digested with bamhi and xbai and ppr-n (dualsystems) was digested with bamhi and nhei and ligated with the similarly treated pcr products of dbp , dbp and dbp . the pcr products of plant helicases were obtained as follows: total rna was isolated from a. thaliana and used for rt-pcr with primers # and # for atrh ; # and # for atrh ; # and # for atrh ; and # and # for atrh . the obtained pcr products were digested with bamhi and sali in the case of atrh , atrh , atrh , fal and tif and bglii and sali in the case of atrh . plasmid pyc-his was digested with bamhi and xhoi and plasmids pmalc- x, ppr-n, pet- c(+) (for atrh and atrh ), and pgd- s (for atrh , atrh , atrh and atrh ) were digested with bamhi and sali and ligated to similarly treated pcr products of atrh , atrh , atrh and atrh , fal and tif . overexpression plasmid pgd-rh was obtained as follows: atrh sequence was amplified using primers # and # and pmal-rh [ ] as a template. the obtained pcr product was digested with bamhi and spei and inserted into pgd- s plasmid, which was digested with bamhi and xbai. the plasmids pgbk-his-cup-flag /gal-di- expressing flag-tagged p of cucumber necrosis virus (cnv) and the tbsv di- reprna [ ] , pgad-cup-flag [ ] , pgd-cnv and pgd-p [ ] were described earlier. cultures of agrobacterium tumefaciens c c strain carrying pgd-rh , pgd-rh , pgd-rh (individually) with pgd-cnv and pgd-p were prepared and infiltrated into leaves of n. benthamiana as described earlier [ ] . agrobacterium culture carrying empty pgd- s plasmid was used as a negative control. during multiple overexpression, we used the agrobacterium cultures with the following density: . od for pgd-cnv, . for pgd-p and . for one of pgd-rhx (or empty pgd) or . for each of pgd-rhx when combination of two was applied to the same leaf. plant samples from infiltrated leaves were taken hours after infection. rna was isolated and northern blot analysis was performed using previously described [ , ] . for selected samples, proteins were isolated and total proteins level was adjusted based on coomassie-blue staining. for western blot analysis, anti-p antibody was used (a generous gift of herman scholthof, texas am university). pictures of infected plants were taken days after agroinfiltration. recombinant mbp-tagged helicase proteins, the mbp-tagged tbsv p and p replication proteins and several truncated mbp-tagged p c derivatives (described earlier) were expressed in e. coli and purified as published earlier with modifications [ , ] . briefly, the expression plasmids were transformed into e. coli strain bl (de ) codonplus. protein expression of the selected helicase proteins was induced by isopropyl-b-d-thiogalactopyranoside (iptg) for h at uc and in the case of viral proteins p and p at uc. after the collection of cells by centrifugation ( , rpm for min), the cells were resuspended and sonicated in low-salt column buffer ( mm hepes-koh ph . , mm nacl, mm edta, mm b-mercaptoethanol). to remove cells debris, the lysate was centrifuged at , rpm for min, followed by supernatant incubation with amylose resin (neb) for min at uc. after careful washing of the columns, the proteins were eluted with mbp-elution buffer [column buffer containing . % (w/v) maltose]. purification of his -tagged atrh and his -atrh (using plasmids pet -rh or pet -rh ) was carried out using probond (invitrogen) resin (washed with column buffer, containing mm imidazole and eluted with column buffer [lacking b-mercaptoethanol], containing m imidazole), following otherwise the same protocol as for the mbp-tagged proteins. purified proteins were aliquoted and stored at uc. proteins used for the replication assays were at least % pure, as determined by sds-page (not shown). for in vitro pull-down assay, purified his -tagged helicase proteins ( mg) were loaded onto mbp columns, containing bound mbp-tagged p c derivatives and incubated with mixing for min at uc [ ] . the columns were washed three times with cold column buffer and the bound protein complexes were eluted with mbp-elution buffer. the eluates were analyzed for the presence of his -tagged proteins by sds-page, followed by coomassie blue staining or western blotting with an anti-his antibody. pcr products for ''+bridge'' and ''dbridge'' constructs ( fig. e) were prepared as follows: pgbk-his-cup-flag /gal-di- was used as a template for pcr with primer pairs # and # , or # and # , respectively. the generated pcr products were used to obtain +bridge rna ( nt in length) or dbridge rna ( nt in length), each starting from position in di- . the rna transcripts were synthesized on the pcr templates using t -based transcription [ ] . the rna transcripts used in cfe-based replication or replicase assays were purified as described earlier [ ] . the p-labeled or unlabeled four separate regions (ri-iv, fig. a ) and the full-length di- (+) and ( )rnas were produced as published [ ] . full-length fhv-derived di- (+) or ( )rna was produced as described [ ] . the amounts of transcripts were quantified by uv spectrophotometer (beckman). partial dsrna duplexes [( )r /(+)di- and (( )r /(+) di- ] for in vitro replicase assay were prepared as follows: approximately pmol of p-labeled ( )r or ( )r were annealed to unlabeled di- (+) rna in ste buffer ( mm tris, ph . , mm edta, and m m nacl) by slowly cooling down the samples (in ml) from uc to uc in min. complete di- dsrna duplexes were prepared using replicator rnai kit (finnzymes). briefly, di- (+)-strand rna, which was synthesized with t transcription, was used as a template for synthesis of di- dsrna by phi rna polymerase in vitro. purity of dsrnas was tested with agarose gel-electrophoresis. gel mobility shift assay (emsa) and strand separation assay emsa was performed as described previously [ ] , with modifications: the binding assay was done in the presence of mm hepes (ph . ), mm nacl, mm mgcl , mm dtt, mm edta % glycerol, u of rnasin and . mg trna in a ml reaction volume. approximately . pmol of plabeled rna probes, . mg of purified recombinant proteins and . or . mg of unlabeled rna were used in template competition assay. for the assay, we used . mg mbp-p c, . pmol of p-labeled slr rna (the stem-loop sequence from rii) [ ] and mbp-atrh (or mbp-atrh ), in . , . , . or . mg amounts. strand separation assay was performed as published [ ] . briefly, about pmol of p-labeled riii ( ) or ri/ii/iii( ) were annealed to unlabeled di- (+)rna in ste buffer by slowly cooling down the samples (in ml) from uc to uc in min. . mg of purified recombinant helicase proteins (in mbp elution buffer) or mbp as a negative controls were added separately to the partial dsrna duplex in the rdrp buffer. mm of atp was added to the reaction. reaction mixtures were incubated for min at room temperature and loaded onto % nondenaturing polyacrylamide gel as described previously [ ] . some samples were treated with proteinase k after the assay. the incubation with proteinase k lasted for min at uc using . ml of proteinase k from stock of mg/ml (dissolved in mm tris-hcl ph . , supplemented with . mm cacl ), followed by loading onto % nondenaturing polyacrylamide gel. the uv-cross-linking assay was performed as described [ ] . the ml reaction mixture contained mg purified mbp-tagged atrh or atrh proteins, respectively, . nm p-utp-labeled rna probe, mm hepes, ph . , mm kcl, mm mgcl , % glycerol, and mg trna. unlabeled rna transcripts of riii( ) or ''+bridge'' and ''dbridge'' constructs (all rnas were comparable in length) were used as competitors in . to . mg amounts in the competition assay. after the formation of rnaprotein complexes during incubation of the reaction mixtures at room temperature for , min, we transferred the reaction mixtures to a -well plate on ice. to cross-link the rna and protein, we irradiated the reaction mixtures on ice at nm wave-length for min using an uv stratalinker (stratagene). then, we digested the unprotected rnas by mg/ml rnase a for min at uc. samples were mixed and boiled for min in sds loading dye. analysis was performed using sds-page and phosphorimaging [ ] . yeast strain ddbp was transformed with plasmids pgbk-his-cup-flag /gal-di- , pgad-cup-flagp and pyc-gal- hisrh or pyc-gal- hisrh to co-express the cellular helicases with the viral replication proteins in yeast cells actively replicating the tbsv reprna. the transformed yeast strains were selected on sc-ulh plates and then pre-grown overnight at uc in selective media containing % glucose [ ] . after that yeast strains were pelleted by centrifugation at , rpm for min, we washed the pellet with sc-ulh media containing % galactose and mm cuso , yeast were grown for hours in sc-ulh media containing % galactose at uc. pelleted yeasts (about mg) were used for affinity-purification of flag-p and flag-p with anti-flag m agarose as published previously [ , ] . flag-p was detected with anti-flag antibody ( / , dilution) and ap-conjugated anti-mouse antibody ( / , ). his -atrh or his -atrh proteins were detected with anti-his antibody from mouse ( / , dilution) and ap-conjugated anti-mouse ( / , ) followed by nbt-bcip detection [ , ] . the split-ubiquitin assay was based on the dualmembrane kit (dualsystems). pgad-bt -n-his , expressing the cnv p replication protein (bait construct), has been published earlier [ ] . yeast strain nmy was co-transformed with pgad-bt -n-his and one of the prey constructs carrying the cdna for a given helicase or ppr-n-re as a negative control or ppr-n-ssa as a positive control [ ] . yeasts were plated onto trp /leu synthetic minimal medium plates. after transformed colonies were picked with a loop and re-suspended in water, we streaked them onto tlh (trp /leu /his ) plates to test for helicase protein-p interactions as described [ ] . to study the effect of over-expression of yeast and plant helicase proteins on di- reprna replication in yeast, we transformed s. cerevisiae strain by with three plasmids: pgbk-his-cup-flag /gal-di- , pgad-cup-flag and one of the following plasmids: pyc-his-rh , pyc-his-rh , pyc-his-rh , pyc-his-rh , pyc-his-dbp , pyc-his-dbp , pyc-his-dbp , pyc-his-fal , pyc-his-tif (empty pyc plasmid was used as a control). after the selection of transformed yeast cells on sc-ulh plates, they were pre-grown in sc-ulh media containing % glucose for h at uc. then cells were centrifuged at , rpm for min, washed with sc-ulh media containing % galactose and resuspended in sc-ulh media containing % galactose and mm cuso . after growing yeast cells for h at uc, they were used for total rna extraction and northern blotting and western blotting as previously published [ ] . after yeast strain tet:fal was transformed with plasmids pgbk-his-cup-flag /gal-di- and pgad-cup-flag , it was pre-grown in sc-ulh media containing % glucose at uc. then yeast cells were centrifuged at , rpm for min, washed with sc-ulh media containing % galactose and resuspended in sc-ulh media containing % galactose and mm cuso in the presence or absence of mg/ml doxycycline. after growing for h at uc, and yeasts were pelleted and the replicase was purified according to a previously published procedure [ ] . briefly, approximately mg of wet yeast cell pellet were resuspended in tg buffer [ in vitro tbsv replication assay in cell-free yeast extract we prepared cell-free extract (cfe) from by or ddbp /ts-fal yeast strains as described earlier [ ] . the cfe-based tbsv replication assays were performed in ml total volume containing ml of cfe, unlabeled . mg di- (+)rna or ri/ii/iv (+ )rna transcripts, ng purified mbp-p , ng purified mbp-p pol and ng purified mbp-tagged helicase proteins. the assays were performed as published [ , ] . fractionation of the assay products was done as follows: after h of incubation at uc, reaction mixtures were centrifuged at , g for min to separate the ''soluble'' (supernatant) and ''membrane'' (pellet) fraction. then the membrane fraction was re-suspended in reaction buffer. both fractions were then treated as separate samples during phenol/chlorophorm extraction, ethanol precipitation. the samples were dissolved in rna loading dye and analyzed by page electrophoresis in % polyacrylamide gel containing m urea with . tris-borate/edta buffer as described [ , ] . for the detection of the p-labeled dsrnas generated in the cfe assays, we prepared the rna samples in rna loading dye (containing % formamide), followed by dividing the samples into two equal fractions; one half was loaded on the gel without heat-treatment, while the other half was heattreated for rna denaturation at uc for min and analyzed by page [ ] . in the presence of . mg of purified recombinant cellular helicases (except . mg in case of atrh ) is shown. note that lanes - show samples from the in vitro replication assays with the combination of two cellular helicases [i.e., atrh ( . mg) plus the shown helicase), while lanes - show samples with only a single helicase in the assay. (c) detection of (+) and ( )-stranded rna products produced by the purified tbsv replicase on the di- rna/rna duplex template in vitro replication assay containing cellular atrh and atrh helicases (lane in panel b). the blot contains the same amount of cold (+) and ( )stranded di- rna, while the p-labeled reprna probes were generated as in panel b. the ratio of (+) and ( )-stranded rna products was estimated. (pdf) figure s cell-free tbsv replication assay supports a role for fal p and dbp p helicases in plus-strand synthesis. (a) scheme of the cfe-based tbsv replication assay. tbsv di- (+)reprna was added to the whole cell extract (cfe) prepared from either wt yeast or dbp p-depleted and ts-fal p-inactivated yeast strain expressing p and p pol replication proteins. the membrane and soluble fractions were separated at the end of the replication assay by centrifugation. (b) detection of single-and doublestranded rna products produced in the cell-free tbsv replication assays. ''t'' total, ''m'' membrane fraction, ''s'' soluble fraction. note that the ssrna present in the ''s'' fraction represents the released (+)reprna products from the membrane-bound vrcs. (c) scheme of the cfe-based tbsv replication assay with purified recombinant cellular helicases. (d) detection of single-and double-stranded rna products produced in the cell-free tbsv replication assays by denaturing page analysis of the p-labeled tbsv reprna products (see panel c). the assay also contained purified recombinant atrh or atrh ( . mg) or mbp (the same molar amount as the helicases) as a control. odd numbered lanes represent replicase products, which were not heat treated (thus both ssrna and dsrna products are present), while the even numbered lanes show the heat-treated replicase products (only ssrna is present). the % of dsrna and ssrna in the samples are shown. note that, in the nondenatured samples, the dsrna product represents the annealed ( )rna and the (+)rna, while the ssrna products represents the newly made (+)rna products. each experiment was repeated three times. (pdf) figure s atrh is a component of the tombusvirus replicase in yeast. (a) the membrane-bound tombusvirus replicase was purified via solubilization of the flag-tagged p f from yeast extracts using a flag-affinity column (lanes - ). yeast not expressing p f was used as a control (lane ). top panel: western blot analysis of flag-tagged p f with anti-flag antibody. bottom panel: western blot analysis of his -tagged atrh with anti-his antibody in the affinity-purified replicase preparations. note that ''soluble'' represents the total protein extract from yeast demonstrating comparable levels of his -atrh in each sample (lanes - ). each experiment was repeated three times. (b) interaction between yeast dead-box helicases and the tbsv p replication protein based on the membrane yeast two hybrid assay (split-ubiquitin assay). the bait p was co-expressed with the prey full-length host proteins in yeast. the yeast ssa p (hsp chaperone), and the empty prey vector (nubg) were used as positive and negative controls, respectively. the image shows fold serial dilutions of yeast cultures. (pdf) figure s interaction between atrh and atrh and the tbsv p replication protein. (a) a schematic representation of viral p and its derivatives used in the binding assay (each mbptagged at the n-terminus). the various domains include: tmd, transmembrane domain; rpr, arginine-proline-rich rna binding domain; p; phosphorylated serine and threonine; s and s subdomains involved in p :p /p interaction. the results of the in vitro binding experiments are summarized (''+'' or '' '', based on two repeats). (b) in vitro pull-down assay of his -tagged atrh (lanes - ) , his -peptide (lanes - ) or his -tagged atrh (lanes [ ] [ ] [ ] [ ] with mbp-p derivatives using amylose resin. top panel: western blot analysis with anti-his antibody of his -tagged helicases pulled down with mbp-p derivatives. lane contains purified his -tagged atrh as a standard. yeast as a model host to explore plant virus-host interactions cytoplasmic viral replication complexes the dependence of viral rna replication on coopted host factors +)rna viruses rewire cellular pathways to build replication organelles yeast as a model host to dissect functions of viral and host factors in tombusvirus replication host factors involved in west nile virus replication viral and cellular proteins involved in coronavirus replication emerging picture of host chaperone and cyclophilin roles in rna virus replication hepatitis c virus replication cycle virus factories: associations of cell organelles for viral replication and morphogenesis virus factories: biogenesis and structural design diverse roles of host rna binding proteins in rna virus replication non-templated functions of viral rna in picornavirus replication yeast as a model host to study replication and recombination of defective interfering rna of tomato bushy stunt virus purification of the cucumber necrosis virus replicase from yeast cells: role of coexpressed viral rna in stimulation of replicase activity role of an internal and two -terminal rna elements in assembly of tombusvirus replicase specific binding of tombusvirus replication protein p to an internal replication element in the viral rna is essential for replication multiple roles of viral replication proteins in plant rna virus replication defining the roles of cis-acting rna elements in tombusvirus replicase assembly in vitro a discontinuous rna platform mediates rna virus replication: building an integrated model for rna-based regulation of viral processes tomato bushy stunt virus co-opts the rna-binding function of a host metabolic enzyme for viral genomic rna synthesis a temperature sensitive mutant of heat shock protein reveals an essential role during the early steps of tombusvirus replication a key role for heat shock protein in the localization and insertion of tombusvirus replication proteins to intracellular membranes in vitro assembly of the tomato bushy stunt virus replicase requires the host heat shock protein proteomics analysis of the tombusvirus replicase: hsp molecular chaperone is associated with the replicase and enhances viral rna replication ubiquitin-conjugating enzyme is a component of the tombusvirus replicase complex and ubiquitinates p replication protein translation elongation factor a facilitates the assembly of the tombusvirus replicase and stimulates minus-strand synthesis translation elongation factor a is a component of the tombusvirus replicase complex and affects the stability of the p replication co-factor synergistic roles of eukaryotic translation elongation factors bgamma and a in stimulation of tombusvirus minus-strand synthesis a co-opted dead-box rna helicase enhances tombusvirus plus-strand synthesis the host pex p plays a role in peroxisomal localization of tombusvirus replication proteins ubiquitination of tombusvirus p replication protein plays a role in virus replication and binding to the host vps p escrt protein a unique role for the host escrt proteins in replication of tomato bushy stunt virus global genomics and proteomics approaches to identify host factors as targets to induce resistance against tomato bushy stunt virus yeast genome-wide screen reveals dissimilar sets of host genes affecting replication of rna viruses identification of essential host factors affecting tombusvirus rna replication based on the yeast tet promoters hughes collection the roles of host factors in tombusvirus rna recombination cpr cyclophilin and ess parvulin prolyl isomerases interact with the tombusvirus replication protein and inhibit viral replication in yeast model host mrna export: rnp remodeling by dead-box proteins bent out of shape: rna unwinding by the dead-box helicase vasa the dead-box protein family of rna helicases rna helicases: emerging roles in viral replication and the host innate response a host rna helicase-like protein, atrh , interacts with the potyviral genome-linked protein, vpg, associates with the virus accumulation complex, and is essential for infection genome-wide analysis of helicase gene family from rice and arabidopsis: a comparison with yeast and human stress response suppressor and stress response suppressor , two dead-box rna helicases that attenuate arabidopsis responses to multiple abiotic stresses sde encodes an rna helicase required for post-transcriptional gene silencing in arabidopsis evolution and taxonomy of positive-strand rna viruses: implications of comparative analysis of amino acid sequences role of rna chaperones in virus replication similar roles for yeast dbp and arabidopsis rh dead-box rna helicases to ded helicase in tombusvirus plus-strand synthesis dbp p, a putative rna helicase in saccharomyces cerevisiae, is required for efficient pre-rrna processing predominantly at site a duplex destabilization by four ribosomal dead-box proteins human eif aiii interacts with an eif g-like partner, nom , revealing an evolutionarily conserved function outside the exon junction complex yeast screens for host factors in positive-strand rna virus replication based on a library of temperature-sensitive mutants proteome-wide overexpression of host proteins for identification of factors affecting tombusvirus rna replication: an inhibitory role of protein kinase c an internally located rna hairpin enhances replication of tomato bushy stunt virus rnas the rna replication enhancer element of tombusviruses contains two interchangeable hairpins that are functional during plus-strand synthesis mechanism of stimulation of plus-strand synthesis by an rna replication enhancer in a tombusvirus rna chaperone activity of the tombusviral p replication protein facilitates initiation of rna synthesis by the viral rdrp in vitro use of double-stranded rna templates by the tombusvirus replicase in vitro: implications for the mechanism of plus-strand initiation internal initiation by the cucumber necrosis virus rna-dependent rna polymerase is facilitated by promoter-like sequences mechanism of di rna formation in tombusviruses: dissecting the requirement for primer extension by the tombusvirus rna dependent rna polymerase in vitro partial purification and characterization of cucumber necrosis virus and tomato bushy stunt virus rna-dependent rna polymerases: similarities and differences in template usage between tombusvirus and carmovirus rna-dependent rna polymerases authentic replication and recombination of tomato bushy stunt virus rna in a cell-free extract from yeast the p polymerase coding region contains an internal rna element required at an early step in tombusvirus genome replication composition of plant virus rna replicase complexes global organization of a positive-strand rna virus genome yeast and human rna helicases involved in ribosome biogenesis: current status and perspectives a genome-wide analysis of the rna helicase gene family in solanum lycopersicum viral rna replication in association with cellular membranes de novo initiation of viral rna-dependent rna synthesis filling a gap(dh) in asymmetric viral rna synthesis characterization of the rna-binding domains in the replicase proteins of tomato bushy stunt virus direct inhibition of tombusvirus plus-strand rna synthesis by a dominant-negative mutant of a host metabolic enzyme, gapdh, in yeast and plants distinct ddx dead-box rna helicases cooperate to modulate the hiv- rev function from promoting to inhibiting: diverse roles of helicases in hiv- replication viruses and the human dead-box helicase ddx : inhibition or exploitation? a mutant allele of essential, general translation initiation factor ded selectively inhibits translation of a viral mrna rna helicase a modulates translation of hiv- and infectivity of progeny virions autogenous translational regulation of the borna disease virus negative control factor x from polycistronic mrna using host rna helicases cyclosporin a associated helicase-like protein facilitates the association of hepatitis c virus rna polymerase with its cellular cyclophilin b identification of rna helicase a as a new host factor in the replication cycle of foot-and-mouth disease virus cellular rna helicase p relocalization and interaction with the hepatitis c virus (hcv) ns b protein and the potential role of p in hcv rna replication affinity capture and identification of host cell factors associated with hepatitis c virus (+) strand subgenomic rna the cellular decapping activators lsm , pat , and dhh control the ratio of subgenomic to genomic flock house virus rnas ddx dead-box rna helicase inhibits hepatitis b virus reverse transcription by incorporation into nucleocapsids the helicase activity of ddx is required for its role in assembly of infectious west nile virus particles kaposi's sarcoma-associated herpesvirus viral protein kinase interacts with rna helicase a and regulates host gene expression dexh-box protein dhx is required for optimal function of the zinc-finger antiviral protein regulating intracellular antiviral defense and permissiveness to hepatitis c virus rna replication through a cellular rna helicase, rig-i dexd/h-box rna helicases as mediators of antiviral innate immunity and essential host factors for viral replication systematic exploration of essential yeast gene function with temperature-sensitive mutants a versatile toolbox for pcr-based tagging of yeast genes: new fluorescent proteins, more markers and promoter substitution cassettes cyclophilin a binds to the viral rna and replication proteins resulting in inhibition of tombusviral replicase assembly the nedd -type rsp p ubiquitin ligase inhibits tombusvirus replication by regulating degradation of the p replication protein and decreasing the activity of the tombusvirus replicase expression of the arabidopsis xrn p - exoribonuclease facilitates degradation of tombusvirus rna and promotes rapid emergence of viral variants in plants comparison of turnip crinkle virus rna-dependent rna polymerase preparations expressed in escherichia coli or derived from infected plants analysis of minimal promoter sequences for plus-strand synthesis by the cucumber necrosis virus rna-dependent rna polymerase nucleolin/nsr p binds to the noncoding region of the tombusvirus rna and inhibits replication the authors thank drs. ching-kai chuang and judit pogany for valuable comments. key: cord- -khrx rnh authors: bordería, antonio v.; isakov, ofer; moratorio, gonzalo; henningsson, rasmus; agüera-gonzález, sonia; organtini, lindsey; gnädig, nina f.; blanc, hervé; alcover, andrés; hafenstein, susan; fontes, magnus; shomron, noam; vignuzzi, marco title: group selection and contribution of minority variants during virus adaptation determines virus fitness and phenotype date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: khrx rnh understanding how a pathogen colonizes and adapts to a new host environment is a primary aim in studying emerging infectious diseases. adaptive mutations arise among the thousands of variants generated during rna virus infection, and identifying these variants will shed light onto how changes in tropism and species jumps can occur. here, we adapted coxsackie virus b to a highly permissive and less permissive environment. using deep sequencing and bioinformatics, we identified a multi-step adaptive process to adaptation involving residues in the receptor footprints that correlated with receptor availability and with increase in virus fitness in an environment-specific manner. we show that adaptation occurs by selection of a dominant mutation followed by group selection of minority variants that together, confer the fitness increase observed in the population, rather than selection of a single dominant genotype. the extreme mutation rates of rna viruses and the highly diverse populations they generate in few replication cycles are considered the basis for their rapid adaptation to new environments [ , ] . such adaptive steps result in the emergence of new variants capable of escaping immune responses, resisting antiviral approaches, altering tissue tropism or crossing species barriers. in the past, experimental evolution of viruses in different host environments has proven to be a useful tool in quantifying fitness increases and the dynamics of adaptation. by classic sequencing techniques, some of the key genetic determinants responsible have been identified [ , ] , but until the advent of deep sequencing, analysis of the mutational composition of rna virus populations was hampered by lack of depth of sequence coverage. the potential to describe the whole virus mutant spectrum and detect variants that otherwise would be overlooked by conventional sequencing is fundamental to studying virus evolution and understanding emergence [ ] . recent work shows that deep sequencing can identify the emergence of escape mutants in experimental and clinical samples [ , ] , and can be used to characterize the entire mutant spectrum of a virus population [ ] . one of the goals in the field of emerging infectious diseases is to determine whether adaptation to novel hosts (species, tissues or cell types) can be identified for a recently introduced pathogen that is confronted with a less than optimal host environment [ ] [ ] [ ] . viruses are well-suited for studying adaptation and evolution for several reasons: i) high mutation rates ii) short generation time and iii) large population sizes. we used coxsackie virus b (cvb ) as a model, since the genetics of this virus and the interactions between the cell receptors and viral capsid proteins (vp , vp and vp ) are well characterized. cvb enters the cell through a primary receptor, the coxsackie and adenovirus receptor (car) [ ] , while certain strains may use as co-receptor the decay accelerating factor (daf) [ , ] , also known as cd . to study expansion of host tropism, we passaged virus in two cellular environments, a highly permissive one and a less permissive one. by deep sequencing longitudinal samples of experimentally evolved populations, we identify the emergence of host environment-specific mutations undergoing positive selection. we show that coxsackie virus adapts differently to two cell types according to receptor and co-receptor availability in a multi-step adaptation sequence that involves group selection of minority variants. importantly, we reveal the significant contribution of several minority variants to the overall fitness of the entire population. our results underscore the importance of characterizing rna virus quasispecies during adaptation and virus evolution. increase of fitness and genetic diversity of cvb during adaptation to permissive and less permissive host environments to monitor the evolution of cvb towards novel and less permissive host environments, we selected human lung a cells, which gave similar final virus yields as the highly permissive hela cells, but after two days rather than one day of infection. cvb was thus serially passaged times in six biological replicate series in both cell types. virus yields were constant throughout the passage series suggesting that no significant genetic drift or accumulation of detrimental mutations through population bottlenecking had occurred (fig a and b) . the time required to reach peak titers was reduced in a cells over the first ten passages from hours to hours, suggesting that fitness increases and adaptation occurred in this novel environment. we measured the relative fitness of the passaged viruses by competing the passage , and populations from each replicate with a genetically marked neutral cvb virus in a quantitative fitness assay [ ] . increases in fitness were observed in both cell types, with the most significant increase found by passage in a cells (fig c and d ), suggesting that adaptation had occurred in this less permissive cell type. whole-genome deep sequencing of these populations revealed significant increases in overall genetic variation, throughout the genome, between the st, th and th passages in both hela ( fig e) and a ( fig f) cells (p< . ). the total number of minority variants within the p structural coding region also significantly increased over the passage series (fig g) , yet there were no significant differences between the numbers observed in hela and a cell passage. the vast majority (> %) of these variants were low frequency (< . % of the total population), suggesting that moderate genetic drift, rather than positive selection, was responsible for most of this variance. the increase in diversity in both cell types is likely the result of general population expansion in sequence space, since all passage series were started from homogenous in vitro transcribed rna derived from an infectious clone. the data also confirmed that severe bottlenecking did not occur during the passage series where expansion of diversity should have been stalled or lost. the mutations that arise from adaptive passage map to receptor binding sites in a cell-type specific manner although the hela and a cell passage series presented similar mean genetic diversity at passage (fig e- g , p = . ), we mined the deep sequence data for all minority variants above % frequency that might explain adaptation in each condition (table ). in both cases, several mutations recurring in multiple replicates mapped to residues known to be part of the car receptor and daf co-receptor footprints [ ] ; however, the distribution of these mutations was different for the hela-and a -adapted viruses. in hela cells, the most abundant variants involved mutations strictly in the car footprint (vp - , - % frequency), in the car/daf shared footprint (vp - , - %), and in the daf footprint (vp - , - % frequency; vp - , - % frequency). in contrast, viruses passaged in a cells presented no variants strictly involved in the car footprint. instead, in addition to car/daf residue vp - ( - % total), a larger cluster of exclusively daf-footprint variants were identified (vp - , - % frequency; vp - , - % frequency and vp - , - % frequency). strikingly, the most abundant mutation in every replicate passaged in a cells, and not observed in hela cell passage, was vp - ( - % total). this residue is not known to participate in either footprint. permissive and less permissive cell environments present different expression profiles of the primary and secondary receptors considering these car/daf-specific differences, we hypothesized that expression levels of these molecules must vary between these cells. there were no significant differences in car expression in either cell by both flow cytometry (fig a) or western blot (fig c) . on the other hand, while daf was very highly expressed in hela cells (fig b and d) , expression was one order of magnitude lower in a cells by flow cytometry (fig b) and barely detectable by virus fitness and genetic diversity increases during serial passage in both hela and a cells. hela (a) or a (b) cells were infected with cvb for serial passages (x-axis) and virus progeny titers (yaxis) were determined for each of six replicate series. the relative fitness of each population from the hela (c) and a (d) series was compared to a non-passaged neutral competitor. box plots show mean values and s.e.m., bars indicate minimum and maximum values, p values are indicated, student's paired t test, n = . the genetic diversity of each replicate was determined for passage , and populations in hela (e) and a (f) cells by calculating the variation rates at every nucleotide position. the mean variation western blot (fig d) . normalized quantification of western blot signals revealed that daf expression was -fold higher in hela than in a cells (fig e) . to characterize the localization of car and daf in these cell types, confocal microscopy was performed. in hela cells, car ( fig f) and daf (s fig) were expressed throughout the surface of the cell. interestingly, in a cells car predominantly localized at cell-to-cell contacts (fig g) , while daf was diffused throughout the surface of the cell (s b fig) , albeit at considerably lower levels than in hela cells (see also, s and s movies). it has been shown that adaptation is often a multi-step process, especially in asexual populations like viruses. usually, until the mutation with the largest beneficial effect becomes fixed, rates ± s.e.m. are shown. ***p< . , mann whitney test, n = . (g) the total number of minority variants in passage , and hela and a populations found within the p structural protein-coding region. mean ± s.e.m. are shown. differences between passage numbers are all significant, p< . ; no significant differences between cell type at each passage number, student's paired t test, n = . secondary beneficial mutations with less effect are unable to compete, thereby rendering the adaptation process sequential [ ] [ ] [ ] . given the different frequencies of minority variants observed at passage , we examined the patterns of emergence and sequential adaptation to novel environments by deep sequencing every second passage in the a cell series (fig and s dataset). a number of step-wise trends were observed across the replicates. in all six replicates, the vp -e g mutation was the first mutation to emerge (already above . % at passage ) and peak by passage to become the most abundant mutation in the population (between and %). furthermore, the increase in frequency of e g over the first passages (fig ) correlated with the increases in fitness over the same period (fig a) , underscoring a considerable contribution of this single mutation to population fitness. indeed, the e g variant was generated by reverse genetics and found to confer significantly enhanced fitness in a cells ( fig b) . although the vp - residue was not previously identified as a contact between virus and receptor, this residue maps to the icosahedral three fold region of the capsid ( fig c) . in the three dimensional virus-receptor structure, there is an interaction between the c-terminal -his tags of three symmetry related daf molecules [ ] . this interference by the his-tag may have restricted the normal interactions of daf scr / with the virus surface, masking a potential role for residue . in a biolayer interferometry assay using blitz technology [ ] ( fig d) , e g bound daf with the same affinity as wildtype virus, but no increased binding could be detected. despite the clear fitness advantage conferred by the e g mutation alone (approximately -fold increase with respect to wildtype), it could not account for the more significant fitness increases observed in the passaged populations, particularly at late passage stages (nearly -fold increases). following the fixation of this first mutation, the frequency of vp -e g plateaued until a second increase after passage , in each of the six replicates, coinciding with the emergence of different combinations of mutations, all mapping to the daf footprint ( fig ) . closer examination of the patterns of emerging mutations revealed potential synergistic and antagonistic epistasis among variants. despite the advantage afforded by extreme depth in coverage, the illumina technology used here set a limit of nucleotides for read length. in principle, this limitation forfeits the possibility of linking more distant mutations and identifying haplotypes. however, the longitudinal deep sequence data revealed that several mutations increase with parallel kinetics and frequency, suggesting that: i) each mutation appeared in individual genomes and the variants were selected as a group and/or ii) the mutations are accumulating in the same genome, resulting in the selection of single haplotypes. to distinguish between the above cases, phylogenetic trees describing the mix of haplotypes at each passage were inferred from the longitudinal data using maximum likelihood estimation in a bayesian model. the best-fit, predicted haplotypes were then generated by reverse genetics and their relative fitness values were measured. as expected, this analysis illustrated the quick rise of the e g genotype that continues to coexist with, although generally dominating over, the original wt genotype (fig a- f ), with a significant increase in relative fitness ( fig g) . for residue vp - , mutations were predicted to arise on either the wt background ( fig c and f ) or the e g genotype (fig a and e ). mutations in residue vp - were also predicted to arise on either wt ( fig b) or e g (fig c, d and f) genotypes at approximately the same time in the passage series as residue . however, their frequencies on the wt background tended to remain lower than on the e g background where they seemed to be better experiments. (e) the relative expression of car and daf with respect to gapdh was quantified by imaging quantification using imagej. localization of car, in red, in hela (f) or a (g) by confocal microsocopy. nuclear (blue) staining was done with dapi. doi: . /journal.ppat. .g tolerated. to confirm these modelled predictions, we generated each mutation on both backgrounds and measured the relative fitness. indeed, the d g mutation alone on the wt background conferred nearly a ten-fold drop in fitness and the q k mutation conferred up to -fold drop in fitness (fig g) , while the fitness costs of these mutations on the e g background resulted in neutral fitness relative to wt. the data suggest that epistasis between these mutations and the e g mutation rescues the fitness of these double variants and permits their positive selection in the viral population. interestingly, the d g and q k variants seemed to entirely exclude one another from the population (fig a, b , d and e), and the mle analysis suggested that if both are present in the population, they must occur on separate genomes. we thus generated the e g-d g-q k haplotype and confirmed that the triple each set of linear and log graphs represents the same data from one replicate, all six replicate passage series presented in this work are shown. doi: . /journal.ppat. .g the trees indicate the presence of wildtype genotype (lowest line on tree) along with the predicted haplotypes as they emerge during the passage series (x-axis). solid lines indicate haplotypes with highest mle scores, dashed lines indicate alternative haplotypes that could exist, with lower scores. the thickness of grey area indicates the expected frequency of each haplotype in the population, according to deep sequence data. (g) relative mutant bears a significant fitness cost, rendering this haplotype less fit than the original wt genotype ( fig g) . finally, shortly after the appearance of residue and variants, a third mutation appeared during the passage series at residue (fig a, b and e ). once again, computational modelling predicted that it too exists on the e g background, and on separate haplotypes than the position and mutations. furthermore, data from two replicates (fig a and e ) suggested that the e g-n y double variant bears higher fitness than e g-q k, as its frequency increased towards the end of the passage series as the other's decreased. indeed, the residue variants presented higher relative fitness than both the and variants on the e g background ( fig g) . although we could not determine whether residue plays a role in receptor interaction directly, and the role of accompanying mutations are inferred from previously published studies, to rule out that these mutations impact virus fitness on activities downstream of receptor binding and entry, we transfected cells with in vitro transcribed rnas corresponding to some of these variants and assayed virus production at hours (before a new round of infection can occur). the data revealed that the fitness advantages (e g, e g-n y) and disadvantages (wt-q k, e g-q k) observed during infection of cells (fig g) do not appear to exist when the binding and entry step is bypassed (fig h) . population fitness is determined by the group contribution of minority variants, rather than the selection of a dominant, single haplotype although we initially expected selection of fitness-increasing adaptive mutations to occur by step-wise accumulation of mutations on a single genotype, our computational analysis followed by fitness measurements of double and triple mutation-bearing genotypes suggested otherwise. importantly, none of these single, double and triple mutations conferred the same fitness increases as those observed in the passage virus population used to identify mutant composition (fig g) . since previous work suggested that overall population fitness results from cooperative interactions among key variants in the mutant swarm, we examined whether a reconstituted composition of the most predominant variants within the passage population could manifest comparable fitness. we thus generated artificial quasispecies presenting mixtures of position , and mutations on the e g background and tested their relative fitness. interestingly, artificial quasispecies presenting : , : or : combinations at : ratios all presented fitness increases as high as, or higher than, the individual values for each variant (fig g) . on the other hand, none of these combinations resulted in the highest fitness values that were observed for the passage populations of each replicate used to identify these individual mutations (fig g, p a-f) . to address whether the minority variant composition may dictate observed population fitness, we reconstituted an artificial quasispecies based on the average frequency of each variant in passage populations. in contrast to the individual variants or the : combinations fitness of the wildtype (wt), single, double and triple variants, followed by combinations of single variants, followed by passaged population samples (p replicates a-f) and reconstituted quasipecies. reconstituted quasispecies is composed of : : : of e g, e g+n y, e g+d g, and e g+q k. vertical dashed lines are placed to separate the aforementioned groups to facilitate the reader. horizontal line is placed to facilitate reading of neutral fitness (value ). the relative fitness (y-axis) is the ratio of the viral rna quantification at h and h, mean values with sem, n = . (h) a cells were transfected with in vitro transcribed infectious rna corresponding to dawildtype (wt), single and double variants. at hours posttransfection, the progeny virus was quantified by tcid assay. no significant differences were observed between wt and variants (p = . , . , . , . , respectively, n = - , two-tailed mann whitney test). described above, a mixture of : : : of the four most predominant genotypes e g, e g-n y, e g-d g, and e g-q k, reproducibly reached the same fitness values as passage samples themselves (fig g) , demonstrating that the global fitness of a virus population is determined by cooperative contribution of minority variants, rather than the dominant genotype alone. taken together, the sequence data and in vitro characterization of car/daf expression provide a model for the adaptive dynamics of cvb to the two host environments presented here (fig ) . in hela cells, where both car and daf are highly and ubiquitously expressed on the surface, adaptive mutations mapped to both footprints. it is not clear whether the car-specific mutations (e.g. vp -k m) observed in hela cells increased interactions with car, or conversely, decreased interactions to facilitate the appearance of other mutations related to the schematic of cvb adaptation to differently permissive environments. in the permissive hela cell type, where the both car and daf are highly and ubiquitously expressed, cvb accumulates car-and car/daf-specific minority variants. in the less permissive a cells, where car is sequestered at cell-cell junctions and daf is poorly expressed at the surface, cvb first fixates the e g daf-specific mutation, with the strongest single contribution to fitness, and in later stages, group selection of other daf-footprint minority variants occurs. doi: . /journal.ppat. .g daf footprint. because the particular strain of cvb nancy used to initiate the passage series already contains some daf-specific binding residues not found on other nancy clones, we cannot speculate with confidence which way evolution would go in hela cells. in retrospect, inclusion of a car-exclusive binding strain of coxsackie virus in the passage series would have helped discern the direction of evolution in hela cells. in a cells, on the other hand, the principal receptor car is mainly located in tight cell-cell contacts and likely inaccessible to the virus during initial stages of infection; while daf is present throughout the surface, but at relatively low levels compared to hela cells. thus, the focus of selection is entirely on residues involved in daf binding (vp - , vp - ) or shared in the car-daf footprints (vp - ) . the car-specific mutations observed at residue vp - in hela cells, were not observed in a cells. the dominant mutation, vp -e g, occurred in all six a -passaged replicates and in none of the hela passages-a residue that was not identified as a determinant by conventional structural studies of receptor usage [ ] . in the original structural studies, the interaction of residue vp - could not be determined due to steric hindrance of the his-tag between the daf short consensus repeat domains, scr / , and the virus surface. when car is not accessible, daf is thought to facilitate translocation of the virus to tight junctions containing car [ , ] . the interaction with car mediates the transition to the a-particle, a required entry intermediate of expanded structure relative to native virus [ ] . nevertheless, it is possible that e g improves the fitness of the virus in aspects not related to receptor binding, even if we could not identify these mechanisms when comparing replication cycles. our work reveals the power of deep sequencing to monitor the population dynamics of virus adaptation in new environments. by sanger sequencing, only the e g mutation would have been identified in each replicate. the remainder of mutants found to be positively selected during adaptation to host environment were all minority variants that could have otherwise been missed in multiple replicates. an issue inherent to deep sequencing technology is the estimated error of the chemistry ( . % for illumina sequencing), which has been regarded as a caveat to properly describe rna virus quasispecies or mutant spectra. this problem was recently resolved by elegant molecular biology techniques to remove background error [ , ] . by applying more stringent bioinformatic treatment in our study, between and individual, statistically significant point mutations were identified in the structural protein region of the six replicates of a -adapted populations; yet only mutations in positions related to the daf footprint underwent selection and amplification over time (s dataset). we thus show that robust bioinformatic treatment coupled with longitudinal data can circumvent error-related issues by identifying changes in variant frequency that would indicate positive or negative selection. this is particularly relevant to in vivo or clinical samples where the quantity of rna genomes may be too low for more direct sequencing approaches and would require pcr amplification. similar, relatively simple experimental studies could be designed to understand the population dynamics and evolution of pathogens in new environments during host adaptation and host switching, by identifying the selection over time of one or more mutations as single or multiple genotypes. however, one must keep in mind that although in vitro studies, such as ours, using immortalized cell lines may facilitate studying the dynamics of adaptation; but the specific mutations that are identified may not be indicative of the panel of mutations that would arise in vivo. in this work, adaptation to a novel host environment was a multistep process involving the emergence of an initial mutation (e g), followed by selection of a number of minority variants on the e g background. initially, we expected mutations to accumulate in a step-wise manner in a single genotype; however, fitness assays revealed that genotypes harboring double and triple combinations of these mutations presented fitness decreases relative to the wildtype and/or e g backgrounds. instead, computational inference of haplotypes suggested that such variants existed as a heterogeneous population of distinct genotypes. an intriguing observation was that no single variant (nor combination of two variants) conferred the high fitness values observed in the passage populations. strikingly, only the combination of the four most predominant genotypes, as an artificial quasispecies with the same frequencies observed in the passage populations, was able to confer the same fitness values as the p replicate samples. this phenomenon is reminiscent of previous work that suggested that minority variants within an rna virus quasispecies may contribute significantly to phenotype [ ] . in that study, a high-fidelity poliovirus with restricted quasispeces composition was unable to infect the central nervous system (cns); while the same virus stock that was chemically mutagenized to present wildtype numbers of minority variants restored the ability to disseminate to the cns. unfortunately, deep sequencing technology was not yet available and the authors could not uncover the identity of these presumed minority variants by sanger technology to confirm their hypothesis. here, we succeed in identifying the variants involved. importantly, we provide evidence for group selection within the virus population and show that only the group contribution of these positively selected minority variants confers the fitness phenotype observed in the original samples. our results thus illustrate the significant role of minority genomes in the fitness and phenotype of a virus population, providing further evidence for the quasispecies behavior of rna viruses under certain conditions. it is important to note, however, that the group selection observed in this study resulted from relatively large population size in passages that reached high moi by the time cell monolayers were lysed for subsequent passage. it is possible that in conditions where moi is low, or when population bottlenecks occur (particularly in vivo), that the emergence of such minority variants would be delayed or impeded. hela and a cells (american type culture collection) were maintained in dmem medium with % new-born calf serum. coxsackie virus b (nancy strain) was recovered from a pcb -nancy infectious cdna plasmid [ ] that was linearized with sal i and in vitro transcribed using t rna polymerase. it should be noted that unlike other nancy strains, this infectious clone already has vp - d and vp - q residues, known to facilitate binding to daf. μg of transcript were electroporated into x vero cells that were washed twice in pbs and resuspended in pbs at cells/ml. for a cells, μg of transcript were electroporated into x cells. electroporation conditions were as follows: . mm cuvette, μf, v, maximum resistance, exponential decay in a biorad genepulser xcell electroporator. cells were recovered in dmem- % ncs. for each passage ( passages total), virus was titrated by tcid and μl of medium containing x tcid was used to infect x hela or a cells in well plates using a multiplicity of infection (moi) of . . cells were incubated with virus for minutes with frequent rocking, the supernatant was removed and monolayers were washed twice with ml pbs, then replenished with ml of complete medium. for each passage, virus was harvested at total cytopathic effect (cpe) by one freeze-thaw cycle, representing - viral generations. six biologically independent stocks and passage series were generated. ten-fold serial dilutions of virus were prepared in -well flat-bottom plates in dmem. dilutions were performed in octuplate and μl of dilution were transferred to vero cells plated in μl of dmem- % ncs. after days living cell monolayers were colored by crystal violet. tcid values were determined by the reed and muensch method. no significant differences were observed between tcid values when using vero, hela or a cells as the cellular substrate. sequencing x virion from passaged samples were rna extracted and rt-pcr amplified by rt (superscript iii) and pcr (phusion) using primers sets that covered the whole genome, in - kb fragments. for consensus sequencing, the resulting pcr products were purified, sequenced and analyzed using lasergene software (dnastar inc). for deep sequencing, pcr fragments were purified via the nucleospin gel and pcr clean-up kit (macherey-nagel) and total dna was quantified by nano-drop. pcr products were then fragmented (fragmentase), linked to illumina multiplex adapters, clusterized and sequenced with illumina cbot and gaiix technology. sequences were demultiplexed by casava with no mismatches permitted. clipping was performed using the fastq-mcf tool, removing common adapter contaminants and trimming low quality bases (phred< ). clipped reads were aligned to the coxsackie virus b nancy sequence as reference with a maximum mismatches per read, and no gaps, using bwa v . . . alignments were processed using samtools to obtain a pileup of the called bases at each position. an in-house pipeline, termed vivan (viral variant analysis) [ ] was used to identify statistically significant variants above the background noise due to sequencing error, in every sufficiently covered site (> x). briefly, for each position throughout the viral genome, base identity and their quality scores were gathered. each variant was determined to be true using a generalized likelihood-ratio test (used to determine the total number of minority variants) and its allele rate was modified according to its covering read qualities based on a maximum likelihood estimation. additionally, a confidence interval was calculated for each allele rate. in order to correct for multiple testing, benjamini-hochberg false-discovery rate of % was set. the total allele rates passing these criteria, across the whole genome, were used to calculate the mean variation rates (diversity) at different passages. the variation rate at position i is defined as the proportion (f) of significant non-reference alleles (k) and is denoted v i : the region-wide variation rate is the averaged variation rate across all covered positions in the genome (denoted n): hela and a cells were plated onto coverslips, fixed with % paraformaldehyde for minutes at room temperature, and then washed with pbs. before staining, non-specific staining was blocked by incubating the cells with % fcs and . % saponin in pbs during minutes. staining antibodies were diluted also in this buffer, and it was used for washes between antibodies. cells were incubated with either car (santacruz) or daf (abcam) primary antibody, washed, stained with a secondary antibody coupled to the appropriate fluorophore and washed. cells were analyzed using a zeiss lsm- confocal microscope. d reconstruction of the images was performed using the imaris software. one million cells of hela and a cells were lysed using a buffer containing % triton-x and % sodium deoxycholate with protease inhibitors (sigma). a fraction of the lysate was run for hour on a - % gradient gel (biorad) on denaturalizing conditions. after the run, we performed the transfer to a nitrocellulose membrane. we washed the membrane with pbs-t (pbs x and . % tween- ) and blocked for hour in pbs-t plus % milk. after the blocking we washed again with pbs-t and left overnight with each one of the antibodies, anti-car and anti-daf (santacruz biotechnology). we washed the membrane and we added the secondary fluorescent antibodies (dylight and dylight conjugated, thermo scientific) for hour. we washed the membrane one last time and measure the fluorescence in the odyssey system (li-cor). in order to prepare cells for cell cytometry analysis, cells were washed twice with pbs x and trysinized, washed again twice in pbs. cells were stained for minutes with either car-pe or daf-fitc antibodies (millipore and abcam) on ice. unbound antibody was discarded and cells were washed again with pbs x. cells were resuspended in % parafolmaldehyde (pfa, electron microscopy sciences) and kept in the dark for minutes. after the incubation pfa was discarded and cells were resuspended in μl of pbs x. cells were kept at c until analysed. for each cell type used (hela and a ) specific instrument settings were set according to the size and complexity of the cell type, as well as antibodies fluorescence. samples were analysed using the macsquant flow cytometer (miltenyi biotec) using well plates and obtaining , events per sample. mock samples were also used in each plate to setup the baseline. results were analysed using flowjo software v . relative fitness values were obtained by competing each virus population with a marked reference virus that contains four adjacent silent mutations in the polymerase region introduced by direct mutagenesis. co-infections were performed in triplicate at an moi of . using a : mixture of each variant with the reference virus for hours. the proportion of each virus was determined by real time rt-pcr on extracted rna from the infection supernanant, using a mixture of taqman probes labelled with two different fluorescent reporter dyes. mgb_cvb _wt detects wt virus (including the fidelity variants) with the sequence cgcatcgtacccatgg and labelled at the ' end with a fam dye ( -carboxyfluorescein) and mgb_cvb _ref containing the four silent mutations: cgctagctacccatgg was labelled with a ' vic dye. each μl-reaction contained ul of rna, nm of each primer (forward primer, '-gatcgcatatggtgatgatgtga- '; reverse primer, '-agctt cagcgagtaaagatgca- ') and nm of each probe. the relative fitness was determined by the method described by carrasco et al. [ ] . briefly, the formula w = [r(t)/(r ( ))] ( /t), represents the fitness, w, of each mutant genotype relative to the common competitor reference sequence, where r( ) and r(t) represent the ratio of mutant to reference virus densities in the inoculation mixture and t days post-inoculation, respectively. the fitness of the normal wildtype to reference virus was . , indicating no significant differences in fitness due to the silent mutations engineered in the reference virus. blitz assay cvb parental strain, and cvb -e g were propagated in hela cells and purified as described previously [ ] . the membranes of infected cells were broken by three freeze-thaw cycles. virus was concentrated by pelleting through sucrose and purified by tartrate step gradient ultracentrifugation. the virus bands were collected and exchanged into pbs and virus concentration and quality was estimated by measuring the absorbance at , , and nm. binding assays were performed by biolayer interferometry (bli) measured by the blitz from fortebio [ ] . briefly, bli measures binding by sending white light down a glass fiber-based biosensor, which is reflected back up to the instrument from two interfaces: ) the interface between the glass fiber and the biosensor, and ) the interface between the surface chemistry and solution. since the two reflections come from the same white light source in the instrument, they both contain the same wavelengths. when molecules bind to the surface of the biosensor, the path length of the reflection (the one reflecting from the interface between surface and solution) increases while that of the other reflection remains the same. . % bsa and . % tween was added to virus and daf to prevent non-specific binding to the sensor during the assay. purified, his-tagged daf was diluted to . mg/ml and attached to a ni-nta sensor. the daf loaded sensor was then dipped into virus diluted to . mg/ml. let x be a phylogenetic tree, taken to include relative population sizes of the branches, and let x t = (s t p t ) t be the state of the tree at time t = , ,. . .,t. here s t denotes the structure of the tree, i.e. the set of branches existing at time t and p t is a vector with the relative population sizes of all existing branches. furthermore, let y be the set of measurements, where y t ∊ z + xn is the number of reads supporting each of the codons at each of the n different positions in the genome. by bayes' theorem, the output probability p(y | x) follows a multinomial distribution for each position. the dynamics of the phylogenetic tree are naturally assumed to be markovian, i.e. pðx t jx tÀ Þ; where the transition probability can be expressed using the structural and population dynamics parts separately, pðx t jx tÀ Þ ¼ pðp t js t ; s tÀ ; p tÀ Þpðs t js t À ; p tÀ Þ ¼ pðp t js t ; p tÀ Þpðs t js tÀ ; p tÀ Þ: a simple random walk model is used for the population dynamics p(p t | s t , p t- ), the population change from t− to t is taken to be lognormally distributed with standard deviation σh where h is the length of the time step. the structural part, p(s t | s t− , p t− ), depends on the mutation frequency of the virus and the population size of the haplotype in which the mutation occurs. the posterior probability is thus, assuming that s and p are known, pðp t js t ; p tÀ Þpðs t js tÀ ; p tÀ Þ to make a maximum likelihood estimation (mle) of x from the posterior tractable, some approximations are made. the attention is limited to a small set of variants, greatly reducing the number of possible tree structures. the most prevalent variants in the data should be included as they dominate the dynamic behaviour, but minority variants of particular interest can also be added. every possible tree structure matching the selected set of variants is generated. the time of appearance for each variant is set to where it is first seen in the measurements, i.e. the first time the frequency is above a small threshold. an mle of p(p t | s t , y t ) is then computed for each tree and time point. the rationale is that there is very little freedom for the population sizes to deviate from a point which can explain the output data. by construction of the tree structure, the number of non-reference haplotypes equals the number of variants at each time point in every tree. hence, there is at most one convex combination of the haplotypes that match the event probabilities of the multinomial distribution for the output data. due to the high number of reads in the deep sequencing data, moving away from the optimal point will cause a quick drop in the posterior probability. dependencies between variants that are close enough to be covered by a single read are included in the model (amino acid residues and ). the mle of x can be found by evaluating the posterior for each generated tree and picking the most likely. all generated trees contain the same number of mutations. since the value of the posterior probably only needs to be known up to a multiplicative constant, the effect of the overall mutation frequency of the virus on p(s t | s t− , p t− ) cancels out. hence, p(s t | s t− , p t− ) is simply proportional to the population size of the haplotypes in which the mutations occur. supporting information s fig. expression of daf in hela and a by confocal microscopy. localization of daf, in green, in hela (a) or a (b) cells by confocal microscopy. nuclear staining was done with dapi (blue). (b) daf could only be detected when the exposure was increased times compared to the hela settings. (pdf) s movie. expression of car in hela cells. hela cells were plated onto coverslips and processed for confocal microscopy analysis, as described in materials and methods. z stack images acquired for fig f and g were mounted as a video using the imaris software. (avi) s movie. expression of car in a cells. a cells were plated onto coverslips and processed for confocal microscopy analysis, as described in materials and methods. z stack images acquired for fig f and g were mounted as a video using the imaris software. (avi) s dataset. minority variants in replicate passage series in a cells. the following tables reveal all of the minority variants identified by bioinformatic analysis that are present at frequencies above . ( . % of total population). the limit of detection where variants could not be distinguished from background error was always above . frequency, but for clarity, variants at . to . frequencies are not shown. the total number of variants in each population are quite similar, ranging from to . the majority of these are very low frequency variants that do not change over the passage series. as would be expected by the generally neutral nature of synonymous mutations, nearly half of low frequency variants are synonymous. in some cases however, a number of synonymous mutations are found at increasing frequency from passage to (eg. nt. position in replicate ), or at significantly higher, yet relatively constant, frequency over the passage series (eg. nt. position in replicate ). their presence may indicate variants altering rna structure, codon usage or other factors influencing fitness, but may also result from stochastic fixation of neutral mutations since they generally appear in only one of six replicates. some variants with amino acid changes are present at considerable frequencies, without significant increase or decrease over the passage series (eg. vp position - ). their contribution to the fitness of the population has not been studied here, but lack of increase in frequency suggests that they do not play a role in adaptation to a cells. data from these tables was used to generate panels in fig . (xlsx) nucleotide sequence heterogeneity of an rna phage population evolvability of an rna virus is determined by its mutational neighbourhood multihost experimental evolution of a plant rna virus reveals local adaptation and host-specific mutations intrahost evolutionary dynamics of canine influenza virus in naive and partially immune dogs closing the gap: the challenges in converging theoretical, computational, experimental and real-life studies in virus evolution ultradeep sequencing analysis of population dynamics of virus escape mutants in rnai-mediated resistant plants whole genome deep sequencing of hiv- reveals the impact of early minor variants upon immune recognition during acute infection mutational and fitness landscapes of an rna virus revealed through population sequencing a decade after sars: strategies for controlling emerging coronaviruses experimental adaptation of an influenza h ha confers respiratory droplet transmission to a reassortant h ha/h n virus in ferrets substitutions near the receptor binding site determine major antigenic change during influenza virus evolution isolation of a common receptor for coxsackie b viruses and adenoviruses and coxsackievirus b adapted to growth in rd cells binds to decay-accelerating factor (cd ) interaction of decayaccelerating factor with coxsackievirus b a real-time rt-pcr assay for quantifying the fitness of tobacco etch virus in competition experiments single amino acid changes in the virus capsid permit coxsackievirus b to bind decay-accelerating factor clonal interference and the evolution of rna viruses beneficial mutations and the dynamics of adaptation in asexual populations clonal interference is alleviated by high mutation rates in large populations the crystal structure of a coxsackievirus b -rd variant and a refined -angstrom cryo-electron microscopy reconstruction of the virus complexed with decay-accelerating factor (daf) provide a new footprint of daf on the virus surface label-free detection of biomolecular interactions using biolayer interferometry for kinetic characterization the coxsackievirus and adenovirus receptor is a transmembrane component of the tight junction interaction with decay-accelerating factor facilitates coxsackievirus b infection of polarized epithelial cells virus-induced abl and fyn kinase signals permit coxsackievirus entry through epithelial tight junctions high-throughput dna sequencing errors are reduced by orders of magnitude using circle sequencing quasispecies diversity determines pathogenesis through cooperative interactions in a viral population complete nucleotide sequence of infectious coxsackievirus b cdna: two initial ' uridine residues are regained during plus-strand rna synthesis deep sequencing analysis of viral infection and evolution allows rapid and detailed characterization of viral mutant spectrum we would like to thank dr. isabel puigdomenech for her help with protein expression analysis and for helpful discussions. we would like to dedicate this paper in her memory, a promising young scientist and friend, who left us abruptly and far too soon. key: cord- -iq z authors: marsh, glenn a.; de jong, carol; barr, jennifer a.; tachedjian, mary; smith, craig; middleton, deborah; yu, meng; todd, shawn; foord, adam j.; haring, volker; payne, jean; robinson, rachel; broz, ivano; crameri, gary; field, hume e.; wang, lin-fa title: cedar virus: a novel henipavirus isolated from australian bats date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: iq z the genus henipavirus in the family paramyxoviridae contains two viruses, hendra virus (hev) and nipah virus (niv) for which pteropid bats act as the main natural reservoir. each virus also causes serious and commonly lethal infection of people as well as various species of domestic animals, however little is known about the associated mechanisms of pathogenesis. here, we report the isolation and characterization of a new paramyxovirus from pteropid bats, cedar virus (cedpv), which shares significant features with the known henipaviruses. the genome size ( , nt) and organization of cedpv is very similar to that of hev and niv; its nucleocapsid protein displays antigenic cross-reactivity with henipaviruses; and it uses the same receptor molecule (ephrin- b ) for entry during infection. preliminary challenge studies with cedpv in ferrets and guinea pigs, both susceptible to infection and disease with known henipaviruses, confirmed virus replication and production of neutralizing antibodies although clinical disease was not observed. in this context, it is interesting to note that the major genetic difference between cedpv and hev or niv lies within the coding strategy of the p gene, which is known to play an important role in evading the host innate immune system. unlike hev, niv, and almost all known paramyxoviruses, the cedpv p gene lacks both rna editing and also the coding capacity for the highly conserved v protein. preliminary study indicated that cedpv infection of human cells induces a more robust ifn-β response than hev. henipaviruses were first discovered in the s following investigation of serious disease outbreaks in horses, pigs and humans in australia and malaysia [ , ] and comprise the only known biosafety level (bsl ) agents in the family paramyxoviridae [ ] . depending upon the geographic locations of outbreaks, and the virus and animal species involved, case mortality is between % to % in both humans and animals [ , ] , making them one of the most deadly group of viruses known to infect humans. the genus henipavirus in the subfamily paramyxovirinae currently contains two members, hendra virus (hev) and nipah virus (niv) [ ] . fruit bats in the genus pteropus, commonly known as flying foxes, have been identified as the main natural reservoir of both viruses although serological evidence suggests that henipaviruses also circulate in non-pteropid bats [ , , , ] . the discovery of henipaviruses had a significant impact on our understanding of genetic diversity, virus evolution and host range of paramyxoviruses. paramyxoviruses, such as measles virus and canine distemper virus, were traditionally considered to have a narrow host range and to be genetically stable with a close to uniform genome size shared by all members of paramyxovirinae [ ] . henipaviruses shifted this paradigm on both counts having a much wider host range and a significantly larger genome [ ] . identification of bats as the natural reservoir of henipaviruses also played an important role in significantly increasing international scientific attention on bats as an important reservoir of zoonotic viruses, including ebola, marburg, sars and melaka viruses [ , , , ] . since the discovery of the first henipavirus in , much progress has been made in henipavirus research, from identification of functional cellular receptors to the development of novel diagnostics, vaccine and therapeutics [ , , , , , , , , , , ] . by contrast, there is little understanding of the pathogenesis of these highly lethal viruses. this is due in part to the requirement of a high security bsl facility for any live infection studies and in part to the limited range of research tools and reagents for the current small animal models. research into the mechanisms of henipavirus pathogenesis is also hampered by the lack of related, but non-pathogenic or less pathogenic viruses, thus preventing targeted comparative pathogenetic studies. early serological investigations in australia and more recent studies in other regions (e.g., china) indicated the presence of cross-reactive, but not cross-neutralizing, antibodies to henipaviruses in bats of different species [ ] . these findings were further supported by the detection of henipavirus-like genomic sequences in african bats [ ] . discovery and isolation of these related viruses will be highly important to our further understanding of henipavirus evolution, mechanism of cross-species transmission, and pathogenesis in different animal species. here we report the isolation and characterization of a new bat henipavirus which, based on preliminary infection studies, is nonpathogenic in two of the small animal infection models currently used in henipavirus research. we believe that this new virus will provide a powerful tool to facilitate our future study into different aspects of henipaviruses, especially in the less advanced area of pathogenesis. as part of our on-going field studies on hev genetic diversity and infection dynamics in the queensland flying fox populations, urine samples were collected on a regular basis for pcr and virus isolation. since the establishment of the pteropus alecto primary cell lines in our group [ ] , we have intensified our effort to isolate live virus from these urine samples by routinely inoculating separate primary cell lines derived from kidney, spleen, brain, and placenta, as well as vero cells. syncytial cpe was observed in kidney cell (paki) monolayers days post inoculation (dpi) with two different urine samples (fig. s ) collected in september from a flying fox colony in cedar grove, south east queensland (see fig. s for map location). no cpe was observed in any of the four other cell lines. supernatant harvested dpi was used to inoculate fresh paki cell monolayers. after two passages in paki cells, the virus was able to infect and cause cpe in vero cells. however, the cpe morphology of cedpv infection in vero cells was different from that of hev infection. further analysis using hev-specific pcr primers indicated that the new bat virus was not an isolate of hev. considering the formation of syncytial cpe by this new virus and the previous success in isolating paramyxoviruses from bat urine [ , , ] , paramyxovirus family-specific and genus-specific primers were used to determine whether this new virus was a member of the family paramyxoviridae. positive pcr fragments of the expected sizes were obtained from the paramyxovirinae and respirovirus/morbillivirus/henipavirus primer sets developed by tong et al [ ] . sequencing of the pcr products indicated that it was a new paramyxovirus most closely related to hev and niv. based on these preliminary data, the virus was named cedar virus (cedpv) after the location of the bat colony sampled. full length genome sequence was determined by a combination of three different approaches, random deep sequencing using technology, sequencing of pcr products obtained using degenerate primers designed based on known henipaviruses, and race to determine the precise genome terminal sequences. as shown in fig. , the genome of cedpv is , nt in length most similar to that of hev in the family. the full genome sequence has been deposited to genbank (accession no. jq ). the genome size is a multiple of , hence abiding by the rule-of-six observed for all known members of the subfamily paramyxovirinae [ ] . it has a -nt intergenic sequence of ctt absolutely conserved at all seven positions and highly conserved gene start and stop signals similar to those present in hev and niv (fig. s ) . also similar to the hev genome is the presence of relatively large non-coding regions in the cedpv genome ( fig. and table ). the overall proteincoding capacity of the cedpv genome is . % which is significantly lower than the average of . % for other family members but higher than hev at . %. as the genome size of cedpv and hev is very similar, the increased coding capacity of cedpv is attributed to an increase in protein sizes for five of the six major proteins, with the l protein being -aa larger (table ) . at , aa, the cedpv l protein is the largest, not only in the family paramyxoviridae but also for all known viruses in the order mononegavirale. phylogenetic analysis based on the full length genome sequence and the deduced amino acid sequences of each structural protein confirmed the initial observation that cedpv is most closely related to henipaviruses in the family. a phylogenetic tree based on the deduced sequences of the nucleocapsid protein (n) is presented in fig. . phylogenetic tree based on whole genome sequences gave similar results (fig. s ). cedpv is more closely related to hev and niv than henipavirus-like sequences detected in african bats [ , ] as shown in a phylogenetic tree based on the only sequences available of a -nt l gene fragment (fig. s ) . a phosphoprotein (p) gene lacking rna editing and coding capacity for the v protein first discovered for the parainfluenza virus (piv , previously known as simian virus ), almost all members of paramyxovirinae have a p gene which produces multiple proteins through an rna editing mechanism by addition of non-templated g residues leading to production of n-terminal co-linear proteins from different reading frames downstream from the editing site [ , ] . these multiple gene products are known to play a key role in antagonizing the innate response of susceptible hosts [ ] . a search of cedpv for open reading frames (orf) in the p gene revealed a -aa p protein and a -aa c protein, but failed to find the highly conserved, cysteine-rich v orf present in most other paramyxoviruses. the rna editing site with the sequence of aaaaggg, which is absolutely conserved in all known hev and niv isolates discovered to date, is also missing from the cedpv p gene sequence. to further verify that there are no multiple mrnas produced from the cedpv p gene, direct sequencing of p gene transcripts was conducted from cedpv-infected vero cells using multiple sets of primers generating overlapping fragments covering the entire coding region of the p gene. each produced hendra and nipah viruses are highly pathogenic paramyxoviruses that have emerged from bats within the last two decades. both are capable of causing fatal disease in both humans and many mammal species. serological and molecular evidence for henipa-like viruses have been reported from numerous locations including asia and africa, however, until now no successful isolation of these viruses have been reported. this paper reports the isolation of a novel paramyxovirus, named cedar virus, from fruit bats in australia. full genome sequencing of this virus suggests a close relationship with the henipaviruses. antibodies to cedar virus were shown to cross react with, but not cross neutralize hendra or nipah virus. despite this close relationship, when cedar virus was tested in experimental challenge models in ferrets and guinea pigs, we identified virus replication and generation of neutralizing antibodies, but no clinical disease was observed. as such, this virus provides a useful reference for future reverse genetics experiments to determine the molecular basis of the pathogenicity of the henipaviruses. uniform trace files indicating a lack of rna editing activities, which is very different from the mixed peaks generated by hev and niv immediately after the editing site (fig. s ). to our knowledge, cedpv is the first member of paramyxovirinae that lacks both rna editing and any v-related coding sequence in its p gene. further investigation is required to exclude the possibility that the p-gene editing in cedpv is cell-or tissue-specific and not present or present at an extremely low level in the current viruscell system. the striking similarity in genome size and organization and the presence of highly conserved protein domains among the n, m and l proteins between cedpv and henipaviruses prompted us to investigate the antigenic relatedness of these viruses. staining of cedpv-infected vero cells using rabbit anti-henipavirus antibodies indicated the presence of cross-reactivity. this cross-reactivity was further confirmed in reverse by staining of hev-infected vero cells using a rabbit serum raised against a recombinant cedpv n protein (fig. ) . however, analysis by virus neutralization test using either polyclonal or monoclonal antibodies found that henipavirus-neutralizing antibodies were unable to neutralize cedpv. similarly, cedpv-neutralizing antibodies obtained in our infection studies (see below) also failed to neutralize either hev or niv. it can therefore be concluded that cedpv and henipaviruses share cross-reactive antigenic regions, but not crossneutralizing epitopes. to further investigate the relationship between cedpv and recognized henipaviruses, we investigated the use of the henipavirus receptors, the ephrin-b and -b host cell proteins, as potential receptors for cedpv infection. our previous studies have demonstrated that the ephrin-b and -b expression negative hela-usu cell line could support henipavirus infection and formation of syncytial cpe only when either the ephrin-b or -b gene was transiently expressed in the cells [ , ] . for cedpv, similar observations were made with respect to the ephrin-b receptor. as shown in fig. , cedpv failed to infect hela-usu, but was able to infect and cause syncytial cpe when the human ephrin-b gene was expressed. in contrast, when ephrin-b molecule was introduced, there was no evidence of infection. ferrets, guinea pigs, and mice exhibit differing responses to the previously described henipaviruses hev and niv, with ferrets and guinea pigs, but not mice developing severe disease characterized by systemic vasculitis [ , , , , ] . in contrast, ferrets and guinea pigs exposed to cedpv by, respectively, oronasal and intraperitoneal routes remained clinically well although neutralizing antibody was detected in serum between to days pi ( table ) . balb-c mice exposed to cedpv by the oronasal route remained clinically well and did not develop neutralizing antibody in serum by day pi. in ferrets electively euthanized at earlier time-points, there was reactive hyperplasia of tonsillar lymphoid tissue, retropharyngeal and bronchial lymph nodes, accompanied by edema and erythrophagocytosis. cedpv antigen was detected in bronchial lymph node of one animal euthanized on day pi, consistent with viral replication in that tissue; cross-reactive immunostaining against anti-niv n protein antibodies was also noted (fig. ) . no other significant histological lesions were identified. viral rna was detected in selected lymphoid tissues of (of ) ferrets sampled day to pi, including pharynx, spleen, and retropharyngeal and bronchial lymph nodes, as well as the submandibular lymph node of the ferret euthanized on day pi. this pattern of lymphoid involvement suggests that there may be transient replication in the upper and lower respiratory tracts although cedpv genome was not recovered from nasal washes, oral swabs, pharynx or lung tissue of affected animals. virus isolation was unsuccessful for all pcr positive tissues. as a first step towards the understanding of the pathogenicity difference between cedpv and hev, we examined the ifn responses in human hela cells upon virus infection. as shown in fig. , while the induction of ifn-a was similar in cells infected with hev or cedpv, there was a significant difference of ifn-b production upon infection by hev or cedpv, with cedpvinfected cell producing a much higher level of ifn-b. to investigate the cedpv exposure status of pteropid bats in queensland and potential co-infection (either concurrent or consecutive) of cedpv with hev, we tested flying fox sera collected previously for other studies for antibody against the two viruses. due to the cross-reactivity observed above, virus neutralization tests were conducted to obtain more accurate infection data for each virus. overall, % of the sera were cedpv-positive and % hev-positive (table s ). co-infection was reflected in % of the sera tested. the emergence of bat-borne zoonotic viruses (including hev, niv, ebola, marburg, and sars) has had a significant impact on public health and the global economy during the past few decades. with the rapidly expanding knowledge of virus diversity in bat populations around the world, it is predicted that more bat-borne zoonotic viruses are likely to emerge in the future. the discovery of a novel ebolavirus-like filovirus in spanish microbats demonstrates that the potential for such spill over events is not limited to africa or asia [ ] . it is therefore important to enhance our preparedness to counter future outbreaks by conducting active pre-emergence research into surveillance, triggers for cross-species transmission, and the science of identification of potential pathogens. henipaviruses represent one of the most important bat-borne pathogens to be discovered in recent history. although cedpv displays some differences from existing members of the genus henipavirus, we propose that cedpv be classified as a new henipavirus based on the following shared features with known henipaviruses: ) it is antigenically related to current henipaviruses; ) its genome size and organization is almost identical to those of hev and niv; ) it has a similar prevalence in flying foxes; and ) it uses ephrin-b as the cell entry receptor. the lack of cross-neutralization between cedpv and hev or niv was not unexpected from the comparative sequence analysis of all the deduced proteins, especially the g protein (see table ). it is clear that the genetic relatedness of cedpv with hev or niv is much lower than between hev and niv. however, the percentage sequence identities of the major viral proteins between cedpv and hev/niv are on average at least % higher than that between hev/niv and any other known paramyxoviruses. also, there was no antigenic cross-reactivity observed between cedpv and representative viruses of the other paramyxovirus genera in the subfamily paramyxovirinae (fig. s ) . like other paramyxoviruses, the p gene of henipaviruses produces multiple proteins which play a key role in viral evasion of host innate immune responses [ , , ] . one of these is the cys-rich v protein: all members of the subfamily paramyxovirinae produce the v protein with the exception of the human parainfluenza virus (hpiv ). although a putative rna editing sequence (aagaggg) is present at the expected editing site of the p gene, the hpiv rna polymerase does not produce an edited mrna of the p gene [ ] . there are remnants of the v orf easily detectable in the hpiv p gene although the predicted -aa orf region is interrupted by multiple in-frame stop codons. of the cys residues conserved between bovine parainfluenza virus and sendai virus, four are still present in the non-functional v orf of hpiv [ ] . in contrast, an extensive orf and sequence homology search of the cedpv p gene only identified one aa coding region with minimal sequence identity to the v orfs of hev and niv (see fig. s ). in this region, out of the cys residues conserved between hev and niv v proteins, only are present in the cedpv p gene. furthermore, the sequence (agatgag) upstream from this putative orf v coding region does not match the consensus rna editing site. it can therefore be concluded that cedpv is the only member of paramyxovirinae which lacks both the functional v mrna/protein and the coding capacity for the rna editing site and orf v. the evolutionary significance of this finding needs further investigation. our in vitro study indicated that ephrin b , but not ephrin b , was able to restore cedpv infection in the ephrin b -deficient hela cells. while this is highly suggestive that ephrin b is the functional entry receptor for cedpv, it should be emphasized that this was not a direct proof that ephrin b is the receptor. further investigation is required to confirm this. in our preliminary studies, it was shown that cedpv was able to replicate in guinea pigs and ferrets, but failed to cause significant clinical diseases, unlike that of the closely related hev and niv. these first infection experiments were conducted with a high dose if virus to establish whether the cedpv could replicate in these animals and determine the degree of any clinical disease. a second experiment was then carried out in ferrets to determine the site of replication and tissue tropism in sequentially sacrificed animals. a lower dose was used to gain better comparison with similar infection experiments using hev and niv [ , ] . although these initial experimental infection studies indicate that cedpv is less or non-pathogenic in these species, it is possible that cedpv may be pathogenic in other hosts, such as horses. we hypothesize that the lack of a v protein may impact on the pathogenicity. in this regard, it was encouraging to observe that infection of human cells by cedpv induced a much more robust ifn-b response than hev. further study is required to dissect the exact molecular mechanism of this observed difference. due to the close relationship between cedpv and hev, it was important to investigate the possibility of co-infection by these two viruses in the australian bat population. based on the detection of neutralizing antibodies at % for cedpv, % for hev and % for both, it can be concluded that the co-infection rate is very close to the theoretical rate of . % (the product of the two independent infection rates). based on this limited preliminary analysis, it appears that infection of bats by one henipavirus neither prevents nor enhances the likelihood of infection by the other. in summary, the discovery of another henipavirus in australian flying foxes highlights the importance of bats as a significant reservoir of potential zoonotic agents and the need to expand our understanding of virus-bat relationships in general. our future research will be directed at determining whether spill-over of cedpv into other hosts has occurred in the past in australia, whether cedpv is pathogenic in certain mammalian hosts, and whether cedpv exists in bat populations in geographically diverse regions. all animal studies were approved by the csiro australian animal health laboratory's animal ethics committee and conducted following the australian national health and medical research council code of practice for the care and use of animals for scientific purposes guidelines for housing and care of laboratory animals. cell lines used this study were vero (atcc), hela-usu [ ] , and the p. alecto primary cell lines derived from kidney (paki), brain (pabr), (spleen) pasp and placenta (papl) recently established in our group [ ] . cells were grown in dulbecco's modified eagle's medium nutrient mixture f- ham supplemented with double strength antibiotic-antimycotic (invitrogen), mg/ml ciprofloxacin (mp biomedicals) and % fetal calf serum at uc in the presence of % co . urine (approximately . - ml) was collected off plastic sheets placed underneath a colony of flying foxes (predominantly pteropus alecto with some p. poliocephalus in the mixed population) in cedar grove, south east queensland, australia and pooled into -ml tubes containing . ml of viral transport medium (spga: a mix of sucrose, phosphate, glutamate and albumin plus penicillin, streptomycin and fungizone). the tubes were temporarily stored on ice after collection and transported to a laboratory in queensland, frozen at uc, and then shipped on dry ice to the csiro australian animal health laboratory (aahl) in geelong, victoria for virus isolation. the samples were thawed at uc and centrifuged at , g for min to pellet debris. urine in the supernatant (approximately . - ml) was diluted : in cell culture media. the diluted urine was then centrifuged at , g for min and split evenly over vero, paki, pabr, pasp and papl cell monolayers in -cm tissue culture flasks. the flasks were rocked for h at uc, ml of fresh cell culture media was added and then incubated for d at uc. the flasks were observed daily for toxicity, contamination, or viral cytopathic effect (cpe). cells showing syncytial cpe were screened using published broadly reactive primers [ ] for all known paramyxoviruses and a subset of paramyxoviruses. pcr products were gel extracted and cloned into pgem t-easy (promega) to facilitate sequencing using m primers. sequences were obtained and aligned with known paramyxovirus sequences allowing for initial classification. whole genome sequence was determined using a combination of sequencing [ ] and conventional sanger sequencing. virions from tissue culture supernatant were collected by centrifugation at , g for min and resuspended in ml of pbs and mixed with ml of freshly made avl for rna extraction using qiaamp viral rna mini kit (qiagen). synthesis of cdna and random amplification was conducted using a modification of a published procedure [ ] . briefly, cdna synthesis was performed using a random octomer-linked to a -mer defined primer sequence: ( -gtttcccagtaggtctcnnn nnnnn- ) and superscript iii reverse transcriptase (invitrogen). ml of ds-cdna was amplified in ml pcr reactions with hot-start taq polymerase enzyme (promega) and -a*g*c*a*c tgtaggtttcccag-taggtctc- (where * denotes thiol modifications) as amplification primers for cycles of uc/ min, uc/ min, uc/ min after an initial denaturation step of min at uc and followed by purification with the qiaquick pcr purification kit (qiagen). sample preparation for roche sequencing ( life sciences branford, ct, usa) was according to their titanium series manuals, rapid library preparation and empcr lib-l sv. to obtain an accurate cedpv genome sequence, generated data (after removing low quality, ambiguous and adapter sequences) was analysed by both de novo assembly and read mapping of raw reads onto the cedpv draft genome sequence derived from sanger sequencing. for read mapping, snps and dips generated with the clc software were manually assessed for accuracy by visualising the mapped raw reads (random pcr errors are obvious compared to real snps and dips especially when read coverage is deep). consensus sequences for both de novo and read mapping assembly methods were then compared to the sanger sequence with the latter used to resolve conflicts within the low coverage regions as well as to resolve homopolymer errors. sequences of genome termini were determined by -and -race using a protocol previously published by our group [ ] . briefly, approximately ng of rna was ligated with adaptor dt (see reference for sequence information) using t rna ligase (promega) followed by cdna synthesis using the super-script iii rt kit (invitrogen) and an adaptor-specific primer, dt . pcr amplification was then carried out using dt and one or more genome-specific primers. pcr products were sequenced directly using either dt or genome specific primers by an in-house service group on the abi sequencer . the clc genomics workbench v . . (clc inc, aarhus, denmark) was used to trim adapter and cdna/pcr primer sequences, to remove low quality, ambiguous and small reads , bp and to perform de novo and read mapping assemblies all with default parameters. clone manager professional ver . (scientific and educational software, cary, nc, usa) was used to join overlapping contigs generated by de novo assembly. phylogenetic trees were constructed by using the neighbor-joining algorithm with bootstrap values determined by , replicates in the mega software package [ ] . quantitative pcr assays (qpcr) were established based on cedpv-specific sequences obtained from the high throughput sequencing. a taqman assay on the p gene was developed and used for all subsequent studies. the sequences of the primer/probe are as follows: forward primer, -tgcat tgagc gaacc catat ac; reverse primer, -gcacg cttct tgaca gagtt gt; probe, -tcccg agaaa ccctc tgtgt ttga-mgb. the coding region for the cedpv n protein was amplified by pcr with a pair of primers flanked by asci ( end) and noti ( end) sites for cloning into our previously described gst-fusion expression vector [ ] . the expression and purification by gel elution was conducted as previously described [ ] . for antibody production, purified protein was injected subcutaneously into different sites of adult (at a dose of mg per animal) new zealand white female rabbits at days and . the csiro's triple adjuvant [ ] was used for the immunization. animals were checked for specific antibodies after days and and euthanized at day for the final blood collection. for immunofluorescence antibody test, vero cell monolayers were prepared in -well chamber slides by seeding at a concentration of , cells/well in ml of cell media and incubating over night at uc. the cell monolayers were infected with an moi of . of cedpv, hev or niv and fixed with % ice-cold methanol at h post-infection. the chamber slides were blocked with ml/well of %bsa in pbs for min at uc before adding ml/well of rabbit sera against cedpv n or niv n diluted : . after incubation at uc for min, the slides were washed three times in pbs-t and incubated with ml/well of anti-rabbit alexafluore conjugate (invitrogen) diluted : at uc for min. the slides were then washed three times in pbs-t and mounted in % glycerol/pbs for observation under a fluorescence microscope. for virus neutralization test, serial two-fold dilutions of sera were prepared in duplicate in a -well tissue culture plate in ml cell media (minimal essential medium containing earle's salts and supplemented with mm glutamine, antibiotic-antimycotic and % fetal calf serum). an equal volume containing tcid of target virus was added and the virus-sera mix incubated for min at uc in a humidified % co incubator. ml of vero cell suspension containing cells/ml was added and the plate incubated at uc in a humidified % co incubator. after days, the plate was examined for viral cpe. the highest serum dilution generating complete inhibition of cpe is defined as the final neutralizing titer. human ephrin b and b genes were cloned into pqcxih (clontech) and the resulting plasmids packaged into retrovirus particles in the gp - packaging cell line (clontech) and pseudotyped with vesicular stomatitis virus g glycoprotein (vsv-g) following the manufacturer's instructions. hela-usu cell line [ ] was infected with the vsv-g pseudotyped retrovirus particles in the presence of mg/ml polybrene (sigma). h post infection, the medium was changed and the cells were allowed to recover for h, allowing time for the retroviral insert to be incorporated into the cell genome and for expression of the hygromycin resistance gene. h post infection, cells transformed by the retrovirus were selected for by the addition of mg/ml hygromycin in the media. stocks of cells that were resistant to hygromycin were prepared and frozen. hela-usu and ephrin-expressing hela-usu cells were seeded in -well tissue culture plates at a density of , cells/well overnight. the viruses (hev and cedpv) were diluted to give an moi of . and inoculated into the wells. the cell monolayers were examined daily for syncytial cpe. animal studies were carried out in the bsl animal facility at aahl. ferrets, guinea pigs and mice were used on the basis of their known and varying responses to exposure to other henipaviruses. firstly, tcid /ml cedpv passaged twice in bat paki cells was administered to male ferrets ( ml oronasally); female guinea pigs ( ml intraperitoneally); and female balb-c mice ( ml oronasally). guinea pigs and mice were implanted with temperature sensing microchips (lifechip bio-thermo, destron fearing) and weighed daily. ferret rectal temperature and weight was recorded at sampling times. animals were observed daily for clinical signs of illness and were euthanized at d postinoculation. sera were collected on days , and to test for neutralizing antibody against cedpv. secondly, on the basis of asymptomatic seroconversion to cedpv noted in ferrets in the first study, further female ferrets were exposed by the oronasal route to a lower dose of tcid . two animals were euthanized on each of days , and post-inoculation and one on day . nasal washes, oral swabs, and rectal swabs were collected on days , , , and and urine was sampled on the day of euthanazia; each specimen was assessed for cedpv genome. a wide range of tissue samples were collected at post mortem examination and assessed by routine histology, immunohistochemistry (using rabbit antibodies raised against recombinant cedpv and niv n proteins, respectively), qpcr (see above) and virus isolation using reagents and procedures previously established in our group [ ] . hela cells were infected with hendra and cedar viruses at an moi . for hours, at which time total cellular rna was extracted and ifn-a and ifn-b mrna levels were quantified by real-time pcr using power sybr green rna-to-ct -step kit (applied biosystems). primers were as previously described [ ] . sera from flying foxes collected during - from queensland, australia were screened for neutralizing antibodies to cedpv. virus neutralization test was conducted as described above (antibody tests). all serum samples were tested at a dilution of : . a morbillivirus that caused fatal disease in horses and humans nipah virus: a recently emergent deadly paramyxovirus paramyxoviridae: the viruses and their replication hendra and nipah viruses: different and dangerous henipavirus vaccine development fields virology nipah virus infection in bats (order chiroptera) in peninsular malaysia antibodies to nipah or nipah-like viruses in bats evidence of henipavirus infection in west african fruit bats pteropid bats are confirmed as the reservoir hosts of henipaviruses: a comprehensive experimental study of virus transmission fruit bats as reservoirs of ebola virus marburg virus infection detected in a common african bat bats are natural reservoirs of sars-like coronaviruses a previously unknown reovirus of bat origin is associated with an acute respiratory disease in humans recombinant nipah virus vaccines protect pigs against challenge feline model of acute nipah virus infection and protection with a soluble glycoprotein-based subunit vaccine a recombinant subunit vaccine formulation protects against lethal nipah virus challenge in cats a recombinant hendra virus g glycoprotein-based subunit vaccine protects ferrets from lethal hendra virus challenge a neutralizing human monoclonal antibody protects african green monkeys from hendra virus challenge a neutralizing human monoclonal antibody protects against lethal disease in a new ferret model of acute nipah virus infection neutralization assays for differential henipavirus serology using bio-plex protein array systems ephrin-b ligand is a functional receptor for hendra virus and nipah virus ephrinb is the entry receptor for nipah virus, an emergent deadly paramyxovirus two key residues in ephrinb are critical for its use as an alternative receptor for nipah virus nipah virus: vaccination and passive protection studies in a hamster model henipavirus rna in african bats establishment, immortalisation and characterisation of pteropid bat cell lines tioman virus, a novel paramyxovirus isolated from fruit bats in malaysia isolation of nipah virus from malaysian island flying-foxes a novel approach for collecting samples from fruit bats for isolation of infectious agents sensitive and broadly reactive reverse transcription-pcr assays to detect novel paramyxoviruses cocirculation of diverse paramyxoviruses in an urban african fruit bat population two mrnas that differ by two nontemplated nucleotides encode the amino coterminal proteins p and v of the paramyxovirus sv functional studies of host-specific ephrin-b ligands as henipavirus receptors chloroquine administration does not prevent nipah virus infection and disease in ferrets experimental hendra virus infectionin pregnant guinea-pigs and fruit bats (pteropus poliocephalus) equine morbillivirus pneumonia: susceptibility of laboratory animals to the virus a golden hamster model for human acute nipah virus infection discovery of an ebolavirus-like filovirus in europe family paramyxoviridae antagonism of innate immunity by paramyxovirus accessory proteins the p gene of human parainfluenza virus type encodes p and c proteins but not a cysteinerich v protein genome sequencing in microfabricated high-density picolitre reactors panmicrobial oligonucleotide array for diagnosis of infectious diseases improved rapid amplification of cdna ends (race) for mapping both the and terminal sequences of paramyxovirus genomes mega : molecular evolutionary genetics analysis (mega) software version . btag: a novel six-residue epitope tag for surveillance and purification of recombinant proteins expression of equine morbillivirus (emv) matrix and fusion proteins and their evaluation as diagnostic reagents a new adjuvant interferon signaling remains functional during henipavirus infection of human cell lines we thank kaylene selleck, jessica haining, lauren dagley, susanne wilson, honglei chen and tony pye for technical assistance. we thank drs. michelle baker and peng zhou for providing critical review of the manuscript. key: cord- -d iuk authors: baquero-pérez, belinda; whitehouse, adrian title: hsp isoforms are essential for the formation of kaposi’s sarcoma-associated herpesvirus replication and transcription compartments date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: d iuk kaposi’s sarcoma-associated herpesvirus (kshv) is an oncogenic herpesvirus associated with various aids-related malignancies. like other herpesviruses, multiple processes required for kshv lytic replication, including viral transcription, viral dna synthesis and capsid assembly occur in virus-induced intranuclear structures, termed replication and transcription compartments (rtcs). here we utilised a novel methodology, combining subcellular fractionation and quantitative proteomics, to identify cellular proteins which are recruited to kshv-induced rtcs and thus play a key role in kshv lytic replication. we show that several isoforms of the hsp chaperone family, hsc and ihsp , are redistributed from the cytoplasm into the nucleus coinciding with the initial formation of kshv-induced rtcs. we demonstrate that nuclear chaperone foci are dynamic, initially forming adjacent to newly formed kshv rtcs, however during later time points the chaperones move within kshv rtcs and completely co-localise with actively replicating viral dna. the functional significance of hsp isoforms recruitment into kshv rtcs was also examined using the specific hsp isoform small molecule inhibitor, ver- . intriguingly, results highlight an essential role of hsp isoforms in the kshv replication cycle independent of protein stability and maturation. notably, inhibition of hsp isoforms precluded kshv rtc formation and rna polymerase ii (rnapii) relocalisation to the viral genome leading to the abolishment of global kshv transcription and subsequent viral protein synthesis and dna replication. these new findings have revealed novel mechanisms that regulate kshv lytic replication and highlight the potential of hsp inhibitors as novel antiviral agents. molecular chaperones represent a large group of proteins that are essential for maintaining cellular homeostasis and survival. as such, the roles of these proteins are numerous; facilitating correct protein folding or unfolding, assembly or disassembly of multimeric protein complexes, participating in translocation of proteins and vesicles into organelles, stabilising a wide range of signalling molecules and preventing aggregation of non-native proteins (reviewed in [ , ] ). heat shock proteins (hsp) are classified according to their molecular weight into several families: hsp , hsp , hsp , hsp , hsp , hsp and the small hsp (less than kda) [ ] . the functional importance of the hsp and hsp families of molecular chaperones is exemplified by their emerging implications in a variety of diseases, including cancer [ , ] , neurodegeneration [ ] or viral infection [ , ] . as such they have gained significant interest recently as potential drug targets. eukaryotes have multiple genes encoding for chaperones of the hsp family, which are amongst the most conserved proteins in evolution [ ] [ ] [ ] . the major hsp isoforms are the constitutively expressed hsc , the stress-inducible hsp (ihsp ), the endoplasmic reticulum resident (grp ) and the mitochondrial form (grp ). all hsp isoforms have an n-terminal domain which harbours a highly conserved atpase and a c-terminal substrate binding domain [ ] . hsp isoforms which comprise the inducible and constitutively-expressed isoforms (hsp α and hsp β respectively), the er resident (grp ) and the mitochondrial form (trap ), also possess a n-terminal atp binding domain, although this has no similarity to the atp-binding domain found in the chaperones of the hsp family [ ] . the presence of atpase pockets in both families of chaperones makes these proteins desirable targets for small molecule inhibitors [ , ] . the therapeutic potential of these compounds is especially evident for several hsp inhibitors, having already reached phase ii and iii clinical trials [ , ] . targeting of hsp isoforms has been more challenging [ ] , but recently specific inhibitors have also undergone clinical trials [ , ] . importantly, the development of highly specific inhibitors for hsp isoforms may have potential for the treatment of a diverse group of viruses as the functional importance of hsp isoforms in the life cycle of numerous viruses has been highlighted over the past few years [ ] . distinct hsp isoforms are usurped to aid in many stages of viral replication as varied as viral entry, uncoating, transcription, envelope protein maturation, morphogenesis or dna replication [ ] . therefore, the importance of these chaperones in the life cycle of such a wide range of viruses suggests the potential of these proteins as targets for broad-spectrum antivirals. kaposi's sarcoma-associated herpesvirus (kshv) is the causative agent of several aidsassociated malignancies, including kaposi's sarcoma (ks), a highly vascular tumour of ne cannot be purified completely due to its multiple subcellular connections. the outer nuclear membrane is continuous with the endoplasmic reticulum and interacts with the cytoskeleton [ , ] while the inner nuclear membrane binds to host chromatin [ ] [ ] [ ] . thus, we took advantage of its incomplete purity so that we could isolate not only the ne and embedded nuclear pore complexes, but also components found in the ne neighbourhood, such as rtcs. utilising this novel approach we demonstrate that cellular chaperones from the hsp family (hsc and ihsp ) are significantly increased in the ne-associated rtcs of reactivated cells. functional dissection further demonstrates that these chaperones were specifically recruited to the periphery of incipient rtcs coinciding in time with their formation. when actively replicated viral dna was synthesised the chaperones were recruited within rtcs. strikingly, inhibition of hsp isoforms precluded rtc formation, curtailed chaperone redistribution within rtcs and rnapii recruitment to viral promoters. importantly, abrogation of lytic replication occurred without affecting cell viability, suggesting that the cellular housekeeping functions carried out by these chaperones were not compromised. as such, hsp inhibitors may provide a novel therapeutic approach for the treatment of kshv-associated malignancies, in particular it would be interesting to determine the efficacy of combining the potential of inhibiting lytic replication using hsp inhibitors with the previous reported effect of hsp inhibitors to eradicate latent kshv reservoirs [ ] . the cellular chaperones hsc , ihsp and grp were enhanced in the ne-associated rtcs of reactivated hek- t rkshv. cells to investigate differential proteome changes which occur during kshv lytic replication in ne-associated rtcs, we utilised the hek- t cell line containing a latent recombinant kshv virus (rkshv. ) [ ] . this cell line can be reactivated into a full lytic replication cycle via chemical induction. unreactivated cells were grown in isotopically labelled media (r k ), while cells to be reactivated were grown in label-free media (r k ). after isotopic labelling was complete, cells were reactivated for h and nuclear envelopes (nes) were then successfully purified using a recently described method [ ] , with minor modifications. western blot analysis of the ne preparations demonstrated an enrichment of nucleoporins (nups), lamins and histones and a loss of cytoplasmic (gapdh) and nucleolar (b- , c- ) proteins compared with whole cell (wc) lysates (fig ) . the essential kshv mrna processing protein, orf [ ] , and viral rta served as markers for lytic viral replication and rtc enrichment, as both of these proteins are known to be recruited to kshv rtcs [ , ] . the monoclonal antibody (mab ), which recognises the phenylalanine-glycine (fg)-repeat motif present in numerous nups, and the polyclonal antibodies against nup and lamin b were used to assess enrichment of the ne region. ne preparations showed a higher number of nups (closed arrows) than their respective wc preparations; although some nups were lost following . m salt wash (open arrow). following lc-ms/ms analysis and using a minimum of three unique peptides assigned to a single protein, most proteins ( ) remained unchanged in their abundance irrespectively of kshv lytic infection and only five proteins had a significant reduction (ratio cut-off < . ) in nes of reactivated cells. in contrast, proteins showed a significant increase (ratio cut-off > . ) during lytic replication. importantly, multiple cellular proteins that are known or expected to localize within herpesvirus rtcs; such as those associated with kshv ori-lyt, the hcmv transactivator ie -p protein or the herpes simplex virus- (hsv- ) icp protein were found significantly increased in the ne regions of reactivated cells using the less stringent ratio cut-off of . (s table) . some of these cellular proteins included csnk a [ ] , blm [ ] , topoisomerases i and ii [ , , ] and dead box helicases ddx [ ] and ddx [ ] . thus, lc-ms/ms results confirmed the correct isolation of the ne region and accompanying rtcs. importantly, many of the identified proteins most likely represent novel cellular proteins hijacked by kshv, not only due to the subcellular fractionation carried out but also to the uncommon use of urea for protein extraction before lc-ms/ ms. all proteins identified by lc-ms/ms can be seen on s dataset. bioinformatical analysis revealed several upregulated pathways (ratio cut-off > . ) in reactivated cells (s table) . of particular interest was an upregulated pathway which related to protein folding. this included several hsp isoforms and their associated co-chaperones from the hsp (dnaj) family ( table ) . notably, the constitutively expressed chaperone hsc presented a . -fold increase with unique peptides assigned. this protein had the highest fold increase associated with the most unique peptide number of all the proteins identified by lc-ms/ms. this could be due to increased hsc expression or to the redistribution of hsc from the cytoplasm into the ne region during kshv lytic replication. therefore, due to the vital importance of hsp isoforms in the replication cycle of a wide range of viral families, we focussed our studies herein on the roles of the three main hsp isoforms (hsc , ihsp and grp ) during kshv lytic replication. hsc and ihsp are redistributed from the cytoplasm to both the periphery and within kshv-induced rtcs to verify the enrichment of hsc in the ne-associated rtcs of reactivated cells detected by our quantitative proteomic approach, indirect immunofluorescence was used to label endogenous hsc protein in trex bcbl -rta cells, a kshv latently infected b-lymphocyte cell line containing a myc-tagged version of viral rta under the control of a doxycycline-inducible promoter [ ] . hsc protein was equally distributed between the cytoplasm and nucleus of unreactivated cells in a fine punctuate pattern (fig ai) . similar hsc localization was seen during early lytic replication ( h reactivation), when rta protein was diffuse in the nucleus, successful enrichment of the nuclear envelope region and associated kshv rtcs in hek- t rkshv. cells. successful enrichment of ne regions in unreactivated (-) and reactivated for h (+) hek- t rkshv. cells was demonstrated by western blotting. equal amounts of total protein extracted from whole cell (wc) and nuclear envelope (ne) fractions were used. the monoclonal antibody mab specifically recognises the phenylalanine-glycine (fg)-repeat motif present in numerous nucleoporins (nups). closed arrows point to nups that were only detected by mab after ne enrichment. open arrow points to a nucleoporin lost during ne enrichment. several fg nups, together with nup , histone h and lamin b were all markedly enriched in the ne fractions compared with wc fractions. the nucleolar proteins b- and c- and the cytoplasmic gapdh protein were all decreased in nes fractions indicating correct subcellular fractionation. the viral rtc-associated orf and rta proteins were used to monitor lytic reactivation and assess rtc enrichment. orf antibody detected both full length orf protein (fl-orf ) and the caspase- -cleaved orf (cl-orf ). prior to rtc formation (fig aii) . in contrast, at later reactivation time points ( h), in which rta was organised into small viral rtcs peripherally located in the nucleus, numerous nuclear hsc foci that were predominantly adjacent to rtcs were observed (fig aiii) . during late reactivation time points ( h), cellular chromatin was marginalised to the nuclear periphery (fig aiv, nucleus highlighted in yellow) and larger hsc foci avidly accumulated within these fully-developed rtcs (fig aiv) . reduced levels of cytoplasmic hsc were also observed at this time point (fig aiv arrows) , suggesting that hsc is redistributed from the cytoplasm to the nucleus during kshv lytic replication. this is further supported by the fact that fractionation of trex bcbl -rta cells into nuclear (n) and cytoplasmic (c) fractions displayed an enrichment of hsc in the nuclei of reactivated cells which occurred without a noticeable increase in hsc protein levels in whole cell (wc) lysates ( fig b) . due to the observed redistribution of hsc to kshv rtcs, further co-localization studies between hsc and the sites of viral dna replication were performed in trex bcbl -rta cells. here, cells were triple-labelled with click-it edu alexa fluor and antibodies specific for rta and hsc . in unreactivated cells, newly synthesized cellular dna during mid-s-phase (edu incorporated) occurred mainly at the nuclear periphery as previously observed in other cell types [ , ] (fig ci) . during early reactivation, hsc was adjacent to rta which was present in small viral rtcs containing actively replicating viral dna (fig cii arrow) . at this stage, a proportion of hsc also co-localised with rta (fig cii') . during late reactivation, when cellular chromatin was marginalised, much larger rtcs were visible and newly synthesized cellular dna was not apparent, here hsc completely co-localised with newly synthesized viral dna and rta (fig ciii and ciii') . the location of the other two main hsp isoforms (ihsp and grp ) during kshv lytic replication was also investigated by indirect immunofluorescence microscopy. ihsp was cytoplasmic in unreactivated trex bcbl -rta cells (s ai fig), in contrast, an increase in nuclear ihsp labelling was observed in reactivated cells, which displayed similar chaperone foci as those seen during early reactivation for hsc (s aii fig). occasionally, cells displayed rtcs completely filled by ihsp (s aiii and s aiv fig, asterisks) . large ihsp foci positioned adjacent to rtcs were also seen in reactivated cells at later time points (s aiv fig arrows) . co-localisation of ihsp with actively replicating viral dna was also observed during late reactivation (s b fig). hsc and ihsp nuclear foci appeared at the same time as kshv rtcs were assembled, suggesting that these chaperones could be involved in rtc assembly. additionally, complete co-localization of hsc and ihsp with viral dna indicated that these chaperones could also participate in viral dna replication and/or capsid assembly. in contrast, the endoplasmic reticulum (er) hsp isoform, grp , was not redistributed in reactivated trex bcbl -rta cells (s fig) , consistent with its er retention signal [ ] . nevertheless, reactivated cells seemed to accumulate larger amounts of grp in the er. to confirm these results, immunoflourescence studies were also performed using hek- t prior to rtc formation, when rta was diffuse in the nucleus (ii). at h reactivation viral rta was assembled into incipient rtcs. hsc was not detected in the cytoplasm and instead numerous nuclear foci that were positioned predominantly adjacent to rtcs were seen (iii). at later reactivation times very large hsc foci were completely recruited within rtcs. hsc cytoplasmic depletion is indicated (iv arrows). nucleus highlighted in yellow shows a typical kshv fully developed-rtc with cellular chromatin marginalised to the nuclear periphery. (b) unreactivated (-) or reactivated for h (+) trex bcbl -rta cells were fractionated into whole cell (wc), cytoplasmic (c) and nuclear (n) fractions. equal amounts of total protein from each fraction were analysed by western blotting. nuclear fractions were characterised by enrichment of lamin b and absence of cytoplasmic gapdh, while cytoplasmic fractions showed the inverse profile. a small decrease in cytoplasmic hsc which correlated with a small increase in nuclear hsc was detected in reactivated cells, further supporting that hsc was redistributed from the cytoplasm to the nucleus. (c) trex bcbl -rta cells remained unreactivated (i) or reactivated for h (iiiii') followed by triple-labelling with antibodies specific for rta and hsc and click-it edu alexa fluor , the latter allowed visualization of newly synthesized dna. during early reactivation, a proportion of hsc protein was adjacent to small viral rtcs. arrow points to a small kshv rtc filled with viral dna (ii). hsc also partially co-localised with viral dna (ii) and rta (ii') in small rtcs. during later reactivation times, hsc completely moved within fully-developed rtcs strongly co-localising with viral dna (iii) and rta (iii'). rkshv. cells, in which the presence of the recombinant virus is tracked by expression of the green fluorescent protein (gfp) from the ef- alpha promoter and lytic reactivation levels are monitored by expression of the red fluorescent protein (rfp) from the kshv lytic noncoding polyadenylated nuclear (pan) rna promoter. unreactivated cells displayed cytoplasmic ihsp and hsc labelling, whereas~ % of reactivated cells ( h reactivation) revealed nuclear ihsp and hsc accumulations that appeared to assemble within small rtcs (s a fig arrows and s b fig arrows respectively) . the incomplete redistribution of ihsp and hsc foci into rtcs was likely due to a more asynchronous progression through the lytic cycle in induced cells by tpa and sodium n-butyrate than in doxycycline-induced trex bcbl -rta cells. similarly to trex bcbl -rta cells, grp was not redistributed in hek- t rkshv. cells, although larger amounts appeared to accumulate in the er of reactivated cells (s c fig), in agreement with the significantly increased amounts of grp detected in the ne region of these cells ( table ) . these results clearly demonstrate that kshv specifically redistributes the molecular chaperones, hsc and ihsp , from the cytoplasm to the nucleus, in contrast to grp , which coincides with the initial formation of kshv rtcs. treatment with the small molecule inhibitor ver- abrogated viral protein synthesis at non-cytotoxic concentrations members of the hsp chaperone family possess an n-terminal nucleotide binding domain with atpase activity which is essential for their function. to examine the implications of hsc and ihsp redistribution into kshv rtcs, a small molecule inhibitor, ver- , (a dibenzyl- -aminoadenosine analog) was utilised. this is the only inhibitor that has been demonstrated to specifically target the highly homologous atpase pocket present in the three main human hsp isoforms [ , , , ] , which is highly divergent structurally from the atpase pocket found in chaperones of the hsp family [ , ] . as such, ver- functions as an atp mimetic that specifically inhibits the atpase activity of members of the hsp family. initially, cytotoxicity of this compound was assessed in unreactivated trex bcbl -rta cells. following h inhibitor exposure, using a non-radioactive mts assay, which quantitatively assesses cell proliferation, a drastic reduction in cell metabolic activity was seen for inhibitor concentrations higher than μm (s fig) , thus concentrations ranging from to μm were used for further cytotoxicity characterization (fig ai) . the inhibitor triggered apoptosis in a dose-dependent manner as demonstrated by the caspase -mediated cleavage of full length poly [adp-ribose] polymerase (fl-parp ) protein into cleaved parp (cl-parp ) (fig aii) . small amounts of cl-parp were seen at μm and μm with a significant increase of this form after μm. these results were confirmed with apotox-glo triplex assay by quantitatively measuring viability, cytotoxicity and activation of effector caspases- / in the same sample well after h inhibitor treatment. a dose-dependent decrease in viability was evident from μm to μm while cytotoxicity and activation of caspases- / were only considerably increased at concentrations higher than μm ( fig b) . next, trex bcbl -rta cells were reactivated for h in the presence of drug vehicle dmso ( . %) or a range of increasing inhibitor concentrations. cells treated with the inhibitor at non-cytotoxic concentrations ( to . μm) revealed a drastic reduction in the levels of early orf and late minor capsid (mcapsid) proteins. a moderate reduction in the immediateearly rta protein was also seen ( fig c) . of note, when detecting the fusion protein rta-myc, which expression is not from the kshv genome, with anti-myc antibody, the decrease in rta-myc was not as dramatic as that seen for viral rta, suggesting that the decrease in viral proteins was not due to a general cytotoxic effect of the inhibitor on the cells, and that viral, but not cellular proteins were specifically affected. as an additional cellular control, protein levels of the large subunit of rnapii, which has a half-life of - h [ ] , was assessed with antibodies specific for the different phosphorylated forms of rnapii. protein levels of these forms were not significantly changed in the presence of the inhibitor, nor were those of hsc or hsp proteins. importantly, in the presence of ver- , ihsp levels were not upregulated. ihsp upregulation is a universal hallmark of hsp inhibition not only in vitro [ ] but also in clinical trials [ ] , pointing to selectivity for hsp isoforms by ver- . hsp and hsp chaperone machineries have been reported to be crucial for the stability and/or maturation of multiple viral proteins [ , , [ ] [ ] [ ] [ ] [ ] . therefore to ascertain whether hsp isoforms could stabilise the essential kshv lytic proteins rta and orf , trex bcbl -rta cells were reactivated for h to allow sufficient viral protein expression followed by addition of dmso control or ver- in conjunction with cycloheximide (chx) at μg/ml to block de novo protein synthesis. protein lysates were then collected at different times after addition of chx. the half-life of rta and orf proteins from inhibitor-treated cells were not altered compared with dmso-treated cells (fig d) . these results indicate that the observed decrease in viral protein synthesis was prior to translation and that neither viral rta nor orf protein were client proteins of the hsp isoforms. as such, this highlights a potentially novel role of hsp isoforms in the kshv replication cycle independent of viral protein stability and maturation. to further corroborate these results, experiments were also repeated in hek- t rkshv. cells. again, cell metabolic activity, parp cleavage, viability, cytotoxicity and activation of caspases- / in unreactivated cells were all assessed at a range of increasing inhibitor concentrations (fig a and b ). on this occasion, the inhibitor did not trigger apoptosis (fig aii and b ) but it caused a pronounced cell cycle arrest at h exposure at concentrations of μm demonstrated by a reduced number of metabolically active cells that exhibited no increased cytotoxicity [ ] (fig b) . it is known that the apoptotic potential of ver- is cell line-dependent and that ver- can cause cell cycle arrest in human colon, breast and lung tumour cell lines [ , ] . hek- t rkshv. cells were also reactivated for h in the presence of drug vehicle dmso ( . %) or increasing inhibitor concentrations. endogenous rta, orf and mcapsid protein levels were moderately reduced in cells treated at an inhibitor concentration of μm and severely reduced at μm while cellular proteins remain unaffected ( fig c) . these were relatively high inhibitor concentrations compared with trex bcbl -rta cells; nonetheless a concentration of μm has been shown before to be necessary for inhibition of hsp isoforms in human carcinoma cell lines [ ] . as previously seen in trex bcbl -rta cells, when blocking de novo protein synthesis with chx at μg/ml in hek- t rkshv. cells, the half-life of rta and orf proteins were not reduced even in the presence of ver- at μm ( fig d) . this supports the findings seen in trex bcbl -rta cells, suggesting that the decrease in viral protein production was due to a pretranslation event. using an mts assay. (ii) a dose-dependent cleavage of full length (fl) parp protein into cleaved (cl) parp was observed in the presence of ver- . (b) unreactivated cells were exposed for h to increasing inhibitor concentrations. concentrations higher than μm resulted in increased cytotoxicity and activation of effector caspases- / as demonstrated by quantification with apotox-glo triplex assay. (c) immunoblot analysis showing that reactivated cells treated with non-cytotoxic inhibitor concentrations ( to . μm) for h revealed a decrease in viral proteins compared with dmso-treated samples while cellular proteins remained unaffected. rnapiio denotes hypophosphorylated rnapii. ser and ser rnapii denote serine -and serine hyperphosphorylated rnapii forms respectively. (d) cells were reactivated for h to allow robust viral protein production. then, dmso control ( . %) or ver- was added in conjunction with cycloheximide (chx) at μg/ml to block de novo protein synthesis. protein lysates were collected at several times post-chx addition ( , , , and h) and analysed by western blotting. ver- did not alter the half-life of rta or orf protein, thus these proteins were not clients of hsp isoforms. the small molecule inhibitor ver- caused a significant reduction in viral transcripts, viral dna and progeny at non-cytotoxic concentrations as the block in kshv protein synthesis occurred pre-translationally, viral gene expression was quantified in the absence or presence of ver- . trex bcbl -rta cells were reactivated for h and two-step quantitative reverse transcription pcr (qrt-pcr) was carried out to quantify a range of viral transcripts. a significant decrease in early (pan, orf , k and vgpcr), late (gl and gb) viral transcripts and ori-lyt transcripts was observed in a dose-dependent manner, with all transcripts with the exception of vgpcr being significantly reduced at an inhibitor concentration of μm (fig a) . to determine whether cellular transcription was negatively affected in the presence of ver- , firstly the stability of gapdh transcript was determined in mrna decay assays using the transcriptional inhibitor actinomycin d (acd) ( . μg/ml) in trex bcbl -rta cells. after h of acd treatment, the amount of gapdh mrna was reduced by half (fig b) , indicating a short stability of gapdh mrna in this cell line. we then plotted the raw cycle threshold (c t ) for gapdh transcript from the same samples in which viral transcripts had been quantified after h of ver- treatment. as the same amount of total rna was converted into cdna for all samples, if cellular transcription was not compromised a very similar c t is expected for all samples. indeed, samples treated with up to μm ver- were all within . c t from the . c t of dmso-treated samples. only after concentrations higher than μm gapdh mrna levels were significantly reduced compared to dmso-treated samples as shown by a significantly higher c t value ( fig c) . this is consistent with the cytotoxicity profile of ver- in trex bcbl -rta cells (fig a and b ) and the clear decrease in rta-myc protein (which expression is not from the kshv genome) at inhibitor concentrations higher than μm ( fig c) . taken together, these results suggest that cellular transcription was compromised at concentrations of ver- higher than μm while at concentrations lower than μm transcription was occurring normally. interestingly, transcription of viral genes which also require host rnapii for their expression was negatively affected even at ver- concentrations lower than μm. next, we assessed whether the inhibitor also caused a reduction in viral dna replication. trex bcbl -rta cells were reactivated for h, total dna was isolated and realtime qpcr was performed using primers specific for orf . while dmso-treated cells reached~ -fold increase in viral dna load, inhibitor concentrations of μm or higher resulted in a significant reduction (> %) in viral dna ( fig d) . moreover, the production of infectious kshv virions in trex bcbl -rta cells was evaluated in the presence of ver- at . μm or vehicle drug dmso. for this, cells were reactivated and treated for h, culture medium was centrifuged and incubated for h with hek- t cells. total rna was then isolated and qrt-pcr carried out. a significant reduction (~ %) in the release of infectious viral progeny was observed in inhibitor-treated cells ( fig e) . viral transcripts were also quantified at h reactivation in hek- t rkshv. cells in the absence or presence of ver- . at non-cytotoxic concentrations of μm there was a drastic decrease for all parp was observed faintly. (b) unreactivated cells were exposed for h to increasing inhibitor concentrations. a reduced number of metabolically active cells accompanied by no increased cytotoxicity was observed in the presence of the inhibitor. there was no activation of effector caspases even in the presence of μm ver- . this phenotype is consistent with cell cycle arrest. results were quantified with apotox-glo triplex assay. (c) immunoblot analysis showing that reactivated cells treated with μm ver- for h resulted in abrogation of viral proteins compared with dmso-treated samples while cellular proteins remained constant. (d) cells were reactivated for h to allow robust viral protein expression. then, dmso control ( . %) or ver- was added in conjunction with cycloheximide (chx) at μg/ml to block de novo protein synthesis. protein lysates were collected at several times post-chx addition ( , , , and h) and western blotting was performed. as previously seen in trex bcbl -rta cells, ver- did not alter the half-life of rta or orf protein. hsp isoforms are essential for kshv lytic replication fig a) . it is intriguing that orf mrna levels did not show a clear dose-response with the inhibitor as seen for orf mrna in trex bcbl -rta cells; however the levels were decreased compared with dmso-control cells. this is the only transcript of all the viral transcripts tested in both cell lines that did not show a dose-response. however, western blotting did reveal a complete reduction of orf protein in cells treated with > μm ver- ( fig c) . this suggests that hsp isoforms may also play a role in the folding of orf protein. in fact, hsc has been previously reported to associate with at least - % of newly synthesized proteins during their biogenesis [ ] , thus, a role for hsp isoforms in folding viral proteins cannot be ruled out. in order to evaluate cellular transcription activity in the presence of ver- , we first determined gapdh transcript stability in mrna decay assays using acd ( μg/ml) in hek- t rkshv. cells. in contrast to trex bcbl -rta cells, gapdh transcripts were very stable, with no significant reduction in their levels after h of acd treatment ( fig b) . the half-lives of two cellular transcripts, srag (a cellular mrna export adapter) and histone h a (h a. ) were also determined in hek- t rkshv. cells. srag transcripts were reduced to % following h acd treatment (fig c) , while the h a. mrna was very unstable with nearly % reduction after h acd treatment ( fig d) . we then plotted the raw c t values for gapdh transcript from samples in which viral transcripts had been quantified after h of ver- treatment; these did not significantly change in the presence of ver- ( fig e) . next, the unstable srag and h a. mrnas were measured in the same samples in which viral transcripts had been quantified after h of ver- treatment. in contrast to viral transcripts, both cellular transcripts were not significantly reduced, indicating that transcription of cellular genes was occurring normally even in the presence of high ver- concentrations ( fig f) . taken together, these results demonstrate that inhibition of hsp isoform function abrogated the expression of viral genes from various temporal classes; however cellular rna-pii-mediated transcription was not compromised when using ver- at non-cytotoxic concentrations. following quantification of viral transcripts in both cell lines, it appeared that the reduction seen in viral gene expression, protein production and infectious virion production could be a consequence of a significant global reduction of viral transcripts in inhibitor-treated cells. this led to the possibility that hsp isoforms could be implicated in activation of viral promoters and subsequent transcription or alternatively hsp isoforms were required for kshv rtc formation. because viral rta and hsc co-localized in trex bcbl -rta cells and rta is (orange colour), late (red colour) and ori-lyt viral transcripts were all significantly reduced at μm ver- compared to the levels found in dmsotreated samples. all samples were normalised to gapdh. results show the mean of three biological replicates with error bar as standard deviation. (b) the stability of gapdh transcript was assessed in unreactivated trex bcbl -rta cells in the presence of the transcriptional inhibitor actinomycin d (acd) ( . μg/ml) or control dmso ( . %). cells were collected over the time points indicated and total rna was extracted followed by qrt-pcr. after h acd treatment, the amount of gapdh transcripts was reduced by more than half (c t = . ) compared with dmso-treated cells (c t = . ). a further reduction was observed at h treatment (c t = . ). the average of two biological replicates with error bar as standard deviation is shown. (c) the amount of gapdh transcripts did not significantly decrease when using ver- for h at concentrations lower than μm compared to dmso-treated samples. in contrast, concentrations higher than μm significantly showed a c t lower than dmso samples, indicating compromised cellular transcription at these inhibitor concentrations. the same samples in which viral transcripts had been quantified were used to plot all c t values. results show the mean of three biological replicates with error bar as standard deviation. (d) cells were reactivated for h, total dna was isolated and real-time qpcr was performed. viral dna load was significantly decreased at μm ver- . results show the mean of three biological replicates with error bar as standard deviation. (e) trex bcbl -rta cells were reactivated for h in the presence of ver- at . μm or vehicle drug dmso ( . %). the culture medium was then incubated for h with hek- t cells followed by total rna extraction and qrt-pcr. a significant decrease in viral progeny was seen in inhibitor-treated cells. results show the mean of three biological replicates with error bar as standard deviation. the master latent-lytic transactivator for multiple kshv immediate-early, delayed-early and late promoters [ ] [ ] [ ] [ ] [ ] , we further investigated the possibility that hsc was required for rta-mediated transactivation. initially we assessed whether an interaction occurred between hsc and rta in the absence of other viral proteins or dna. for this, hek- t cells were transiently transfected for h with control pegfp or prta-egfp and immunoprecipitations were carried out using a gfp-specific antibody. rta-egfp precipitated endogenous hsc in contrast to the control egfp protein (fig a) . in addition, hek- t cells were transfected with control pegfp or prta-egfp for h and examined by immunofluorescence. in cells expressing egfp protein, endogenous hsc remained cytoplasmic (fig bi) , while in egfp-rta-expressing cells hsc strongly co-localised with rta in the nuclei, suggesting rta expression alone is sufficient to redistribute hsc into the nucleus (fig bii) . similar nuclear redistribution was also seen for endogenous ihsp (s fig). next, we determined whether ver- was able to disrupt the interaction between egfp-rta and hsc in hek- t cells. hek- t cells exhibited a very similar cytotoxicity profile to that seen in hek- t rkshv. cells (s fig). hek- t cells were transiently transfected with prta-egfp or control pegfp. to allow maximal protein expression and avoid interference of the inhibitor with the transfection, the inhibitor was added at h post-transfection and incubated for a further h, prior to immunoprecipitations being performed. western blot analysis revealed that the inhibitor did not disrupt the interaction between hsc and rta even at high inhibitor concentrations ( μm) (fig c) , suggesting that the atpase function of hsc is not required for the interaction with rta protein. therefore, to investigate whether hsc was required for rta-mediated transactivation of the rta-responsive orf promoter, a dual-luciferase reporter assay system was utilised. hek- t cells were co-transfected with prta-egfp along with the renilla luciferase vector and either the orf promoter firefly luciferase reporter vector, or the empty reporter vector (pgl -basic). the same co-transfections were performed using pegfp, as a negative control. h post-transfection, cells were exposed for h to increasing concentrations of ver- and luciferase activities were measured. longer exposure times to the inhibitor affected the formation of the control renilla luciferase protein which has a half-life of h, suggesting that hsp isoforms may be required for the folding/maturation of this enzyme. in the presence of egfp-rta and the orf promoter reporter construct, the orf promoter activity was increased~ -fold, while the empty vector had a~ -fold increase. however, orf promoter activity was not significantly decreased in the presence of ver- ( fig d) . to confirm this result, hek- t cells were also transiently co-transfected with ppan-wt, a plasmid encoding the genomic region of wild type pan rna including its promoter region [ ] , and either prta-egfp or control pegfp. h post-transfection either vehicle drug dmso or ver- at μm was added and incubated for a further h followed by qrt-pcr. in quantification by rt-pcr of immediate-early (blue colour), early (orange colour), late (red colour) and ori-lyt viral transcripts showed that all transcripts were significantly decreased at μm ver- compared to the levels found in dmso-treated samples. all samples were normalised to gapdh. results show the mean of three biological replicates with error bar as standard deviation. (b-d) the stability of cellular gapdh, srag and h a. transcripts was determined in mrna decay assays using actinomycin d (acd) ( μg/ml) or control dmso ( %) in hek- t rkshv. cells. cells were collected over the time points indicated and total rna was extracted followed by qrt-pcr. the average of two biological replicates with error bar as standard deviation is shown. (b) gapdh transcripts were very stable, with no significant reduction in their levels after h of acd treatment. (c) at h acd treatment srag mrna levels were reduced by % showing a relatively short stability. (d) h a. mrna levels were very unstable, with nearly % reduction after h acd treatment. (e) gapdh transcript levels remained unchanged following h ver- treatment in reactivated hek- t rkshv. cells. the same samples in which viral transcripts had been quantified were used to plot all c t values. (f) the unstable cellular srag and h a. mrnas were not significantly decreased in the presence of ver- treatment compared with dmso treatment for h indicating that cellular transcription was not compromised even at high concentrations of ver- . the same samples in which viral transcripts had been quantified were used to quantify srag and h a. . all samples were normalised to gapdh. however, no significant decrease in the amount of pan rna was seen in inhibitor-treated cells (fig e) , indicating that rta-mediated promoter transactivation and subsequent synthesis of pan rna was occurring normally in the presence of ver- . these data demonstrate that hsp isoforms did not directly enhance rta-mediated transactivation. to assess the transcription activity of cellular rnapii in the presence of the inhibitor, the half-life of pan rna was determined in hek- t cells co-transfected with ppan-wt and prta-egfp. following h post-transfection, acd ( μg/ml) or dmso control ( . %) was added. after h of transcription inhibition, pan rna levels were reduced to % compared to dmso-treated cells (fig f) . this quick reduction in the stability of pan rna in the absence of orf protein is in agreement with previous reports [ ] . thus, if ver- was blocking general rnapii transcription, a significant reduction in pan rna levels should be observed after h of inhibitor treatment; however, pan rna levels were not reduced in cells treated with ver- for h (fig e) . a dramatic reduction in early, late and ori-lyt transcripts after h treatment with ver- was evident during kshv infection in both cell lines used. however, hsp isoforms were not required for rta-mediated transactivation of kshv promoters in transiently transfected cells. thus, we next monitored kshv rtc formation in the absence or presence of the inhibitor during kshv lytic infection. trex bcbl -rta cells were reactivated and treated with either control dmso or μm inhibitor. at h reactivation cells were fixed and immunofluorescence was performed using rta-and hsc -specific antibodies. dmso-treated cells displayed abundant rtcs and numerous nuclear hsc foci that partially co-localised with rtcs. hsc cytoplasmic depletion was also observed (fig a and a') . in contrast, inhibitortreated cells showed diffuse nuclear rta that was not able to assemble into rtcs (fig b and b' ). in these cells, hsc nuclear foci were still visible, but these were much less numerous and smaller compared with the foci seen in dmso-treated cells. significantly, following ver- treatment, hsc was observed in the cytoplasm of reactivated cells (fig b and b' ). hsc subcellular localization was also analysed in dmso-and inhibitor-treated cells by confocal profiling. profiling was performed by drawing a line long enough (~ μm) to cover the nucleus and cytoplasm at either side of the nucleus. if an hsc pixel intensity data point was equal or greater than to the data point in the previous and subsequent pixel and above the background noise, it was considered as an hsc peak, representing an hsc foci. analysed by immunofluorescence. endogenous hsc was cytoplasmic in cells expressing egfp (i). in contrast, hsc strongly co-localised with egfp-rta in the nucleus, but not the nucleoli (ii). (c) hek- t cells were transiently transfected with control pegfp or prta-egfp. h posttransfection, ver- was added and incubated for a further h. then, immunoprecipitations were carried out using a gfp-specific antibody. even in the presence of μm ver- the interaction between hsc and egfp-rta was not disrupted. (d) hek- t cells were co-transfected with prta-egfp along with the renilla luciferase vector and either the orf promoter firefly luciferase reporter vector, or the empty reporter vector (pgl -basic). the same co-transfections were performed using pegfp as a negative control protein. h post-transfection, cells were exposed for h to ver- and luciferase activities were measured. comparable activation of the rta-responsive orf promoter to that seen in dmso-treated cells occurred in cells treated with μm ver- . the results of three independent transfections were averaged with error bars as standard deviation. (e) hek- t cells were transiently co-transfected with ppan-wt and either prta-egfp or control pegfp. h post-transfection either vehicle drug dmso ( . %) or μm ver- was added and incubated for a further h. total rna was then extracted and qrt-pcr performed. rta-mediated promoter transactivation and subsequent synthesis of pan rna occurred at a similar rate in the presence of ver- or control dmso. the results of three independent transfections were averaged with error bars as standard deviation. (f) the stability of pan rna was determined in hek- t cells that had been co-transfected with ppan-wt and prta-egfp. following h post-transfection, actinomycin d (acd) ( μg/ml) or dmso control ( . %) was added. cells were collected over the time points indicated and total rna was extracted followed by qrt-pcr. the average of two biological replicates with error bar as standard deviation is shown. doi: . /journal.ppat. .g hsp isoforms are essential for kshv lytic replication dmso-treated cells predominantly showed hsc peaks only within the dapi boundaries, that is, within the nucleus (fig a") . inhibitor-treated cells displayed hsc peaks outside the dapi boundaries, that is, in the cytoplasm (fig b" asterisk) more often than control cells. a significant increase in cytoplasmic hsc peaks was seen in inhibitor-treated cells compared with dmso control cells (fig c) . fractionation of reactivated trex bcbl -rta cells in the presence or absence of ver- , also pointed to slightly higher levels of hsc in the cytoplasm and a decrease in nuclear hsc in inhibitor-treated cells compared with dmso control cells (fig d) . cells were also labelled with click-it edu alexa fluor and an antibody specific for hsc (s fig). the percentage of assembled rtcs in dmso-and inhibitor-treated cells was also calculated. in dmso-treated cells~ % of cells presented assembled rtcs while only % of inhibitor-treated cells showed assembled rtcs (fig e) . this demonstrates that chaperone recruitment to the nucleus is essential for the assembly of kshv rtcs and treatment with ver- was sufficient to impair nuclear chaperone recruitment and kshv rtc formation. the subcellular localisation of rnapii was also assessed by indirect immunoflourescent labelling in trex bcbl -rta cells with the monoclonal antibody ctd h , which specifically recognises unphosphorylated and serine- phosphorylated rnapii. in unreactivated cells, rnapii exhibited a nuclear localization, excluding the nucleolus (fig ai arrow) irrespective of the presence of dmso (fig ai) or the inhibitor (fig aii) . however, in reactivated and dmso-treated cells, rnapii was clearly hijacked to rtcs (fig bi) . in contrast, in reactivated cells treated with the inhibitor, rnapii was diffuse throughout the nucleus, but excluding the nucleoli, and formed very small foci (fig bii arrow) multiple kshv rtcs identified by rta-labelling were formed in dmso-treated cells to which large and numerous hsc foci were recruited. in these cells, hsc was depleted from the cytoplasm. (a') confocal images were subjected to profiling analysis using zeiss zen software. profiling was conducted for each cell in two representative confocal images taken with a -times objective; this included profiling a total of dmso-treated cells. a representative line profile (yellow line) and accompanying relative intensities of each channel at every pixel along the line is shown for a dmso-treated reactivated cell. the dapi intensity profile shows values below outside the nucleus boundary. rta profile shows a peak with intensity of that lies within the dapi boundaries corresponding to a kshv replication compartment. hsc peaks lie within the dapi boundaries demonstrating their nuclear location. an hsc intensity peak closely resembles the rta intensity peak (arrows), indicating co-localization of hsc with rta since both peaks occur at the same position along the line. (b) in cells reactivated and treated with μm ver- for h, rta was diffuse in the nucleus and not assembled into kshv rtcs. in addition, hsc nuclear foci were smaller and less abundant than in dmso-treated cells and hsc was not depleted from the cytoplasm. (b'). confocal profiling was performed for each cell in two representative confocal images taken with a -times objective; a total of inhibitor-treated cells were analysed. a representative line profile (yellow line) is shown for an inhibitor-treated cell. an hsc peak is seen outside the dapi boundaries corresponding to an hsc cytoplasmic peak (asterisk). (c) total hsc peaks and total nuclear hsc peaks per cell were combined for each confocal image and experimental condition. data was converted to percentage of nuclear and cytoplasmic peaks. cells exhibited very small rnapii foci and diffuse nuclear edu labelling ( s bii fig). to confirm that inhibition of hsp isoform function was able to abolish rnapii recruitment to viral genomes, we also utilised chromatin immunoprecipitation (chip) assays in trex bcbl -rta a polyclonal antibody against rta and a monoclonal antibody (ctd h ) against rnapii were used for immunoflourescence analysis. rnapii protein was nuclear excluding the nucleoli (arrows) regardless of inhibitor treatment. (b) trex bcbl -rta cells were reactivated and treated with either control dmso ( . %) or μm ver- for h. in dmso-treated cells rnapii and rta were recruited to viral rtcs (i arrow) while in inhibitor-treated cells rta was diffuse in the nucleus and rnapii formed numerous small foci that excluded the nucleoli and resembled prereplicative sites (ii arrow). (c) trex bcbl -rta cells were either reactivated (r) in the presence of dmso ( . %) or μm ver- for h. unreactivated (u) cells treated with dmso ( . %) were used to assess levels of lytic reactivation. chip assays were carried out with either monoclonal ctd h rnapii antibody or mouse control antibody (igg). in the presence of ver- , significantly reduced amounts of rnapii bound to viral promoters were detected. the average of three independent experiments is shown with error bars as standard deviation. cells that were either reactivated for h in the presence of dmso or μm ver- ( fig c) . in unreactivated control cells, there was a clear enrichment of rnapii at the promoter of gapdh gene, while rnapii occupancy at viral promoters was minimal. however, kshv reactivation in dmso-treated cells led to a drastic reduction of rnapii at the promoter of gapdh and a significant increase of rnapii at the viral promoters in agreement with the previous immunofluorescence results, showing rnapii recruitment to rtcs (fig bi) . conversely, upon treatment of the hsp inhibitor, the amount of rnapii bound at the promoters of ori-lyt, k and orf was decreased by~ %,~ % and~ % respectively compared with dmso-treated reactivated cells. importantly, these results indicate for the first time that inhibition of hsp isoforms leads to a severe impairment in rnapii recruitment at multiple viral promoters including that of ori-lyt. to further confirm the essential role of hsc and ihsp in the formation of kshv rtcs, specific individual sirna-mediated depletion of both isoforms was performed in hek- t rkshv. cells. following four days post-sirna transfection, cells were reactivated for h and rna and protein were extracted from the same sample. hsc depletion was evaluated by western blotting and by qrt-pcr, the latter showing~ % hsc mrna knockdown ( fig a) . in contrast, ihsp mrna levels were not affected confirming specificity of the hsc sirna. despite a successful knockdown at the mrna level, significant amounts of hsc protein remained in hsc -depleted cells (fig b) . however, even with this modest amount of depletion at the protein level, all viral transcripts (with the exception of pan) displayed a significant reduction in hsc sirna-treated samples compared with the scramble sirnatreated cells as demonstrated by qrt-pcr analysis (fig c) . orf , orf and gl mrna levels were decreased by~ % following hsc knockdown. ori-lyt and rta transcripts were reduced by~ % and gb levels by~ %. this suggests that depletion of hsc impaired the expression of viral genes from various temporal classes and thus hsc may be necessary for kshv rtc formation. viral dna replication was also assessed following hsc knockdown. for this, after four days post-sirna transfection, cells were reactivated for a further h. there were no significant differences between scramble and depleted cells (fig d) . the production of infectious kshv virions was also evaluated after h reactivation. again, no significant differences were seen between scramble and depleted cells ( fig e) ; however this result is not surprising due to incomplete hsc depletion even after seven days post-transfection ( fig f) . this highlights the remarkable stability of hsc protein in this cell line and it suggests that hsc depletion was enough to cause a reduction in viral transcripts but not enough to cause a reduction in the amount of viral proteins, thus kshv lytic replication remained unaffected. it is also possible that ihsp was able to functionally compensate for hsc . next, specific depletion of ihsp was performed in hek- t rkshv. cells. ihsp depletion at the mrna level reached~ % knockdown (fig a) which correlated with efficient depletion at the protein level ( fig b) . however, in ihsp -depleted cells, the majority of viral gene expression was unaffected, apart from gl and pan transcripts which were decreased bỹ % and~ % respectively (fig c) . importantly, taken together these results indicate that partial depletion of hsc at the protein level is sufficient to cause a reduction in viral transcription, suggesting an essential role of this chaperone in the formation of kshv rtcs, whereas ihsp may have a more subtle effect on viral gene expression. to further support the essential role of hsc during kshv rtc formation, hsc was specifically silenced in trex bcbl -rta cells. for this, nucleofection was carried out. transfection efficiency was monitored co-transfecting the hsc sirna together with pmaxgfp, which encodes maxgfp, a green fluorescent protein from the copepod pontellina p. (fig a) . note that higher transfection efficiency is expected for the sirna due to the smaller size of this compared with the plasmid dna. after four days post-nucleofection, hsc mrna levels showed~ % knockdown (fig b) with a minor depletion at the protein level (fig c) . due to the stability of hsc protein, cells were incubated for six days post-nucleofection followed by a further h reactivation and immunofluorescence for hsc and rta was carried out. rtc formation dramatically decreased after nucleofection. similar impairment in kshv lytic replication has previously been reported in electroporated trex bcbl -rta cells [ ] ; nevertheless, in scramble sirna-treated cells, groups of cells could still be seen displaying rtcs to which hsc was relocated (fig d) . in contrast, in hsc sirna-treated cells, fewer rtcs were visible and these exhibited nuclear hsc (fig e yellow arrow) while cells fully depleted of hsc , as identified by lack of hsc -labelling, did not form rtcs (fig d white arrows) . this result strongly suggests that hsc is an essential chaperone for the formation of kshv rtcs in trex bcbl -rta cells. current quantitative proteomics approaches have become an invaluable tool for large-scale, high-throughput identification of proteins in complex biological samples. moreover, advances in subcellular fractionation offer a way to further reduce the complexity of the samples to be analysed by lc-ms/ms, allowing identification of low abundance proteins. in this present study, we have developed a novel quantitative proteomic approach enhanced by subcellular fractionation that has enabled us to elucidate the cellular protein composition of kshv rtcs. this novel approach led to the identification of several upregulated pathways in reactivated cells associated with the ne fraction (s table) . the first scored pathway was rna post-transcriptional modification, the second highlighted pathway was protein synthesis with different ribosomal proteins identified and the third scored pathway was dna replication, recombination and repair. in addition, the isolation of the ne regions from unreactivated and reactivated cells followed by the uncommon use of urea for protein extraction led to the mass spectrometric identification of several hsp isoforms and their respective co-chaperones at significant levels in ne-associated rtcs of reactivated cells. immunoflourescence analysis confirmed that endogenous hsc and ihsp were redistributed from the cytoplasm to the periphery of kshv rtcs where they formed multiple nuclear foci during early lytic replication. the formation of rtcs coincided in time with the appearance of nuclear chaperone foci. similar virus-induced-chaperone-enriched (vice) domains that form adjacent to hsv- rtcs, have also been observed in hsv- -infected cells and contain sequestered hsc , ihsp , hsp and hsp [ , , ] . hsv- induced-vice domains also accumulate ubiquitinated proteins and components of the proteasome and function to sequester misfolded proteins away from rtcs and serve as protein quality control centers [ , ] . ihsp redistribution very similar to that seen in hsv- induced-vice domains was observed in multiple unreactivated and total rna and protein were extracted from the same sample. despite achieving~ % hsc knockdown at the mrna level (a), significant amounts of hsc protein remained in hsc sirna-treated samples (b) . despite this small knockdown at the protein level, in hsc -depleted cells there was a significant decrease in the amount of multiple viral transcripts from various temporal classes as quantified by rt-pcr analysis (c). viral dna replication was assessed by qpcr following hsc -knockdown. cells were reactivated for h after four days post-sirna transfection. no significant differences were observed between scramble and hsc -treated cells (d). similar virion production was detected in scramble and hsc -treated cells. cells were reactivated for h after four days post-sirna transfection, culture medium was centrifuged and immediately incubated with hek- t cells for h. total rna was then isolated and qrt-pcr carried out (e). incomplete hsc -knockdown at the protein level was observed by western blotting even after seven days post-sirna transfection in hek- t rkshv. cells. this demonstrates the remarkable stability of hsc in this cell line (f). the average of three independent transfections is shown with error bars as standard deviation (a, c-e). doi: . /journal.ppat. .g hsp isoforms are essential for kshv lytic replication fig . the majority of kshv lytic gene expression was unaffected following specific depletion of ihsp in hek- t rkshv. cells. cells were transfected twice with nm ihsp -specific sirna or nm scramble sirna. following four days post-sirna transfection, cells were either reactivated cells undergoing kshv lytic replication, moreover this labelling was also observed for hsc in some cells. hsc and ihsp remain positioned exclusively adjacent to hsv- rtcs. however, in contrast; these chaperones were very dynamic during kshv lytic replication and surprisingly very large chaperone foci were recruited within kshv rtcs when viral dna was actively synthesised (fig c and s b fig) . therefore, our results strongly suggest that these chaperones may also aid replication of kshv genomes during lytic replication. we therefore further suggest that hsp isoforms may have an important role in the assembly and activation of pre-initiation complexes on the origin of dna replication. this is supported by observations in prokaryotes, such as in plasmid p [ , ] , and eukaryotes, such as saccharomyces cerevisiae [ ] , human papillomavirus- [ , ] , hsv- [ ] and bacteriophage λ [ , ] . interestingly, hsp isoforms have also been identified in the kshv virion [ , ] and therefore an additional role of these chaperones in kshv capsid assembly may also be possible. cancer cells greatly rely on members of the hsp and hsp chaperone families for their growth and survival [ , ] , consequently, significant efforts have been invested to design small molecule inhibitors specific for these atpases as novel anticancer therapeutics. this has been successfully achieved for the hsp family with several inhibitors undergoing phase iii clinical trials [ ] , although the clinical efficacy of these inhibitors has been somewhat limited because of the inevitably upregulation of ihsp , when inhibiting hsp [ , ] . the development of hsp inhibitors has substantially lagged behind that of hsp inhibitors due to lack of natural product inhibitors specific for hsp isoforms, due to the highly polar nature of the hsp / hsc atp binding site and the high affinity for atp displayed by these atpases [ ] . nevertheless, in recent years several hsp inhibitors have been designed and tested in pre-clinical or clinical trials [ ] . however, a major challenge remains in finding inhibitors which can specifically discriminate between ihsp and hsc . to date, only peptide aptamers targeting ihsp are selective for a specific hsp isoform [ ] and also recently, methyl blue was reported to specifically inhibit ihsp by oxidizing a cysteine in its atpase domain, the same residue is absent in hsc allowing differential targeting of the isoforms [ ] . because both hsc and ihsp were specifically redistributed to kshv rtcs during lytic replication, we made use of a recently developed small molecule inhibitor, ver- , which targets the atpase pocket of the main three human hsp isoforms. this inhibitor used at non-cytotoxic concentrations was able to effectively abrogate early and late kshv transcription together with viral protein production, viral dna replication and viral progeny. therefore, our study highlights the potential of ver- or other novel hsp inhibitors to prevent kshv lytic replication in kshv-associated tumours. one could expect that hsp inhibition would be directly detrimental not only to cancer cell survival but also the virus-specific functions that are dependent on hsp isoforms. these results may have exciting implications in combination with the recently demonstrated efficacy of atp-competitive hsp inhibitors in blocking kshv latent cycle in vitro and in a xenograft kshv tumour model [ ] . it may be the case that combining hsp inhibitors with hsp inhibitors may lead to enhanced efficacy in eradicating latent kshv reservoirs. excitingly, in our cell culture models ver- abrogated lytic replication without severely affecting cell viability or triggering apoptosis. it is intriguing that inhibition of constitutively expressed hsc chaperone did not result in cell death. however, it is important to highlight previous studies carried out on the susceptibility of tumour cells versus normal cells to hsp inhibitors. in tumour cells, for h or remained unreactivated and total rna and protein were extracted from the same sample. efficient ihsp knockdown was achieved both at the mrna level (~ %) (a) and the protein level (b). a small decrease in the amount of gl and pan transcripts was observed in ihsp -depleted cells (c). the average of three independent transfections is shown with error bars as standard deviation. doi: . /journal.ppat. .g hsp is present entirely in multi-chaperone complexes with high atpase activity; in contrast, in normal cells hsp is in an uncomplexed conformation. these two distinct hsp presentations result in tumour hsp exhibiting a -fold higher binding affinity to an hsp inhibitor than normal hsp [ ] . thus, it is tempting to speculate that the multi-chaperone hsp foci that are recruited to kshv rtcs during lytic replication (fig and s fig) are more sensitive to ver- than the chaperones not assembled in vice domains which carry out the housekeeping functions for cell survival. this would explain why hsp inhibition had a profound effect on kshv lytic replication without affecting cell viability and support the idea that isoform specificity may not be a requirement for treatment of kshv-associated tumours. to establish the essential role of hsp isoforms during kshv lytic replication, we examined several possible functions. it could be hypothesised that hsp isoforms could be implicated in ) stabilization of essential viral proteins, ) clearing stalled rnapii during times of robust viral transcription, ) activation of viral promoters, ) formation of kshv rtcs. initially we hypothesised that hsp isoforms may be necessary for maintaining the stability of the key kshv viral proteins rta and/or orf . this was deemed a possibility as during kshv latency, the essential viral latency associated nuclear antigen (lana) protein has been recently shown to be a client protein of the hsp chaperone and several hsp inhibitors reduced the expression of lana [ ] . in addition, the lytic kshv k glycoprotein has also been reported to be a client protein of hsp [ ] . however, inhibition of hsp isoforms did not alter the half-life of rta or orf (figs d and d) . alternatively, hsc has been observed at the periphery of hsv- rtcs where it is also believed to aid in clearing stalled rnapii from viral genomes during times of active transcription [ ] . indeed, the serine- phosphorylated form of rnapii undergoes ubiquitination and robust proteasomal degradation during hsv- infection [ ] . in contrast, in kshv-reactivated cells, there was only a slight reduction of serine- phosphorylated rnapii in comparison with unreactivated cells even at late times ( h) post-reactivation ( fig c) and a significant decrease in the other rnapii forms was not evident (fig c) . this suggests that although robust rnapii degradation is a feature observed in virus infection, such as hsv- and influenza virus [ , ] , it may not be universally conserved. it is interesting to note that expression of a dominant-negative hsc (k m) that cannot hydrolyze atp during hsv- infection resulted in prevention of serine- rnapii degradation and rtcs formation [ ] . however, inhibition of hsp isoforms by ver- did not prevent the slight degradation of phospho-serine- rnapii protein ( fig c) . using transient transfections, we also demonstrated that hsp isoforms were not directly involved in the activation of viral promoters (fig d and e) . strikingly however, inhibition of hsp isoforms precluded kshv rtcs formation (fig ) and rnapii re-localization to viral promoters (fig ) , thus, blocking kshv rtcs formation led to abolishment of global viral transcription and subsequent protein synthesis and viral dna replication. these results, taken together with the differential hsc and ihsp labelling seen in hsv- and kshv-infected cells, suggest that these chaperones may be playing additional roles, such as participating in viral dna replication and/or capsid assembly, in kshv lytic infection compared with hsv- infection. importantly, in both viruses, hsv- and kshv, inhibition of hsc atpase function leads to a clear impediment in rtcs formation and presents a novel antiviral target for multiple herpesviruses. moreover, as hsv- and kshv belong to different subfamilies of the herpesviridae family (α and γ subfamily respectively), the key role of hsp isoforms in rtc formation may be conserved across all subfamilies. in support of this notion is the fact that hsc and ihsp have also been reported to be incorporated in the virion of the β-herpesvirus hcmv [ ] and the γ-herpesvirus ebv [ ] . our results also support that the finding of hsc and ihsp chaperones in the kshv virion [ , ] a decade ago was not casual, and that these chaperones are essential for kshv rtcs formation during lytic replication. ihsp has also been detected in the hsv- virion [ ] . to support the conserved role of hsp isoforms in herpesvirus infection, a recent study showed that cellular depletion of hsc protein significantly reduced hsv- viral output in cell culture without adversely affecting cell viability. depleting hsc from the hsv- virion also significantly reduced viral production by more than % [ ] . hsp isoforms may be recruited to rtcs for several reasons. firstly, the chaperone may sequester misfolded, modified or unwanted proteins away from rtcs. alternatively, hsp could produce a site for protein remodelling and/or degradation which may regulate or delay cellular pathways, such as the apoptosis cascade. finally, it may aid subtlety to clear stalled rnapii complexes during robust viral transcription and replication. an additional question which is yet to be addressed is the mechanism by which hsp isoforms are recruited to rtcs. one intriguing possibility is observations made during hsv- infection. here, hsv- icp has been shown to interact with hsc and is required for hsc nuclear foci formation [ ] . it will now be interesting to determine if the functional kshv homologue orf , also interacts with hsc and whether its nucleocytoplasmic shuttling ability is essential for hsc nuclear import. in summary, we have identified a new essential role for hsp isoforms during the formation of rtcs in kshv lytic replication. importantly, our results suggest that hsp inhibitors have the potential as novel kshv antiviral agents and it would now be interesting to test these in conjunction with other molecular chaperone inhibitors, specifically hsp inhibitors [ ] , which have the potential to eradicate latent kshv reservoirs in both in vitro and in vivo tumour models. trex-bcbl- -rta cells (kindly provided by dr. jae jung, university of southern california) are a bcbl- -based, primary effusion lymphoma (pel) b cell line that has been engineered to inducibly express exogenous myc-tagged rta by the addition of doxycycline, leading to a robust reactivation of the full kshv lytic cycle [ ] . the rkshv. cell line (kindly provided by dr. jeffery vieira, university of washington, seattle, usa) maintains kshv as a latent infection and was generated by infecting hek- t cells (atcc) with a recombinant kshv that contains a constitutively active puromycin resistance and gfp gene, and an rfp gene that is fused to an rta-responsive lytic cycle (pan) promoter; hence, expression of rfp can be used as a reporter of rta activity [ ] . hek- t rkshv. cells were grown in dmem (life technologies) supplemented with % foetal calf serum (fcs) (life technologies) and % penicillin/streptomycin (p/s). this cell line was kept under puromycin (sigma) selection ( . μg/ml). reactivation into the lytic cycle was achieved by addition of -o-tetradecanoylphorbol -acetate (tpa) ( ng/ml) and sodium n-butyrate (nab) (sigma) ( mm). the trex bcbl -rta cell line was grown in rpmi medium (life technologies) supplemented with % fcs and % p/s. this cell line was kept under hygromycin b (life technologies) selection ( μg/ml) and inductions were performed using μg/ml doxycycline hyclate (sigma) as previously described [ ] . all cells were maintained at °c in a humidified incubator with % co . plasmid transfections were carried out using lipofectamine (life technologies), as previously described [ ] . luciferase assay plasmids renilla luciferase vector prl-tk and firefly luciferase vector pgl -basic were purchased from promega, pegfp-n was obtained from clontech, prta-egfp and ppan-wt have been previously described [ , ] . the monoclonal mouse antibodies to anti-nuclear pore complex proteins (mab ) (ab ), gapdh ( c ), rabbit polyclonal anti-lamin b and anti-ser rnapii were purchased from abcam. the rabbit polyclonal anti-histone h c-terminus ( ) was purchased from active motif. the rabbit polyclonal anti-nup was obtained from bethyl laboratories. monoclonal antibodies to kshv orf ( . ), to hsc (b- ), to grp (a- ), to hsp ( f ), to b- ( ) and to c- (h ) were obtained from santa cruz. the mouse monoclonal (c f a- ) anti-ihsp was from enzo life sciences. the rabbit polyclonal anti-parp was purchased from cell signalling. the mouse monoclonal (ctd h ) anti-rnapii was purchased from millipore. the mouse monoclonal ( e ) anti-c-myc was from sigma. sheep anti-kshv minor capsid protein was purchased from exalpha biologicals, inc. the rabbit polyclonal anti-rta was a gift from professor david blackbourn (university of surrey, uk). the mouse monoclonal (jl- ) anti-gfp was supplied by clontech. the inhibitor for hsp isoforms (ver- ) was obtained from tocris bioscience. for silac, hek- t cells rkshv. were fed with either medium (r k ) or light (r k ) labelled medium (dundee cell products) containing % dialysed fcs (dundee cell products) for six passages to allow incorporation of the isotopes, as previously described [ ] . subsequently, to induce lytic replication three t flasks were reactivated with tpa ( ng/ml) and nab ( mm) for h, while another three t flasks remained unreactivated as control. to isolate nes a protocol published by korfali et al., [ ] was used with minor modifications. million cells were used per experimental condition. cells were washed with pbs and incubated in hypotonic lysis buffer ( mm hepes ph . , . mm mgcl , and mm kcl) for min followed by homogenization with a tight dounce homogenizer. to stabilise and avoid lysing the nuclei after the hypotonic swelling step, cells were resuspended in . m shkm ( . m sucrose, mm hepes ph . , mm kcl, and mm mgcl ) and m kcl. the resuspended cells were then underlayed with % shkm ( . m sucrose, mm hepes ph . , mm kcl, and mm mgcl ) and nuclei were pelleted at , xg for min at °c in a eppendorf centrifuge r. nuclei were resuspended in . m shkm, underlayed with . m shkm and transferred to . -ml ultracentrifuge tubes (beckman coulter). nuclei were then centrifuged at , xg for h at °c in a sorvall discovery se ultracentrifuge. pellets were resuspended in . m shkm ( mm hepes ph . , mm kcl, and mm mgcl ), treated with % triton-x in % shm ( . m sucrose, mm hepes, ph . , mm mgcl and . mm ca cl ) for min and centrifuged at , xg for min. nuclei were then treated with rnase a (thermo scientific) and dnase i (life technologies) for min, pelleted at , xg for min, resuspended in % shm and treated again with rnase a and dnase i for min. nuclei were centrifuged at , xg for min, resuspended and incubated for min in % shm containing . m nacl to remove nucleoplasmic contents. nuclear envelopes were then pelleted at , xg for min. insoluble nuclear envelope proteins were solubilised for min in pbs supplemented with . % triton-x and m urea. samples were centrifuged at , g for min to remove insoluble material and the supernatant containing nuclear envelope proteins was stored at - °c for further western blotting and mass spectrometry analysis. all solutions had freshly added x complete, edta-free protease inhibitors (roche). dtt ( mm) was also freshly added to the solutions specified on korfali's protocol. lc-ms/ms was performed as previously described [ ] . bioinformatical analysis was performed with the ingenuity systems software packet, ipa . (ingenuity systems, inc). protein samples were extracted using lysis buffer containing mm tris (ph . ), mm nacl, % np- and x complete, edta-free protease inhibitors (roche) for min on ice, as previously described [ ] . protein samples were run on sds-page gels and transferred to nitrocellulose membranes (amersham) via wet transfer. membranes were blocked with tbs + . % tween and % dried skimmed milk powder. membranes were probed with relevant primary and secondary antibodies, treated with ez-ecl (geneflow) and exposed to amersham hyperfilm ecl (ge healthcare). secondary antibodies were horseradish peroxidase (hrp)-conjugated polyclonal goat anti-mouse and polyclonal goat anti-rabbit (dako). hrpconjugated polyclonal rabbit anti-sheep was from santa cruz. gfp-trap (chromotek) experiments were performed as previously described [ ] . nuclear/cytoplasmic fractionations were performed as previously described [ ] , with the exception that nuclear pellets were solubilised for min in pbs supplemented with . % triton-x and m urea. vigorous pipetting and vortexing was applied to the nuclear pellet. after urea treatment, insoluble material was removed by centrifugation at , g for min and the supernatant kept for further analysis. cells were cultured overnight on poly-l-lysine (life technologies) coated glass coverslips in -well plates. cells were fixed with % formaldehyde (calbiochem) for min and permeabilised with . % triton x- for min as previously described [ ] . for labelling with grp antibody cells were fixed with ice-cold % methanol for five min. after permeabilization, cells were then incubated in blocking solution (pbs with % bsa) for h at °c. primary antibodies anti-hsc (diluted : ), anti-ihsp ( : ), anti-grp ( : ), anti-rnapii (ctd h ) ( : ) or rabbit rta ( : , ) were incubated for h at °c. coverslips were washed five times with pbs, incubated with appropriate secondary antibody for h at °c, washed five times with pbs again and mounted in vectashield with dapi (vector labs). images were obtained using a lsm meta confocal microscope (carl zeiss) and processed using zen imaging software (carl zeiss) as previously described [ ] . fluorescentlyconjugated secondary antibodies were all obtained from life technologies: alexa flour goat anti-mouse igg, alexa flour goat anti-mouse igg, alexa flour goat anti-rabbit igg, alexa flour donkey anti-mouse igg and alexa flour goat anti-rabbit igg. trex bcbl -rta cells were labelled using the click-it edu alexa fluor imaging kit (life technologies) according to the manufacturer's instructions with minor modifications as follows. cells were seeded onto poly-l-lysine treated coverslips in -well plates followed by induction and incubation at °c for hours. prior to cell fixation, μm edu ( -ethynyl- 'deoxyuridine) was added to each well for min. cells were then fixed for min in % formaldehyde and permeabilised in % triton x- for min. edu detection was carried out adding the click-it reaction cocktail for min and immunofluorescent labelling for rta and hsc was performed as above. cells were mounted in vectashield with dapi (vector labs). total rna from cells was extracted using trizol (life technologies) according to the supplier's protocol. dna-free dna removal kit (ambion) was used to remove any contaminating dna from rna samples. reverse transcription was performed with protoscript ii (neb) and oligo(dt) primers and . μg of total rna. negative control reactions were performed in the same manner but without reverse transcriptase. quantitative pcr (qpcr) reactions ( μl) included x sensimix sybr green master mix (bioline), . μm of each primer and μl template cdna (used at : dilution in rnase-free water). cycling was performed in a rotor-gene q machine (eppendorf). the cycling programme was a min initial preincubation at °c, followed by cycles of °c for sec, °c for sec and °c for sec. after qpcr, a melting curve analysis was performed between and °c (with . °c increments) to confirm amplification of a single product. relative expression compared to control cells was calculated using the ΔΔc t method as previously described [ ] . for each gene of interest and housekeeping gene (gapdh) a standard curve was constructed using a pool of cdna derived from unreactivated and reactivated cells. six different dilutions of the standards were quantified, these included : , : , : , : , : , and : , dilution. the slope of the standard curve was used to calculate the amplification efficiency (ae) of the primers using the formula: ae = ( − /slope ). the mean cycle threshold (c t ) was determined from three independent biological replicates. all genes of interest were normalised against the housekeeping gene gapdh (Δc t ). ΔΔc t was calculated subtracting Δc t of unreactivated cells from Δc t of reactivated cells and the fold change was then determined using ae (-ΔΔc t ) . statistical significance was validated by student's t-test. unreactivated trex bcbl -rta cells treated with dmso ( . %) were used as control to assess viral reactivation. reactivated cells were exposed to doxycycline for h. total dna was then isolated with the use of a qiaamp dna mini kit (qiagen) as per the manufacturer's instructions. qpcr was carried out as described above. ng of template dna and primers specific for the orf gene were used. quantification of gapdh gene was used to normalize between samples and the mean cycle threshold (c t ) was determined from three independent biological replicates. relative levels of viral dna compared with unreactivated cells were calculated using the ΔΔc t method as previously described [ ] . trex bcbl -rta cells that had been seeded on -well plates were reactivated and treated with control dmso ( . %) or ver- . unreactivated cells treated with dmso ( . %) were used as control to evaluate viral reactivation. after h reactivation, μl of the rpmi culture medium was centrifuged at g for five min, immediately mixed with μl of dmem supplemented with % fcs and % p/s and incubated for a further h with hek- t cells that had been seeded in -well plates the previous day. total rna was then extracted with trizol (life technologies) and qrt-pcr carried out as described above. relative expression compared to control cells was calculated using the ΔΔc t method as previously described [ ] . determination of the cellular metabolic activity was performed using a non-radioactive celltiter aq ueous one solution cell proliferation assay (mts) (promega), according to the manufacturer's manual. , cells trex bcbl -rta or , hek- t rkshv. cells were seeded in triplicate in a flat -well culture plate (corning). after h inhibitor exposure, celltiter aqueous one solution reagent was added and cells were incubated for h in a humidified incubator in % co at °c. absorbance was measured at nm using an infinite f (tecan) plate reader. background control had culture medium without cells and the signal from this was subtracted to all other absorbance values. this assay allows evaluation of viability, cytotoxicity and effector caspases activation within a single assay well. the assay was carried out as specified on the supplier's manual. , trex bcbl -rta cells or , hek- t rkshv. cells were seeded in triplicate in tissue culture treated black microplates (greiner bio-one). no-cell control (background) contained only culture medium and the signal from this was subtracted to all other absorbance and luminescence values. fluorescence and luminescence readings were collected using a glomax system (promega) (kindly provided by dr. john boyle, university of leeds, uk). luciferase activity was detected using the dual-luciferase reporter assay system (promega) as previously described [ ] . hek- t cells were seeded in triplicate in flat -well culture plate (corning) at a density of , cells per well. following the respective plasmid transfections and inhibitor exposure, media was removed from the culture wells and cells washed gently with μl pbs. μl x passive lysis buffer was added to the cell monolayer which was rocked for min and then μl of each lysate was transferred to tissue culture treated white microplates (greiner bio-one). luciferase measurements were carried out in a fluostar optima microplate reader (bmg labtech ltd), with injectors and being used to dispense μl of luciferase assay reagent ii and stop & glo reagent respectively. firefly luciferase activity was normalized to renilla luciferase activity. formaldehyde-crosslinked chromatin was prepared using the pierce chromatin prep module (thermo scientific) following the manufacturer's protocol. x cells were used per experimental sample and digested with six units of micrococcal nuclease (mnase) per μl of mnase digestion buffer in a °c water bath for min. these conditions resulted in optimal sheared chromatin with most chromatin fragments ranging from - base pairs. immunoprecipitations were carried out using ez-chip kit (millipore) according to the supplier's instructions and as previously described [ ] . immunoprecipitations were done overnight at °c and contained μl of digested chromatin ( x cells), μl of chip dilution buffer and . μg of rnapii antibody (clone ctd h ) (millipore) or isotype antibody, normal mouse igg (millipore). both antibodies were provided with the ez-chip kit. prior to qpcr analysis, eluted dna was subjected to a dna clean up step using ultraclean pcr clean-up kit (mo bio laboratories) according to the supplied protocol with the exception of using μl of spinclean buffer instead of μl. qpcr reactions were performed as described above and using either μl of chip'ed dna or μl of input dna as template. sirna knockdown hek- t rkshv. cells seeded on -well plates were reverse transfected with either nm of the specific silencer select sirna (life technologies) or nm allstars negative control sirna (qiagen) using μl of siport neofx transfection agent (life technologies) per transfection. the sirna id for hsc and ihsp were s and s respectively. s sirna targets the two major ihsp proteins (hsp - and hsp - ). two days post-transfection, cells were transfected again in the same manner. four days after the first transfection, cells were reactivated and incubated for the desired time. proteins and total rna were isolated with trizol (life technologies) and subsequent western blot and qrt-pcr were performed. × trex bcbl -rta cells were transfected once with μl of nucleofector solution v (lonza) to which μm sirna (scramble or hsc ) was added. in addition, to monitor transfection efficiency, μg of the control plasmid pmaxgfp was also co-transfected. cells were transfected using program t- of an amaxa nucleofector i (lonza). after nucleofection cells were maintained in six-well plates. medium was freshly replaced every day. confocal images were subjected to profiling analysis using zeiss zen software. this involved drawing a line in a confocal image to measure the relative intensity of each channel at every pixel along the line. profiling was conducted for each cell in two representative confocal images taken with a -times objective. these data were then analysed using microsoft excel . firstly, a function was used to define whether the relative intensity of a pixel could be defined as a "peak" and thus an hsc foci. this function asked whether the data point for one specific pixel of the rhodamine channel was , this was set as the arbitrary threshold to eliminate background noise. if this condition was met, the function then asked whether the data point was greater than or equal to the data point in the previous and subsequent pixel. if these conditions were true, then this data point was counted as a peak. this was performed for every data point measured in the line profile providing the total number of hsc peaks in the profile of one cell. next, another function was used to determine whether the relative intensity in the dapi channel was , a threshold determined from visualising the line profiling data as a graph. if a pixel was shown to exceed the threshold in the dapi channel and also in the rhodamine channel, then it was counted as a nuclear hsc peak. these measurements were conducted for each pixel in each profile allowing counting hsc nuclear peaks in each profile. hsc peaks outside the nucleus corresponded to cytoplasmic hsc peaks. oligonucleotide primer sequences are available upon request. all primers were purchased from sigma (uk). supporting information s table. cellular proteins previously reported to localise to herpesvirus rtcs and found significantly increased in the ne of reactivated cells. (xls) s fig. ihsp was redistributed from the cytoplasm to both the periphery and within kshv-induced rtcs. (a) trex bcbl -rta cells remained unreactivated or reactivated for either h or h. in unreactivated cells ihsp was cytoplasmic (i). in contrast, at h reactivation an increase in nuclear ihsp labelling was seen with numerous small ihsp foci found mainly adjacent to viral rtcs (ii). some cells displayed ihsp completely recruited within rtcs (iii and iv asterisks), while other cells accumulated large ihsp adjacent to rtcs (iv arrows). (b) trex bcbl -rta cells remained unreactivated (i) or reactivated for h (ii and iii) followed by triple-labelling with antibodies specific for rta and ihsp and click-it edu alexa fluor . complete co-localisation between ihsp , rta and actively replicated viral dna (edu-labelled) was observed in both incipient rtcs (ii) and in fully-developed rtcs (iii). note that in these cells ihsp was not depleted from the cytoplasm. trex bcbl -rta cells remained unreactivated or reactivated for h in the presence of control dmso ( . %) or μm ver- followed by labelling with click-it edu alexa fluor and an antibody specific for rnapii (clone ctd h ). (a) a high proportion of unreactivated trex bcbl -rta cells replicated their cellular dna (edu-labelled) in the presence of control dmso ( . %) or μm ver- . normal rnapii localization was observed in these cells, with nuclear rnapii excluding the nucleoli. (b) in contrast, reactivated cells entered cell cycle arrest as demonstrated by fewer edu-labelled cells. in the presence of dmso, multiple rtcs were formed with some replicating viral dna (white arrows). in cells treated with ver- , multiple pre-replicative sites were seen labelled by rnapii antibody and edu-labelling was more diffused in the nucleus compared with dmso-treated cells. (tif) chaperone machines for protein folding, unfolding and disaggregation folding of newly translated proteins in vivo: the role of molecular chaperones chaperones: general characteristics and classifications. the chaperonopathies hsp in cancer: back to the future hsp and the chaperoning of cancer hsp molecular chaperones and parkinson's disease broad action of hsp as a host chaperone required for viral replication recruitment of hsp chaperones: a crucial part of viral survival strategies evolution of hsp gene and its implications regarding relationships between archaebacteria, eubacteria, and eukaryotes the heat shock proteins phylogenetic analysis of kd heat shock protein sequences suggests a chimeric origin for the eukaryotic cell nucleus atpases as drug targets: insights from heat shock proteins and targeting hsp the second potentially druggable heat shock protein and molecular chaperone? update on hsp inhibitors in clinical trial advances in the clinical development of heat shock protein (hsp ) inhibitors in cancers phase-ii study of deoxyspergualin in metastatic breast cancer. invest new drug tau aggregation inhibitor (tai) therapy with rember arrests disease progression in mild and moderate alzheimer's disease over weeks kshv and the pathogenesis of kaposi sarcoma: listening to human biology and medicine kshv infection and the pathogenesis of kaposi's sarcoma kaposi's sarcoma and its associated herpesvirus x box binding protein xbp- s transactivates the kaposi's sarcoma-associated herpesvirus (kshv) orf promoter, linking plasma cell differentiation to kshv reactivation from latency a novel mechanism inducing genome instability in kaposi's sarcoma-associated herpesvirus infected cells inefficient establishment of kshv latency suggests an additional role for continued lytic replication in kaposi sarcoma pathogenesis herpesvirus saimiri open reading frame (rta) protein reactivates the lytic replication cycle in a persistently infected a cell line reactivation of kaposi's sarcoma-associated herpesvirus infection from latency by expression of the orf transactivator, a homolog of the ebv r protein dna virus replication compartments a dominant-negative herpesvirus protein inhibits intranuclear targeting of viral proteins: effects on dna replication and late gene expression annexation of the interchromosomal space during viral infection herpes simplex virus replication compartments can form by coalescence of smaller compartments architecture of replication compartments formed during epstein-barr virus lytic replication kaposi's sarcoma-associated herpesvirus ori-lyt-dependent dna replication: involvement of host cellular factors proteomics of herpes simplex virus replication compartments: association of cellular dna replication, repair, recombination, and chromatin remodeling proteins with icp using silac and quantitative proteomics to investigate the interactions between viral and host proteomes nucleolar proteomics and viral infection use of sequential chemical extractions to purify nuclear membrane proteins for proteomics identification coupling of the nucleus and cytoplasm: role of the linc complex role of anc- in tethering nuclei to the actin cytoskeleton dynamic interactions of nuclear lamina proteins with chromatin and transcriptional machinery nuclear lamins: their structure, assembly, and interactions histones h /h form a tight complex with the inner nuclear membrane protein lbr and heterochromatin protein hsp inhibitors are efficacious against kaposi sarcoma by enhancing the degradation of the essential viral gene lana, of the viral co-receptor epha as well as other client proteins use of the red fluorescent protein as a marker of kaposi's sarcoma-associated herpesvirus lytic gene expression kaposi's sarcoma-associated herpesvirus orf protein: exploiting all stages of viral mrna processing neddylation is essential for kaposi's sarcoma-associated herpesvirus latency and lytic reactivation and represents a novel anti-kshv target interaction network of proteins associated with human cytomegalovirus ie -p protein during infection: a proteomic analysis human cytomegalovirus induces expression of cellular topoisomerase ii herpes simplex virus activates cdc to recruit topoisomerase ii alpha for post-dna synthesis expression of late genes global changes in kaposi's sarcomaassociated virus gene expression patterns following expression of a tetracycline-inducible rta transactivator dynamic organization of dna replication in mammalian cell nuclei: spatially and temporally defined replication of chromosome-specific alpha-satellite dna sequences the intra-s-phase checkpoint affects both dna replication initiation and elongation: single-cell and -dna fiber analyses a c-terminal signal prevents secretion of luminal er proteins functional analysis of hsp inhibitors adenosine-derived inhibitors of kda glucose regulated protein (grp ) atpase: insights into isoform selectivity a novel, small molecule inhibitor of hsc /hsp potentiates hsp inhibitor induced apoptosis in hct colon carcinoma cells influenza virus infection causes specific degradation of the largest subunit of cellular rna polymerase ii gene expression profiling of human colon cancer cells following inhibition of signal transduction by -allylamino- -demethoxygeldanamycin, an inhibitor of hsp molecular chaperone phase i pharmacokinetic and pharmacodynamic study of -allylamino, -demethoxygeldanamycin in patients with advanced malignancies herpes simplex virus type dna polymerase requires the mammalian chaperone hsp for proper localization to the nucleus hsp inhibitors reduce influenza virus replication in cell culture evolutionary constraints on chaperone-mediated folding provide an antiviral approach refractory to development of drug resistance hsp inhibitors exhibit resistance-free antiviral activity against respiratory syncytial virus antiviral activity and rna polymerase degradation following hsp inhibition in a range of negative strand viruses update on in vitro cytotoxicity assays for drug development ver- , a small molecule inhibitor of hsp with potent anti-cancer activity on lung cancer cell lines in vivo newly translated polypeptides are sequestered in a protected folding environment auto-activation of the rta gene of human herpesvirus- kaposi's sarcomaassociated herpesvirus transcription activation of polyadenylated nuclear rna by rta in human herpesvirus /kaposi's sarcoma-associated herpesvirus identification of direct transcriptional targets of the kaposi's sarcoma-associated herpesvirus rta lytic switch protein by conditional nuclear localization comparative study of regulation of rta-responsive genes in kaposi's sarcoma-associated herpesvirus/human herpesvirus a comprehensive analysis of recruitment and transactivation potential of k-rta and k-bzip during reactivation of kaposi's sarcoma-associated herpesvirus a kaposi's sarcoma virus rna element that increases the nuclear abundance of intronless transcripts stability of a long noncoding viral rna depends on a -nt core element at the rna ' end to interact with viral orf and cellular pabpc cell cycle arrest by kaposi's sarcoma-associated herpesvirus replication-associated protein is mediated at both the transcriptional and posttranslational levels by binding to ccaat/enhancer-binding a viral nuclear noncoding rna binds relocalized poly(a) binding protein and is required for late kshv gene expression nuclear sequestration of cellular chaperone and proteasomal machinery during herpes simplex virus type infection hsc focus formation at the periphery of hsv- transcription sites requires icp virus-induced chaperone-enriched (vice) domains function as nuclear protein quality control centers during hsv- infection function of dnaj and dnak as chaperones in origin-specific dna-binding by repa three escherichia coli heat shock proteins are required for p plasmid dna replication: formation of an active complex between e. coli dnaj protein and the p initiator protein similarities between the dna replication initiators of gram-negative bacteria plasmids (repa) and eukaryotes (orc p)/archaea (cdc p) human hsp and hsp chaperone proteins facilitate human papillomavirus- e protein binding to the origin and stimulate cell-free dna replication chaperone proteins abrogate inhibition of the human papillomavirus (hpv) e replicative helicase by the hpv e protein activation of the herpes simplex virus type- origin-binding protein (ul ) by heat shock proteins heat shock protein-mediated disassembly of nucleoprotein structures is required for the initiation of bacteriophage lambda dna replication ordered assembly of nucleoprotein structures at the bacteriophage lambda replication origin during the initiation of dna replication host and viral proteins in the virion of kaposi's sarcoma-associated herpesvirus virion proteins of kaposi's sarcoma-associated herpesvirus the hsp family and cancer peptides and aptamers targeting hsp : a novel approach for anticancer chemotherapy cysteine reactivity distinguishes redox sensing by the heat-inducible and constitutive forms of heat shock protein a high-affinity conformation of hsp confers tumour selectivity on hsp inhibitors hsp and hsp /erdj are required for the expression and anti-apoptotic function of kshv k icp interacts with the c-terminal domain of rna polymerase ii and facilitates its recruitment to herpes simplex virus transcription sites, where it undergoes proteasomal degradation during infection identification of proteins in human cytomegalovirus (hcmv) particles: the hcmv proteome proteins of purified epstein-barr virus comprehensive characterization of extracellular herpes simplex virus type virions analysis of virion-incorporated host proteins required for herpes simplex virus type infection through a rna interference screen an interaction between kshv orf and uif provides mrna-adaptor redundancy in herpesvirus intronless mrna export identification of a cis-acting element within the herpesvirus saimiri orf promoter that is responsive to the hvs.r transactivator kaposi's sarcoma-associated herpesvirus (kshv) rta and cellular hmgb proteins synergistically transactivate the kshv orf promoter merkel cell polyomavirus small t antigen mediates microtubule destabilization to promote cell motility and migration the activation domain of herpesvirus saimiri r protein interacts with the tata-binding protein the cellular interactome of the coronavirus infectious bronchitis virus nucleocapsid protein and functional implications for virus biology the carboxy terminus of the herpesvirus saimiri orf gene contains domains that are required for transactivation and transrepression a γ- herpesvirus nucleocytoplasmic shuttle protein interacts with importin α and α nucleolar disruption impairs kaposi's sarcoma-associated herpesvirus orf -mediated nuclear export of intronless viral mrnas merkel cell polyomavirus small t antigen targets the nemo adaptor protein to disrupt inflammatory signaling we are grateful to members of the whitehouse laboratory for helpful discussions, especially to alexander j. coleman for guidance with confocal profiling. the authors would like to thank key: cord- - e yufo authors: breiman, adrien; ruvën-clouet, nathalie; le pendu, jacques title: harnessing the natural anti-glycan immune response to limit the transmission of enveloped viruses such as sars-cov- date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: e yufo nan domain [ ] . the recently emerged sars-cov- responsible for covid- shows overall conservation of the s protein glycosylation sites. the primary target organ of human coronaviruses, including both sars and sars-cov- , is the lung and both viruses use angiotensin converting enzyme (ace ) as receptor [ ] . being expressed on lung alveolar epithelial cells, chiefly type pneumocytes, [ , ] , it is to be expected that the glycosylation of sars-cov and sars-cov- should be similar. using a cellular experimental model, our group showed that the interaction between sars-cov s protein and ace could be specifically blocked in a dose-dependent manner by anti-a blood group antibodies when the s protein was synthesized by cells that expressed the a histo-blood group antigen following transfection by the appropriate glycosyltransferases cdna [ ] . these observations suggested that, when produced in cells that express the a or b blood group enzymes, infectious sars virions are decorated by the corresponding glycan antigens and that the presence of anti-a and anti-b antibodies in blood group o individuals could prevent infection by blocking virus attachment and entry. moreover, blood group o individuals were at a much lower risk of being infected than non-o individuals in a hong kong sars hospital outbreak [ ] , and a similar trend has just been observed for covid- in china [ ] . accordingly, blood group o individuals would be at a lesser risk of being infected than non-o individuals due to blocking of potential transmission events from either a, b, or ab individuals, providing anti-a or anti-b titers are of sufficient magnitude (fig ) . mathematical modeling of the consequences of this potential limitation of virus transmission suggested that the hong kong sars hospital outbreak had been slowed down to some extent thanks to the abo genetic polymorphism and the ensuing neutralizing anti-a and anti-b antibodies. it further indicated that if anti-blood group a and/ or b titers had always been high, transmission of the virus, in the absence of any containment measure, would be largely impaired and the outbreak slowed to a considerable extent [ ] . we therefore hypothesize that as they are produced in cells coexpressing the ace receptor and either the αgal, neugc, or a/b blood group antigens, both sars-cov and sars-cov harbor the corresponding glycan epitopes. because of the natural immune response against these epitopes, the αgal and neugc xenoantigens would contribute to prevent cross-species transmission from nonprimate mammals to humans, while a/b blood group antigens would contribute to decrease and slow between-human transmission. nonetheless, owing to the presence of individuals with low anti-αgal titers, occasional cross-species transmission may occur. interestingly, a recent genomic analysis across vertebrates revealed that two bats lineages, including rhinopholus bats suspected to have originated the sars-cov- closest ancestor, lost their cmah gene function, similar to humans [ ] . the lack of neugc xenoantigen on the virions produced by these bats might have facilitated cross-species transmission. likewise, impairment of transmission by the anti-blood group antibodies may not work to its full potential because of their variable titers in the population and of the high affinity of the sars-cov for ace [ ] , rendering its neutralization more difficult. this leaves room to amplify these innate mechanisms of protection in preparation for the next emergence and mitigation of the virus impact once emergence has occurred. if the antibody blocking effect can be documented in vitro, and possibly in vivo, it will become important to consider raising the anti-αgal, as well as the anti-a and anti-b antibodies titers in human populations. that could be achieved as previously described either by immunizing against inactivated harmless bacteria that harbor the αgal, a, and b epitopes or by immunizing against the corresponding synthetic oligosaccharides linked to an immunogenic scaffold [ , ] . raising the anti-a and anti-b titers in the whole population carries the risk of complicating incompatible platelet transfusion as well as increasing the risk of hemolytic disease of the newborn in case of mother-infant abo incompatibility. these issues should be carefully dealt with. raising the anti-neugc titers might be more problematic since meat and dairy products consumption allows incorporation of neugc onto human glycans, and this may contribute to the promotion of inflammation and cancer progression as experimentally demonstrated [ , ] . by contrast, raising the anti-αgal titers should not carry any risk since the antigen is entirely absent from human tissues. blood groups a and b might also be harnessed to increase the efficacy of sars-cov- vaccines. indeed, the virus spike proteins, which are the main target of currently designed vaccines, might be produced in cells that are enzymatically equipped to synthetize a and b antigens so that the vaccine glycoprotein will carry these epitopes. in addition to generating neutralizing anti-s protein, the vaccine would stimulate anti-a and anti-b responses that may contribute to the vaccine efficacy in all cases of abo incompatible transmissions. in conclusion, we propose to enhance the innate anti-viral protection conferred by natural anti-glycan antibodies in order to lower both the risk of emergence of coronaviruses, or other enveloped viruses, from a nonprimate mammalian species and the risk of transmission within the human population. this could add-up to other protection and containment measures, mitigating the impact of the epidemic. virions produced by blood group o individuals are devoid of a or b antigens and can be fully transmitted regardless of the recipient blood type (full arrows). viruses produced by a and b blood groups individuals are decorated by a or b blood group epitopes (red and green spikes, respectively) and viruses produced by blood group ab individuals are decorated by both a and b epitopes. transmission of such viruses will be decreased by the presence of either anti-a and/or anti-b of the recipient (dashed arrows). transmission between individuals of the same subtype will always be maximal (circular arrows). in the presence of high-titered anti-a and anti-b antibodies, transmissions represented by dashed arrows should be completely ablated. https://doi.org/ . /journal.ppat. .g evolution of carbohydrate antigens-microbial forces shaping host glycomes? catastrophic-selection" interplay between enveloped virus epidemics, mutated genes of enzymes synthesizing carbohydrate antigens, and natural anticarbohydrate antibodies serum sickness" to "xenosialitis": past, present, and future significance of the non-human sialic acid neu gc absence of neu gc and presence of anti-neu gc antibodies in humans-an evolutionary perspective abo research in the modern era of genomics blood group isoantibody stimulation in man by feeding blood-group active bacteria genetic regulation of the expression of abh and lewis antigens in tissues quantitation and characterization of anti-galalpha - gal antibodies in sera of healthy persons human b cells are the main blood group a-specific b cells that have a moderate correlation with anti-a antibody titer unexpected receptor functional mimicry elucidates activation of coronavirus fusion functional assessment of cell entry and receptor usage for sars-cov- and other lineage b betacoronaviruses tissue distribution of ace protein, the functional receptor for sars coronavirus. a first step in understanding sars pathogenesis single-cell rna expression profiling of ace , the putative receptor of wuhan -ncov inhibition of the interaction beteen the sars-cov spike protein and its cellular receptor by anti-histo-blood group antibodies abo blood group and susceptibility to severe acute respiratory syndrome relationship between the abo blood group and the covid- susceptib phylogenetic distribution of cmp-neu ac hydroxylase (cmah), the enzyme synthetizing the proinflammatory human xenoantigen neu gc cryo-em structure of the -ncov spike in the prefusion conformation induction of cytolytic anti-gal antibodies in α- , -galactosyltransferase gene knockout mice by oral inoculation with escherichia coli o : b bacteria development of a-gal-antibody conjugates to increase immune response by recruiting natural antibodies a red meat-derived glycan promotes inflammation and cancer progression the authors are grateful to hanane el kenz (brugmann university hospital, brussels, belgium) and france pirenne (henri mondor university hospital, créteil, france) for fruitful discussions and helpful remarks. key: cord- - d esp authors: walker, peter j.; firth, cadhla; widen, steven g.; blasdell, kim r.; guzman, hilda; wood, thomas g.; paradkar, prasad n.; holmes, edward c.; tesh, robert b.; vasilakis, nikos title: evolution of genome size and complexity in the rhabdoviridae date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: d esp rna viruses exhibit substantial structural, ecological and genomic diversity. however, genome size in rna viruses is likely limited by a high mutation rate, resulting in the evolution of various mechanisms to increase complexity while minimising genome expansion. here we conduct a large-scale analysis of the genome sequences of animal rhabdoviruses, including genomes which we determined de novo, to identify patterns of genome expansion and the evolution of genome complexity. all but seven of the rhabdoviruses clustered into well-supported monophyletic groups, of which eight corresponded to established genera, seven were assigned as new genera, and two were taxonomically ambiguous. we show that the acquisition and loss of new genes appears to have been a central theme of rhabdovirus evolution, and has been associated with the appearance of alternative, overlapping and consecutive orfs within the major structural protein genes, and the insertion and loss of additional orfs in each gene junction in a clade-specific manner. changes in the lengths of gene junctions accounted for as much as . % of the variation in genome size from the smallest to the largest genome, and the frequency with which new orfs were observed increased in the ’ to ’ direction along the genome. we also identify several new families of accessory genes encoded in these regions, and show that non-canonical expression strategies involving turbs-like termination-reinitiation, ribosomal frame-shifts and leaky ribosomal scanning appear to be common. we conclude that rhabdoviruses have an unusual capacity for genomic plasticity that may be linked to their discontinuous transcription strategy from the negative-sense single-stranded rna genome, and propose a model that accounts for the regular occurrence of genome expansion and contraction throughout the evolution of the rhabdoviridae. rna viruses are among the most structurally and ecologically diverse of all life forms [ ] . their genomes may consist of positive (+) sense, negative (-) sense or ambi-sense singlestranded (ss) rna, or double-stranded (ds) rna, and may take the form of a single or multiple segments that are packaged in single or multiple particles. rna viruses also employ a plethora of strategies for replication and gene expression, and encode a vast array of structural and nonstructural proteins, many of which are unique and have multiple, highly specialized functions [ ] . despite their diversity, rna virus genomes are ubiquitously small, averaging only kb, and with a maximum size of~ kb for some members of the order nidovirales [ , ] . this size limitation has been linked to high mutation rates (a mean rate of~ mutation /genome /replication) due to replication with an error-prone rna-dependent rna polymerase that lacks proofreading capability [ , ] . high error rates are thought to limit genome sizes because, as size increases, the number of deleterious mutations also increases to levels beyond which reproduction of the fittest variant cannot be guaranteed [ , ] . due to this fundamental evolutionary constraint, rna viruses have employed various mechanisms of genome compression, such as the use of alternative or overlapping open reading frames (orfs) and the evolution of multiple functions for individual proteins [ , , ] . for some rna viruses, increases in genome size have been associated with increases in the size of replicative proteins [ ] and the presence of helicase and proof-reading exonuclease domains [ , [ ] [ ] [ ] . however, the mechanisms and evolutionary context that would favour increased genome size and complexity, given constraints on replication efficiency, are currently unknown [ , ] . the rhabdoviridae is one of the most ecologically diverse families of rna viruses. rhabdoviruses have been identified in a very wide range of plants and animals, including mammals, birds, reptiles, and fish with many transmitted by arthropod vectors [ , ] . the family includes rabies virus (rabv), which causes over , human deaths annually [ ] , vesicular stomatitis indiana virus (vsiv), which has served as an important model for the study of many aspects of mammalian virus replication and virus-host interactions, and many other important pathogens of humans, livestock, farmed aquatic animals and food crops. the nonsegmented [-] ssrna rhabdovirus genome is packaged within a characteristic bullet-or rodshaped particle comprising five structural proteins-the nucleoprotein (n), polymeraseassociated phosphoprotein (p), matrix protein (m), glycoprotein (g) and rna-dependent rna polymerase (l) [ ] . the genome features partially complementary, untranslated leader (l) and trailer (t) sequences and five orfs arranged in the order '-n-p-m-g-l- '. each orf is flanked by relatively conserved transcription initiation (ti) and transcription termination/ polyadenylation (ttp) sequences which orchestrate expression of the five corresponding capped and polyadenylated mrnas [ ] . rhabdovirus genomes may also contain additional orfs encoding putative proteins, which are mostly of unknown function. these may occur as alternative or overlapping orfs within the major structural protein genes or as independent orfs flanked by ti or ttp sequences in the regions between the structural protein genes [ ] , some of which appear to have arisen by gene duplication [ , [ ] [ ] [ ] [ ] [ ] . here we undertake the first large-scale analysis of the evolution of genome size and complexity in a family of [-] ssrna viruses. we demonstrate that remarkable changes in genome size and complexity have occurred in rhabdoviruses in a clade-specific manner, primarily by extension and insertion of additional transcriptional units in the structural protein gene junctions, followed by occasional losses. we also show that rhabdoviruses have evolved a large number of accessory proteins and that the use of non-canonical gene expression strategies appears to be common, particularly amongst vector-borne rhabdoviruses. our data set comprised the complete or near-complete genome sequences of animal rhabdoviruses, including viruses isolated from various vertebrates and arthropods for which we determined the sequences de novo (s table) . incomplete genomes lacked only the extreme terminal sequences. all rhabdovirus genomes contained the five canonical structural protein genes (n, p, m, g and l); however, there was remarkable diversity in the number and location of other long orfs. across the data set, we identified additional orfs nt in length of which shared no detectable protein sequence similarity with any other protein in our data set or with those in public databases (s table) . these additional orfs were located either within the structural protein genes or in additional transcriptional units located in regions between these genes (fig. ) . the additional transcriptional units were annotated by using relatively conserved ti and ttp motifs. the core ti sequence (uugu) was conserved with some minor variations (cugu, uugc, uuga, ucgu, ugau) employed in some viruses. the ttp motif g[u] was also conserved, with the variation a[u] occurring only in several genes of one virus (chov). due to the large number and diversity of additional orfs, we adopted a standard nomenclature that does not necessarily reflect structural homology. unless previously assigned a distinctive name (e.g., befv g ns , α , α , β and γ proteins), all orfs nt were assigned names according to the following rules: i) each additional transcriptional unit was designated u (unknown) followed by a number as they appeared in order in the genome presented in positive polarity (i.e., u , u , u , etc); ii) the first orf within each transcriptional unit was assigned the same designation as the transcriptional unit; and iii) each subsequent orf within any transcriptional unit (alternative, overlapping or consecutive) was designated by letter (i.e., u x, u y, u z) (s table) . alternative orfs are defined here as those which occur in a different frame within another longer orf; overlapping orfs are alternative orfs which extend beyond the end of the primary orf; and consecutive orfs are those which do not overlap but follow consecutively within the same transcriptional unit. the arbitrary cut-off of nt ( aa) was selected on the basis that two small basic proteins of and amino acids (c and c') have been shown to be expressed from an alternative orf within the vsiv p gene [ , ] . these are the smallest known rhabdovirus proteins. to determine the evolutionary history of the rhabdoviruses studied here, we inferred a phylogenetic tree using conserved regions of the l protein of all viruses in our data set as well as the recently described north creek virus (norcv) [ , ] (fig. ) . all but two of these rhabdoviruses (norcv and mouv) clustered into well-supported monophyletic groups (bootstrap proportion [bsp] ); however, many of the deeper nodes were unresolved throughout the phylogeny. eight of the well-supported clades corresponded to the eight established genera (lyssavirus, vesiculovirus, perhabdovirus, sigmavirus, ephemerovirus, tibrovirus, tupavirus and sprivivirus) and we assigned a further seven clades as proposed new genera (almendravirus, bahiavirus, curiovirus, hapavirus, ledantevirus, sawgravirus and sripuvirus). the taxonomic assignment of the two remaining clades was considered to be ambiguous (s table) . for simplicity of expression we refer here to all as 'genera', whether existing or proposed, but we recognise that taxonomic proposals require consideration and ratification by the international committee on taxonomy of viruses (ictv). although the analysis was limited by the availability of single isolates of most viruses, apparent structure by geographic location or reservoir host was not observed in the phylogeny. however, multiple genera appeared to be primarily associated with bats (i.e., ledanteviruses, lyssaviruses), fish (i.e., perhabdoviruses, spriviviruses) or ungulates (i.e., ephemeroviruses, tibroviruses, vesiculoviruses). vector-borne rhabdoviruses were present in of the groups, dominating the dimarhabdovirus supergroup, but were largely absent from clades associated with bats (lyssavirus), flies (sigmavirus) and fish (perhabdovirus, sprivivirus) (fig. ) . the exception to this trend was the tupavirus clade, which comprised viruses that have not yet been associated with a vector species, and for which little is known about their ecology or distribution. each of the seven newly proposed rhabdovirus genera formed an independent, well-supported monophyletic group in the l protein phylogeny (bsp ), and comprised viruses with similar genome organization ( fig. ; fig. ). in several instances, viruses clustered closely with other members of a genus, yet we considered them to be unassigned species due to major differences in genomic architecture (see below). for example, the newly proposed genus curiovirus comprises a monophyletic group of four viruses isolated from biting midges (culicoides sp.), sandflies (lutzomyia spp.) and mosquitoes (coqillettidia and trichoprosopon spp.) from the forests of south america and the caribbean (s table) . the genomes of curv, irirv, rbuv and itav all have one or more orfs located between the m and g genes, and the g and l genes. in contrast, the closely related aruv and inhv lack additional genes between the m and g and for this reason we have excluded them from the genus curiovirus at this time. we also recognize the previous suggestion that curv and itav should be assigned to a new genus for which the name bracorhabdovirus (brazilian amazonian culicoides rhabdoviruses) was proposed [ ] . however, our analysis clearly indicates that this monophyletic group has a broader host range and geographic distribution than this regionally-derived name suggests. five of the novel viruses (comprising four putative new species) identified in this study were assigned to established genera. two of these, koolv and yatv, clustered within the existing ephemerovirus clade, (bsp ) and possessed the characteristic genome organization of ephemeroviruses, including a non-structural glycoprotein gene (g ns ) followed by a viroporin newly proposed genera are indicated by a † symbol. cytorhabdovirus, novirhabdovirus and nucleorhabdovirus outgroup sequences were excluded from the tree as they were too divergent to establish a reliable rooting. the tree is therefore rooted arbitrarily on one of two basal clades (genera almendravirus and bahiavirus) that comprise viruses isolated from mosquitoes. (α ) and several other small proteins ( fig. ; fig. ). similarly, two novel viruses isolated from biting midges (culicoides insignis), swbv and bav, clustered within the genus tibrovirus (bsp ) and exhibited the conserved n-p-m-u -u -g-u -l genome organisation ( fig. ; fig. ; s table) . swbv was assigned as a new species (sweetwater branch virus), but bav is closely related to tibv and may be regarded as the same species (tibrogargan virus). finally, a novel tupavirus (klav) identified from two species of vole (microtus and clethrionomys spp.), clustered with the tupv and durv clade in the l protein phylogeny ( fig. ; s table) . a more detailed rationale for the assignment of viruses to existing and proposed new genera is provided as supplementary text. we identified a . % variation in genome size from the smallest genome (fukv, ledantevirus; , nt) to the largest in our data set (koolv, ephemerovirus; , nt). all genomes, including those for which extreme terminal sequences were unresolved, appeared to fall within this range. variations in genome size were associated with: i) variation in the length of intergenic regions (igrs) between transcriptional units; ii) variation in the length of ' and ' untranslated regions (utrs) within individual transcriptional units; iii) the presence of additional transcriptional units containing long orfs; and iv) the presence of overlapping or consecutive long orfs within individual transcriptional units. an examination of genome size across the phylogeny revealed a general trend towards larger genomes in the lower third of the tree, which is comprised of the hapaviruses, curioviruses, tibroviruses and ephemeroviruses, as well as several unassigned viruses (s fig.) . although this may indicate that an enhanced capacity for genome expansion is a property specific to this group, variation in genome size can also be observed between viruses in the majority of genera in the data set. several clade-specific patterns were evident when the lengths of the transcriptional units and igrs were compared within and between rhabdovirus genera (table ) . ledantevirus genomes were smallest on average ( . × the length of the l) whereas ephemeroviruses genomes were the largest ( . × the length of the l, table ). interestingly, although substantial variation in the length of gene junctions was observed in several genera (including ephemeroviruses and lyssaviruses), most variation in genome size occurred as the result of the presence of new, non-canonical orfs in the regions between the structural protein genes (table ) . although new orfs were observed in each igr across the phylogeny (n-p, p-m, m-g and g-l) their location was primarily restricted to a single igr within each genus. for example, while hapavirus genome expansion occurred primarily in the p-m junction, genome expansion in the ephemeroviruses occurred at the g-l junction and tibrovirus and curiovirus genomes contained additional orfs primarily in the m-g junction (table ). this suggests that once a new orf arises at a particular gene junction within a lineage, further expansion is more likely to continue at the same gene junction, rather than begin anew elsewhere in the genome. whilst the genome architecture in some viruses was highly compact, others featured long stretches of sequence with non-ascribed function that occurred primarily as 'utrs and 'utrs within transcriptional units (fig. ) . the proportion of untranslated sequences within or between transcriptional units ranged from . % (fukv; nt) to . % (wcbv; nt) and did not correlate with genome size. furthermore, although all lyssaviruses (such as wcbv) featured a high proportion of untranslated sequences (primarily evident as a very long 'utr in the g gene), there was no consistent association between the proportion of untranslated sequences and genus assignment (fig. ). for example, in the genus hapavirus, the proportion of untranslated sequences in the two largest genomes varied from . % (ngav) to . % (ljv). similarly, in the genus ephemerovirus the proportion of untranslated sequences varied from . % in the smallest genome (yatv) to . % in the largest genome (koolv). the presence of long stretches of untranslated sequence, which occurred primarily within transcriptional units, suggests these regions may be functional. however, it is unclear at this time why they are present in some rhabdoviruses and not in others. gene duplication. previous studies have provided evidence of gene duplication in the rhabdoviridae, involving the g and g ns genes [ , ] and the β and γ genes [ ] in the ephemeroviruses, and the u , u and u genes in the hapaviruses flav and wonv [ , , ] . to identify further examples of gene duplication, we conducted a blast analysis of all proteins in our database (e-value < e- ) and used clustalx alignments to confirm sequence similarity. by this analysis, orfs located between the p and m genes of most hapaviruses encode proteins which share detectable sequence similarity. this family of homologous p-m intergenic region proteins (pmips) includes the u , u and u proteins of ljv, wonv, pcv, orv, ljav, manv, mqov, flav, hpv, kamv and mosv (s fig. and s fig.) , as well as the u x proteins of manv and glov which are encoded in orfs overlapping their respective u orfs (s fig.) . although pairwise alignments provide clear evidence for homology, the hapavirus pmips share generally low levels of sequence identity and no universally conserved motifs, indicating considerable structural and functional divergence from their ancestral homolog. proteins encoded in the p-m region in other hapaviruses (i.e., joiv u , ngav u , u x and ngav u ) failed to display significant similarity with the pmips or evidence of gene duplication but this may be due to further structural divergence. additional evidence of gene duplication included the u and u proteins of joiv (encoded in orfs located between the g and l genes), and the n-terminal regions of the p proteins and the upstream u accessory proteins of the sripuviruses chov and smv, each of which share significant sequence similarity (s fig.) . these data suggest that the u protein of the sripuviruses originated from a duplication of the p gene, with the downstream copy of the gene retaining the parental function. similarly, in the curioviruses there is extensive amino acid sequence similarity between the u proteins of curv and irirv and the n-terminal region of the g proteins, suggesting evolution of u through partial duplication of the g gene, which lies immediately downstream. putative accessory genes were found to be abundant and varied greatly in number and location in each genome (fig. ) . a complete list of orfs > nt is annotated in s table. in most cases, homology searches detected no significant amino acid sequence identity with entries in genbank. however, various rhabdovirus accessory gene families were identified based on amino acid sequence identity in our custom blast searches, or common structural characteristics. viroporins. viroporins are small hydrophobic proteins that oligomerize in host cell membranes to form hydrophilic pores, disrupting various cellular processes and promoting virus replication [ ] . orfs encoding viroporin-like proteins were found in more than one-third of the rhabdoviruses in the data set, either as overlapping or consecutive orfs within the g gene, or in additional transcriptional units following the g (or g ns ) gene (fig. ) . orfs encoding putative viroporins were evident in the genomes of all ephemeroviruses, tibroviruses, hapaviruses, bahiaviruses, almendraviruses and curioviruses, as well as the unassigned species aruv and inhv (fig. ) . several of these proteins have been identified previously [ , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] . like the befv α protein for which viroporin activity has been confirmed experimentally, these proteins have the structure characteristics of class ia viroporins, including a central transmembrane and a highly basic c-terminal domain. however, although located in similar positions in the genomes, they are generally too divergent in sequence to establish orthology [ , ] . other small transmembrane proteins. small proteins with a predicted central transmembrane domain but lacking other characteristics of class a viroporins were identified in several other rhabdoviruses (s fig.; s table) . transmembrane proteins with an n-terminal ectodomain are encoded in the gx orf of sripuviruses and the u orf of one curiovirus (rbuv). however, in other curioviruses (curv and irirv), transmembrane proteins are encoded in the u orf and are predicted to have the reverse membrane topology to the rbuv u protein. sequence alignments further suggest these proteins are not orthologous. there is also a small double-membrane spanning protein with a predicted short ectodomain loop encoded in an alternative orf in the fukv m gene that is not present in other ledanteviruses. other small hydrophobic (sh) proteins. small highly hydrophobic proteins ( . - . kd) lacking predicted transmembrane domains are encoded in all tupaviruses (as independent transcriptional units following the m gene) and sripuviruses (as overlapping orfs within the m gene) (s fig.; s table) . all have similar hydropathy profiles with a highly hydrophilic n-terminal domain extending to the centre of the sequence, but sequence identity indicative of orthology is restricted to closely-related viruses. several of these sh proteins have been identified previously but their function remains unknown [ ] [ ] [ ] [ ] . large class i transmembrane glycoproteins. all ephemeroviruses encode a class i transmembrane glycoprotein (g ns ) in the orf following the g gene [ , , , ] . ngav (assigned to the proposed new genus hapavirus) also encodes a g ns protein with similar structural characteristics [ ] . however, as we found no evidence to support recombination between ngav and any ephemerovirus, the ngav g ns gene is likely to have arisen by an independent duplication event of the upstream g gene with which it shares amino acid sequence identity. orf u immediately following the mcov g gene (genus hapavirus) also encodes a large class i transmembrane glycoprotein but lacks the set of conserved cysteine residues that are characteristic of g and g ns proteins, and our homology searches failed to identify similarity with any known protein (s fig.) . other genus-specific accessory gene families. orthologous sets of accessory genes occur in genus-specific patterns in each of the structural protein gene junctions ( fig. ; s table) . in addition to the hapavirus pmip genes, these include genes in the n-p junction of sripuviruses chov and smv (u proteins), the m-g junction of curioviruses (u and u x proteins) and tibroviruses (u and u proteins), and the g-l junction of curioviruses (u x proteins) and ephemeroviruses (α , β, γ and δ proteins) (s fig. to s fig.) . some of these orthologous gene sets have been described previously [ ] . most encode proteins without remarkable structural characteristics and of unknown function (s table) . several general architectural patterns in the arrangement of orfs were evident, implicating several mechanisms of non-canonical gene expression. non-cannonical expression mechanisms are used commonly in other families of rna viruses to increase genome complexity without significantly increasing genome size [ ] . the patterns we observed in this data set were associated with consecutive, overlapping of alternative orfs within individual transcriptional units. consecutive orfs and turbs motifs. consecutive long orfs with termination and initiation codons that are either overlapping (e.g., uaaug) or separated by a short stretch of nucleotides were common in several groups of rhabdoviruses (fig. ) . as previously observed for flav, this 'stop-start' arrangement is commonly preceded by a 'termination upstream ribosome-binding site' (turbs), which contains a short sequence motif that is complementary to the loop region of helix of s ribosomal rna [ , ] . the turbs may also contain flanking anti-complementary sequence motifs that are predicted to form a stem-loop structure. this arrangement was found in the m transcriptional unit in the sripuviruses, the g transcriptional unit of several hapaviruses (flav, hpv, manv, mqov, kamv, mosv and glov) and the transcriptional unit between the p and m genes of glov. the 'stop-start' arrangement also occurs in the transcriptional unit between the g and l genes of aruv, allowing expression of the u orf, but in this case the turbs appears to be further upstream of the stopstart site. finally, the α gene transcriptional unit in most ephemeroviruses contains consecutive orfs encoding a viroporin (α ) and a second protein of unknown function (α ). in kotv, a tubrs is evident upstream of the stop-start site but in other ephemeroviruses the turbs appears to be more cryptic. overlapping orfs and ribosomal-frame shift (rfs) sites. overlapping orfs are common in rhabdovirus genomes and represent a second common architectural arrangement requiring non-canonical gene expression. overlapping orfs occur within the n transcriptional unit (wonv, orv, pcv, mcov, manv), the g transcriptional unit (wonv, orv, pcv, bgv, harv) or within additional transcriptional units between the p and m genes (manv, ngav) or the m and g genes (curv, irirv, rbuv). expression of the second orfs in these arrangements would require either internal initiation in an alternative reading frame or another mechanism such as rna editing or a ribosomal frame-shift (rfs) to extend the first orf. use of alternative initiation codons has been reported in the m and p genes of vsv and the p gene of rabv, and rna editing has been described in the p gene of paramyxoviruses [ , [ ] [ ] [ ] [ ] . although not described previously in mononegaviruses, potential rfs sites were identified in some of these rhabdovirus gene overlap regions, featuring the 'slippery' sequence motifs uaruuuuuuca (bgv, harv, msv) or ccnuuuuuuga (wonv, orv, pcv) followed by a predicted stem-loop structure (s fig.) . these sequence motifs and associated stem-loop structures most closely resemble the- rfs that allows expression of gag-pol in hiv- and other lentiviruses [ , ] . alternative orfs and leaky ribosomal scanning. the third architectural arrangement involves the use of alternative orfs within a longer orf. this arrangement was described previously in vsiv, in which two small basic proteins of and amino acids (c and c') are expressed from an alternative orf within the p gene [ , ] . on this basis, we scanned the rhabdovirus genome data set for alternative orfs of various size ranges and observed that the frequency varied from~ . /genome for orfs in the range of - nt ( - amino acids) tõ . /genome for range - nt ( - amino acids) (fig. ). alternative orfs amino acids occurred in each of the structural protein genes (n, p, m, g and l) and in the additional transcriptional units between the p and m genes. they were most common in the p and least common in the m genes. as observed in other viruses, expression of these alternative orfs could occur by leaky ribosomal scanning, allowing initiation of transcription by a proportion of ribosomes on the alternative start codon [ ] . although, it is not known which (if any) of these alternative orfs are expressed, several factors are likely to be important in determining the probability and level of expression: i) the kozak contexts of the first and alternative initiation codons; ii) the length of the alternative orf (longer orfs are less likely to occur by chance); iii) the location of the alternative orf (distally located orfs are less likely to be expressed in long transcripts); and iv) the expression level of the transcript (l gene transcripts are likely to be the least abundant). for example, short orfs with initiation codons in poor kozak context at the distal end of the l gene are not likely to be expressed at significant levels, if at all. however, in some cases, closely related viruses were found to contain alternative orfs at the same genome location, with initiation codons in good context and encoding predicted polypeptides with high levels of sequence identity (s table) . such arrangements occurred in the n table. doi: . /journal.ppat. .g genes of hpv and flav, the p genes of manv and mqov, the u and m genes of kamv and mosv, and near the start of the g genes of the sripuviruses (niav, sriv, chov and smv); these proteins are considered very likely to be both expressed and functional. we have conducted a detailed analysis of the structural organisation and genome evolution of a family of negative-sense rna viruses-the rhabdoviridae. previous studies have surveyed known rhabdoviruses for biological and genomic diversity, revealed phylogenetic relationships, and considered factors that may have determined their rates of evolution [ , , , ] . in this study, we greatly expanded the repertoire of rhabdovirus genome sequences, which demonstrate extensive variation in genome size and complexity, allowing the assignment of seven proposed new genera. we also identified patterns of accessory gene evolution and expression, and showed that changes in rhabdovirus genome length and composition have occurred throughout the evolutionary history of the family, primarily through the generation and loss of new transcriptional units. this observation is especially striking given the obvious constraints on viral genome size [ ] . the most remarkable aspect of this analysis is the number and variety of additional orfs identified in rhabdovirus genomes, which provides a very different perspective of the family and its evolution than had been obtained from studies of the traditional prototype members (vsiv and rabv). as many of these orfs occur as additional transcriptional units complete with conserved transcriptional control sequences, there is a high likelihood that they would be expressed in infected cells. expression of orfs located in additional transcriptional units has been demonstrated previously for several ephemeroviruses and for the hapavirus wonv [ , , , , , ] . others occur as either alternative or overlapping orfs. further studies are required to determine which of these orfs may be expressed, but we suggest that expression is likely when both the encoded amino acid sequence and the translational context are conserved in related species. notably, very few of the additional orfs detected in this analysis encode proteins with identifiable sequence similarity to other known proteins. sequence similarity, when detected, occurred only between closely related viruses assigned to a genus and, although some accessory protein families were identified, these were more commonly related by shared structural characteristics, such as charged or transmembrane domains, than by sequence. this has been observed previously for so-called orphan ('orfan') proteins in other viruses and bacteria. it has been suggested that the uniqueness of orphan proteins, or their restriction to a single species or genus, is the result of creation de novo, rather than by recombination or lateral gene transfer, and that they play an 'accessory' role in viral pathogenicity or transmission instead of having functions in virion structure or replication [ ] [ ] [ ] . it has also been observed that many orphan proteins are predicted to be highly disordered in structure or, when ordered, structural resolution has revealed unique folds [ ] . as such, future determination of the biological activities of the plethora of novel proteins identified here will require functional studies that may well provide important insights into aspects of infection and immunity as well as fundamental cellular processes and pathways. substantial variation in genome size and complexity was also observed in many rhabdovirus genera, suggesting that the length of the genome is not heavily constrained in all members of the family. indeed, the presence of new orfs and/or very long stretches of non-coding sequence within or between transcriptional units was noted frequently. previous observations have demonstrated that foreign genes of up to~ kb can be inserted into the vsiv genome without significant disruption to viral replication in vitro [ , ] . expanded vsiv genomes were morphologically similar but proportionally longer than wild-type viruses, suggesting that the unique morphology of the rhabdovirus particle may more readily accommodate genome expansion than other virion structures. a significant body of evidence suggests that genome size in rna viruses is likely to be constrained by low replication fidelity [ , ] , and a relationship between genome size and error rate has been observed in a diverse array of organisms [ ] . however, if the genome sizes of rhabdoviruses are constrained by selective pressures other than (or in addition to) those imposed by the background mutation rate, genome expansion may not require a concomitant reduction in polymerase error rates. as the mutation rate of rhabdoviruses has only been determined experimentally for vsiv thus far (~ × - subs/ nucleotide/replication), it is impossible to assess whether the increases in genome size observed here have been associated with concomitant reductions in mutation rate [ ] . it is also striking that while some rhabdovirus genomes appear to have undergone major changes in length and complexity, others contain only the ' and ' promoter regions and five canonical transcriptional units with minimal ' and 'utrs. this suggests that the acquisition and loss of new genes and intergenic regions may be a regular feature of rhabdovirus evolution. previous studies of rna viruses have concluded that constraints on genome size imposed by polymerase error have led to various strategies to minimize genome size while increasing functional complexity, such as gene overlaps and protein multi-functionality [ , ] . given these size constraints, it is unclear why long non-coding regions would arise both within and between transcriptional units and be maintained throughout the evolution of some rhabdovirus genera. it has been known for many years that a long '-utr of unknown function (ψ region) in the g gene of rabv is unnecessary for efficient replication in cell culture or in mice, but may play a role in neuroinvasion [ ] [ ] [ ] . indeed, the retention of similar ψ regions in all lyssaviruses and the existence of long utrs and igrs in other rhabdoviruses suggests that they must provide some fitness advantage in vivo, such as stabilising rna secondary structure, serving as a source of, or targets for, micro rnas, or attenuating transcription of downstream genes to achieve the most effective balance of gene expression. indeed, an analysis of patterns and rates of sequence evolution in the rhabdoviridae and other families in the mononegavirales revealed that, although non-coding regions are less conserved than those that encode proteins, their evolutionary rates are associated with relative genomic position, suggesting that they impact on gene expression [ ] . additional orfs and non-coding sequences occurred at all junctions of the canonical structural protein genes (i.e., n-p, p-m, m-g, and g-l), although there was variation in both the frequency of insertion and the extent of expansion. notably, insertions at the n-p junction are rare, with a single additional orf present in the closely related sripuviruses chov and smv, and short overlapping orfs present within the n gene transcriptional unit in some hapaviruses. it has been reported previously in a study of vsiv recombinants that only the n-p gene junction was refractory to the stable expression of an inserted transcriptional unit, and resulted in a virus with significantly reduced replication efficiency [ ] . in contrast, transcriptional units inserted at other gene junctions were stably expressed, maintained through repeated passages and had no effect on replication efficiency. as the insertion of additional transcriptional units attenuates expression levels of all downstream genes, this may be associated with the importance of maintaining precise control of n and p protein ratios in infected cells to ensure efficient switching between the transcription and replication modes of the ribonucleoprotein complex [ , ] . the relationships, locations and contexts of additional orfs in various viruses lead us to propose a general model for rhabdovirus genome plasticity, which can account for both gains and losses in genome size and complexity (fig. ) . in each of these viruses, small orfs of various lengths occur within most transcriptional units; and although only those nt have genomic evolution in rhabdoviruses been catalogued here, there are numerous other smaller orfs throughout most genomes. it is reasonable to assume that, although the polypeptides encoded in many of these orfs may not be expressed at all during infection, some may be expressed through leaky ribosomal scanning. these are likely to represent a rich genetic resource for the evolution of new functional genes in rna viruses [ ] , triggering the rapid evolution of highly specialised functions. contemporarily, the evolution of a suitable kozak context, turbs motifs and ribosomal frame-shift sites would allow optimal expression within the parental transcriptional unit. ultimately, these new orfs may become uncoupled from the parental gene through gene (sequence) duplication [ ] . as observed previously, this process would allow unconstrained evolution of the new orf and loss of the redundant copy of the parental orf [ , ] . alternatively, new genes may also evolve independently of existing orfs. in some rhabdoviruses in our data set, very long non-coding regions (up to nt) were present either within or between transcriptional units that could serve as a resource to spawn genes de novo in the absence of the evolutionary constraints imposed on alternative or overlapping orfs. this is most likely to occur when orfs are present in transcribed non-coding regions (utrs) such as the ψ region of wcbv in which, uniquely amongst lyssaviruses, an orf of nt has been identified [ ] . the creation of new genes de novo in non-transcribed igrs, such as those present in the g-l gene junctions of ljv, kotv and koolv, almost certainly would require prior or simultaneous evolution of new or modified transcriptional control sequences to allow their expression. we recognise that other mechanisms of genome expansion are also possible. in central american isolates of vsiv, for example, imprecise reiterative insertions of up to nt in the '-utr of the g-gene (variations of '-uuuuuaa- ') have been attributed to non-templated extension by polymerase stutter at the ttp sequence [ , ] . although homologous recombination appears to be very rare in mononegaviruses [ ] , and we found no evidence of lateral gene transfer, we cannot exclude their involvement in rhabdovirus genome expansion. it is also evident that although there is an overall trend toward an expansion of genome size and complexity in the rhabdoviruses, gene loss is also likely to have occurred periodically throughout the evolution of the family. for example, the ephemerovirus γ proteins appear to have been lost in arv and obov, and the hapavirus pmips are entirely absent only from mcov (fig. ) . although our data suggests that gene gain is a more frequent process than gene loss, we acknowledge that, if loss is very frequent, we might not be able to observe it given the available data. this may be resolved in the future with the acquisition of significantly more genomes sampled more closely in time. indeed, as defective-interfering particles are known to occur commonly in rhabdoviruses, a mechanism for purging redundant sequences appears to be readily available [ ] [ ] [ ] . nevertheless, it is evident that a remarkable capacity for genomic plasticity through the gain and loss of accessory functions has been a central theme of rhabdovirus evolution. although our analysis was limited to the rhabdoviridae, similar mechanisms of genome expansion appear to occur in other families of non-segmented (-) ssrna viruses (mononegavirales). for example, amongst the paramyxoviridae genome length varies by . % from human metapneumovirus ( , nt) to beilong virus ( , nt) , and paramyxoviruses also contain novel accessory genes in transcriptional units inserted at various gene junctions [ ] . the apparent propensity for genome expansion in mononegaviruses may be due to their discontinuous transcription strategy which generates multiple viral mrnas. sequence insertions within and between the individual transcriptional units of mononegaviruses are less likely to disrupt gene expression than in (+) ssrna viruses in which the genome commonly encodes a single polyprotein which is processed post-translationally. finally, this study has also provided an important advance in rhabdovirus taxonomy, allowing the assignment of six new species to existing genera and the assignment of species to seven proposed new genera as well as the identification of six new unassigned species. there are currently no formal criteria for genus demarcation in rhabdoviruses. a system of genetic classification (demarc) that allows demarcation of viral taxa based on pairwise evolutionary distances has been proposed and, for picornaviruses, was shown to be comparable to expertbased taxonomic classification [ , ] . however, the application of this approach to the rhabdoviridae would likely require a larger set of sequenced genomes at lower taxonomic levels [ ] , and would be compromised by extensive rate variation among lineages (as this leads to biases in genetic distance measurements). in the taxonomy of higher organisms, to be descriptively useful, a genus should be monophyletic, reasonably compact, and ecologically, morphologically, or biogeographically distinct [ ] . our assignment of new genera in the rhabdoviridae has been based primarily on the identification of well-supported monophyletic groups using unambiguously aligned regions of the l gene, together with a consideration of common features of genome organisation and known aspects of viral ecology. genome organisation has proven here to be a useful taxonomic marker as similar arrangements of accessory genes and other conserved elements of genome architecture appear to be the result of significant evolutionary events that provide resolution between the family and species levels. for some of the new genera, host and/or vector associations have also been relatively informative but in many cases, only single isolates of a species are available and else little is known of their ecology. it is likely that the proposed assignments of viruses to genera and the placement of the proposed unassigned species will evolve into a more complete taxonomic description as more viruses are discovered and as ecological data accumulates. details of the viruses included in this study, including taxonomic status, sources and dates of isolation, and genbank accession numbers of genome sequences are given in s table. all but three viruses sequenced in this study were obtained from the world reference center for emerging viruses and arboviruses (wrceva), located at the university of texas medical branch, galveston. of the remaining viruses, fukv and koolv were obtained from the collection held at the csiro australian animal health laboratory, geelong, and joiv was obtained from the qimr collection held at the queensland university of technology, brisbane, and kindly provided by dr john aaskov. viruses sequenced in this study were prepared as described previously [ ] . with the exception of hpv, itav, curv, glov, inhv, nmv, mebv, yatv, ldv, garv, cntv, irirv, rbuv, barv, ljav, keuv, mcov, smv, chov, pcv and bav, which were sequenced directly from infected suckling mouse brain, viruses were sequenced from viral preparations grown in bhk-bsr, c / or vero cells monolayers. sequencing was performed using either the illumina hiseq or miseq platforms. viral rna was fragmented by incubation at °c for min in . l of fragmentation buffer (illumina ). a sequencing library was prepared from the sample rna using an illumina truseq rna v kit following the manufacturer's protocol. samples were sequenced using the × paired-end protocol. reads in fastq format were quality-filtered and any adapter sequences were removed using trimmomatic software [ ] . the de novo assembly program abyss [ ] was used to assemble the reads into contigs using several different sets of reads and k values from to . the longest contigs were selected and reads were mapped back to the contigs using bowtie [ ] and visualized with the integrated genomics viewer [ ] to verify that the assembled contigs were correct. total reads ranged from . to million and the percentage of reads mapping to the virus genome in each sample ranged from . % to %. details are available upon request. assembly of full genome sequences was performed as previously described [ ] and predicted orfs > amino acids in length were identified across each genome using geneious . . (biomatters ltd). for each non-canonical orf > amino acids in length, we sought to identify putative homologues by first comparing the protein sequence to the complete non-redundant protein sequence database available on genbank using the blastp and psi-blast search algorithms, as well as to the uniprot database using the hidden markov model alignment-based algorithm hhblits [ ] . for these searches, we investigated all matches with an evalue < . we then created a custom protein database containing all orfs > amino acids in length from our data set ( proteins) and performed a custom blast search to identify homologues within this data set. here, an e-value of < e- was considered a significant match. amino acid sequence alignments containing all putative matches to each orf were then created using clustal x and evidence of structural and sequence similarity was investigated by visual inspection. structural predictions for proteins were conducted using compute pi/mw, sig-nalp, tmhmm, tmpred, netnes and netnglyc available through the expasy bioinformatics resource portal (http://www.expasy.org/). to quantify the location and extent of variation in genome size in our data set, we compared the average length of each genomic region within and between rhabdovirus genera. for all viruses, we normalized the length of each gene region (from the ti to ttp sequences, inclusively) and intergenic region by dividing by the length of the corresponding l gene, which varied least across the data set (coefficients of variation: n = . , p = . , m = . , g = . , l = . ). as there was substantial variability in the proportion of the ' and ' utrs that were included in the sequence data set, we considered each genome to begin at the first ti sequence and end at the final ttp sequence for this analysis. to infer evolutionary relationships among animal rhabdoviruses, we compiled sequences of the l (rna-dependent rna polymerase) protein, as this was the most highly conserved protein across the data set. we initially attempted to root the tree using a standard outgroup method. members of the rhabdovirus genera that infect plants (i.e., cytorhabdovirus and nucleorhabdovirus) were excluded as their sequences were highly divergent. we therefore utilized four members of the genus novirhabdovirus (infectious haematopoietic necrosis virus adb ; viral hemorrhagic septicaemia virus bah ; hirame rhabdovirus aco ; and snakehead rhabdovirus np ) as outgroups. unfortunately, these novirhabdovirus sequences were also far too divergent (>> amino acid change per site under multiple amino acid substitution models; results available on request) to establish a reliable rooting for our data set, as three different basal groups were identified using different models of amino acid substitution, although overall tree topologies were similar among substitution models (results available on request). in addition, the use of the novirhabdoviruses as outgroups resulted in excessive numbers of residues being removed following gblocks pruning (see below). based on the observation that most known rhabdoviruses are either insect viruses or replicate in insect vectors, it has been reasonably argued that plant and animal rhabdoviruses may have origins in insects [ ] . we therefore selected the rooting scheme that best fit this theory. to this end, we choose one of the two basal clades from the novirhabdovirus-rooted tree, comprising viruses isolated from mosquitoes (i.e., the almendraviruses), as the most divergent group. we then repeated the phylogenetic analysis (procedure described below) excluding the novirhabdoviruses and rooting it on the almendraviruses. importantly, the choice of outgroup did not influence relationships either between or within the major clades demonstrating strong bootstrap support (bsp ). the alignment used for the final tree inference (i.e., excluding the novirhabdoviruses) was comprised of amino acid sequences aligned using the muscle program [ ] , with ambiguously aligned regions removed using the gblocks program with default parameters [ ] . this resulted in a final sequence alignment of taxa, amino acid residues in length. the phylogenetic relationships among these sequences were determined using the maximum likelihood (ml) method available in phyml . [ ] employing the wag+g model of amino acid substitution and subtree pruning and regrafting (spr) branch-swapping. the phylogenetic robustness of each node was determined using , bootstrap replicates and nearest-neighbour branch-swapping. fig. (a-d) . amino acid sequence alignments of small accessory proteins encoded in the genomes of ephemeroviruses. (pdf) s fig. (a-d) . amino acid sequence alignments of the u , u x proteins, u x and u x proteins of the curioviruses, and of the rbuv u protein with the itav u protein. (pdf) s fig. (a, b) . amino acid sequence alignments of the u and u proteins of tibroviruses. (pdf) s fig. (a-e) . analysis of the potential ribosomal frame-shift sites in the sequence overlap regions of curioviruses and some hapaviruses. (pdf) s table. rhabdoviruses for which genome sequences have been used in this study. the evolution and emergence of rna viruses virus taxonomy. classification and nomenclature of viruses. ninth report of the international committee on taxonomy of viruses the footprint of genome architecture in the largest genome expansion in rna viruses the evolution of genome compression and genomic novelty in rna viruses lack of evidence for proofreading mechanisms associated with an rna virus polymerase rates of spontaneous mutation error thresholds and the constraints to rna virus evolution selforganization of matter and the evolution of biological macromolecules why genes overlap in viruses pacing a small cage: mutation and rna viruses discovery of the first insect nidovirus, a missing evolutionary link in the emergence of the largest rna virus genomes unique and conserved features of genome and proteome of sars-coronavirus, an early split-off from the coronavirus group lineage nidovirales: evolving the largest rna virus genome the rhabdoviruses: biodiversity, phylogenetics, and evolution rhabdovirus accessory genes global and regional mortality from causes of death for age groups in and : a systematic analysis for the global burden of disease study virus taxonomy, ninth report of the international committee on taxonomy of viruses adelaide river rhabdovirus expresses consecutive glycoprotein genes as polycistronic mrnas: new evidence of gene duplication as an evolutionary process gene duplication and phylogeography of north american members of the hart park serogroup of avian rhabdoviruses gene duplication is infrequent in the recent evolutionary history of rna viruses the genome of bovine ephemeral fever rhabdovirus contains two related glycoprotein genes genome organization and transcription strategy in the complex gns-l intergenic region of bovine ephemeral fever rhabdovirus a small highly basic protein is encoded in overlapping frame within the p gene of vesicular stomatitis virus identification of a set of proteins (c' and c) encoded by the bicistronic p gene of the indiana serotype of vesicular stomatitis virus and analysis of their effect on transcription by the viral rna polymerase sunguru virus: a novel virus in the family rhabdoviridae isolated from a chicken in north-western uganda enhanced arbovirus surveillance with deep sequencing: identification of novel rhabdoviruses and bunyaviruses in australian mosquitoes characterization of two new rhabdoviruses isolated from midges (culicoides spp) in the brazilian amazon: proposed members of a new genus viroporins: structure and biological functions arboretum and puerto almendras viruses: two novel rhabdoviruses isolated from mosquitoes in peru kotonkan and obodhiang viruses: african ephemeroviruses with large and complex genomes malakal virus from africa and kimberley virus from australia are geographic variants of a widely distributed ephemerovirus complex genome organization in the gns-l intergenic region of adelaide river rhabdovirus tibrogargan and coastal plains rhabdoviruses: genomic characterisation, evolution of novel genes and seroprevalence in australian livestock genomic characterisation of wongabel virus reveals novel genes within the rhabdoviridae ngaingan virus, a macropod-associated rhabdovirus, contains a second glycoprotein gene and seven novel open reading frames bovine ephemeral fever rhabdovirus α protein has viroporin-like properties and binds importin β and importin niakha virus: a novel member of the family rhabdoviridae isolated from phlebotomine sandflies in senegal characterization of the tupaia rhabdovirus genome reveals a long open reading frame overlapping with p and a novel gene encoding a small hydrophobic protein genetic characterization of k , a strain of oak vale virus from western australia characterization of durham virus, a novel rhabdovirus that encodes both a c and sh protein non-canonical translation in rna viruses translation initiation at alternate in-frame aug codons in the rabies virus phosphoprotein mrna is mediated by a ribosomal leaky scanning mechanism identification of two additional translation products from the matrix (m) gene that contribute to vesicular stomatitis virus cytopathology accessory genes of the paramyxoviridae, a large family of nonsegmented negative-strand rna viruses, as a focus of active investigation by reverse genetics internal initiation of translation on the vesicular stomatitis virus phosphoprotein mrna yields a second protein the where, what and how of ribosomal frameshifting in retroviral protein synthesis phylogenetic relationships among rhabdoviruses inferred using the l polymerase gene phylogenetic relationships of seven previously unclassified viruses within the family rhabdoviridae using partial nucleoprotein gene sequences wongabel rhabdovirus accessory protein u targets the swi/snf chromatin remodelling complex overlapping genes produce proteins with unusual sequence properties and offer insight into de novo protein creation orphans as taxonomically restricted and ecologically important genes finding families for genomic orfans genetically modified vsv(nj) vector is capable of accommodating a large foreign gene insert and allows high level gene expression expression of human immunodeficiency virus type gag protein precursor and envelope proteins from a vesicular stomatitis virus recombinant: highlevel production of virus-like particles containing hiv envelope extremely high mutation rate of a hammerhead viroid the effect of gene overlapping on the rate of rna virus evolution rabies virus glycoprotein gene contains a long ' noncoding region which lacks pseudogene properties infection characteristics of rabies virus variants with deletion or insertion in the pseudogene sequence identification of viral genomic elements responsible for rabies virus neuroinvasiveness level of gene expression is a major determinant of protein evolution in the viral order mononegavirales adding genes to the rna genome of vesicular stomatitis virus: positional effects on stability of expression the transcription complex of vesicular stomatitis virus transcription and replication of nonsegmented negative-strand rna viruses origins of genes: "big bang" or continuous creation? complete genomes of aravan, khujand, irkut and west caucasian bat viruses, with special attention to the polymerase gene and non-coding regions polymerase errors accumulating during natural evolution of the glycoprotein gene of vesicular stomatitis virus indiana serotype isolates full-length genome analysis of natural isolates of vesicular stomatitis virus (indiana serotype) from north, central and south america phylogenetic analysis reveals a low rate of homologous recombination in negative-sense rna viruses defective interfering viruses the origins of defective interfering particles of the negative-strand rna viruses origin and replication of defective interfering particles beilong virus, a novel paramyxovirus with the largest genome of non-segmented negative-stranded rna viruses partitioning the genetic diversity of a virus family: approach and evaluation through a case study of picornaviruses toward genetics-based virus taxonomy: comparative analysis of a genetics-based classification and the taxonomy of picornaviruses genetics-based classification of filoviruses calls for expanded sampling of genomic sequences phylogeny of titmice (paridae): ii. species relationships based on sequences of the mitochondrial cytochrome-b gene trimmomatic: a flexible trimmer for illumina sequence data abyss: a parallel assembler for short read sequence data fast gapped-read alignment with bowtie integrative genomics viewer hhblits: lightning-fast iterative protein sequence searching by hmm-hmm alignment plant and animal rhabdovirus host range: a bug's view muscle: a multiple sequence alignment method with reduced time and space complexity improvement of phylogenies after removing divergent and ambiguously aligned blocks from protein sequence alignments new algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of phyml . key: cord- -h zpjab authors: mcguckin wuertz, kathryn; treuting, piper m.; hemann, emily a.; esser-nobis, katharina; snyder, annelise g.; graham, jessica b.; daniels, brian p.; wilkins, courtney; snyder, jessica m.; voss, kathleen m.; oberst, andrew; lund, jennifer; gale, michael title: sting is required for host defense against neuropathological west nile virus infection date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: h zpjab west nile virus (wnv), an emerging and re-emerging rna virus, is the leading source of arboviral encephalitic morbidity and mortality in the united states. wnv infections are acutely controlled by innate immunity in peripheral tissues outside of the central nervous system (cns) but wnv can evade the actions of interferon (ifn) to facilitate cns invasion, causing encephalitis, encephalomyelitis, and death. recent studies indicate that stimulator of interferon gene (sting), canonically known for initiating a type i ifn production and innate immune response to cytosolic dna, is required for host defense against neurotropic rna viruses. we evaluated the role of sting in host defense to control wnv infection and pathology in a murine model of infection. when challenged with wnv, sting knock out (-/-) mice displayed increased morbidity and mortality compared to wild type (wt) mice. virologic analysis and assessment of sting activation revealed that sting signaling was not required for control of wnv in the spleen nor was wnv sufficient to mediate canonical sting activation in vitro. however, sting-/- mice exhibited a clear trend of increased viral load and virus dissemination in the cns. we found that sting-/- mice exhibited increased and prolonged neurological signs compared to wt mice. pathological examination revealed increased lesions, mononuclear cellular infiltration and neuronal death in the cns of sting-/- mice, with sustained pathology after viral clearance. we found that sting was required in bone marrow derived macrophages for early control of wnv replication and innate immune activation. in vivo, sting-/- mice developed an aberrant t cell response in both the spleen and brain during wnv infection that linked with increased and sustained cns pathology compared to wt mice. our findings demonstrate that sting plays a critical role in immune programming for the control of neurotropic wnv infection and cns disease. introduction through subcutaneous inoculation from the bite of an infected mosquito. a parallel form of infection using sub-cutaneous challenge of wnv in a mouse model has been shown to replicate the progression, tissue involvement, and pathology of wnv infection that occurs in humans [ ] [ ] [ ] [ ] . in the mouse model, viral replication occurs at the subcutaneous site of entry followed by infection of the draining lymph node and splenic infection [ ] . these processes first trigger innate immune activation in peripheral tissues outside of the central nervous system (cns) through viral recognition by the rig-i-like receptors to induce irf activation and the production of types i and iii interferon (ifn) [ ] [ ] [ ] [ ] . innate (rlr) immune defenses triggered by rlr signaling and ifn actions serve to restrict the tissue tropism of wnv and are essential for protection against neuroinvasion [ , , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . type i and iii ifn are essential to inform the innate and adaptive immune interface to balance development of effective immunity, protect the blood-brain barrier, and limit immune-related pathology in the cns [ , , , [ ] [ ] [ ] [ ] [ ] . in particular, type i ifn-dependent cytokine and chemokine signaling cascades are essential for functional development of the cytotoxic cd + t cell response, as well as its regulatory t cell (tregs; foxp + cd + t cells) counterpart [ , , , [ ] [ ] [ ] [ ] . while cd + t cells are required for controlling both peripheral and cns viral load, cd + t cells, specifically tregs, are essential for preventing symptomatic disease in the cns [ ] [ ] [ ] [ ] . the adaptor protein, stimulator of interferon genes (sting), has also been implicated in host defense against wnv [ ] [ ] [ ] . sting was first described as an essential defense mechanism against both rna and dna viruses [ , ] . since then, sting has been recognized for its role in responding to cytoplasmic dna and mediating subsequent innate immune activation and ifn production. however its role in the defense against rna viruses is poorly understood [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . intriguingly, multiple rna viruses, including dengue virus, yellow fever virus, hepatitis c virus and coronaviruses, direct viral evasion strategies to disrupt the sting signaling pathway, reflecting a likely role for sting in host defense against rna viruses [ ] . sting was found to be required for host defense during infection with influenza a virus, as well as dengue virus, a closely related flavivirus to wnv [ ] [ ] [ ] . additionally, during infection with related flavivuses including japanese encephalitis virus (jev) and zika virus, sting deficiency led to increased neuropathology in vivo and in vitro, suggesting a critical role for sting in cns defense [ , ] . the role for sting in the cns has been implicated in multiple other neurodegenerative diseases including aicardi-goutières syndrome, sterile immune mediated cns pathology and during chronic cns diseases [ , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] . in this study, we investigated the hypothesis that sting plays a regulatory role in the immune response against wnv, thereby restricting viral neurotropism and neuropathology. we show that sting is essential for host defense against wnv in a mouse in vivo model of infection. clinical and pathological analyses demonstrate a novel role for sting in conferring cns defense against wnv in vivo. we found that tonic levels of type i ifn were decreased in sting-/-bone marrow derived macrophages (bmdm) and linked with increased susceptibility to wnv infection. following infection, we observed heightened immune responses in vitro and in vivo concomitant with increased viral load. sting deficiency led to the development of an aberrant adaptive immune response, with decreased activation of cd + cells and t regulatory cells (tregs) in the spleen, and decreased cd + t cell numbers resulting in an altered cd /cd t cell ratio in the cns coupled with cns disease. our observations imply an essential role for sting within the interface between the innate and adaptive immune responses for effective immune programming in the control of wnv infection and cns disease. previous studies demonstrated that mice defective in sting signaling experienced increased mortality during wnv infection, yet the linkage of sting to immune response programming for defense against wnv has not been defined [ ] . using genetically knocked-out tmem (sting-/-) mice [ ] , we first performed a survival analysis to confirm the role of sting in host survival during wnv infection (fig a) . c b/ j (b , wt) and sting-/-mice were infected through subcutaneous virus challenge via foot-pad injection and monitored for days post infection (dpi). mice were scored daily for morbidity, marked as loss in body weight ( fig b) and overall increased clinical score (fig c) . consistently, between - dpi, mice either met euthanasia criteria (terminal; t) or went on to survive (survivors; s) through dpi (study end-point) (fig d) . using this model, we confirmed the occurrence of increased susceptibility to wnv infection in the complete absence of sting (figs a and s a), similar to what was previously described in sting gt/gt mice [ ] . we also observed significantly increased clinical severity scores in the sting-/-mice that persisted until the study-endpoint, when wt mice had returned to a base-line clinical score (fig b and c) . additionally, we monitored mice daily for the duration of the experiment until they either met euthanasia criteria or at the study end-point, day post infection. results from each mouse were analyzed to determine if there were differences in clinical signs between wt and sting-/-mice. notably, sting-/-mice displayed increased neurological signs of disease, characterized by loss of balance, reduced muscle tone and reflexes predominantly in the pelvic limbs and increased paresis and paralysis, implicating more severe damage to the hind-brain and spinal cord (fig e and f ). in order to determine if there was a survivor bias in the clinical data, we retrospectively stratified the data into cohorts of mice that met euthanasia criteria (terminal; t) or ones that survived until day post-infection (survivors; s), the pre-determined study end-point ( fig d, g and h ). by doing so, we found that significant differences in body weight loss and clinical scores between wt and sting-/-mice were only observed in the survivor cohort and not in the terminal cohort. while there is an essential role for sting in host survival during acute infection (figs a and s a), these data implicate an additional prolonged requirement for sting in both prevention and recovery from neurological pathology. when we examined cns pathology, we found that in both wt and sting-/-mice, pathological scores were significantly increased in the spines of the survivor cohort, with a trend toward increased scores in the brains and spines of the terminal cohort ( fig d, i and j ). intriguingly, while sting-/-terminal mice displayed increased cns pathology, wt mice that met terminal criteria had unexpectedly low clinical scores, suggesting that they met euthanasia criteria for reasons independent of severe encephalitis. during necropsy, we observed that the gastrointestinal (gi) tract of terminal mice exhibited gross distension or other aberrant phenotypes including stool compaction, disintegration and in some cases severe reduction in size or collapse of the gi tract (s b fig) . pathologic analysis confirmed that terminal mice display increased gi pathology that included microbiome overgrowth and neuronal degeneration and loss in the myenteric ganglia, particularly in sting-/-(s c and s d fig) . previous studies have indicated that gi manifestations during wnv infections exist in both mice and humans, and are positively correlated to increased neurotropism and mortality [ ] [ ] [ ] ] . this outcome may imply that wt mice are meeting euthanasia criteria following wnv infection due to severe gi disease rather than severe cns involvement as previously thought. further, these results demonstrate that sting plays a systemic role in host defense against wnv, with increased frequency of mortality and pathology occurring in the cns and gi tract in sting-/ mice. together, these results show an essential role for sting in host survival and neuropathological defense in the cns during wnv infection. to determine if sting is required for viral control in the cns, we challenged mice with wnv via footpad injection and examined tissue viral load at dpi (peak of peripheral viremia) and dpi (peak of detectible virus in the cns) (fig a) . viral titer of macrodissected brains and extracted spinal cords were examined by plaque assay individually for each mouse in the cohort (fig a) . as expected, virus was not detected at dpi in the cns but by dpi virus was clearly detected in different cns regions. virus was not consistently found in the cns of all mice nor in every tissue examined. there was however, a consistent trend toward increased numbers of infected mice with detectible virus in the cns as well as increased viral titers in the cns of sting-/-mice compared to wt. to determine if there was detectible virus in the brains of terminal vs survivor mice, tissues from retrospectively sorted mice utilized for pathological analysis (fig i and j) were immunostained for the presence of wnv antigen ( fig b) . wnv foci were found in the brains of wt and sting-/-terminal mice but were not apparent in wt or sting-/-survivors, suggesting that either the virus had cleared or that surviving mice did not have cns infection. neuronal death was assessed by tunel stain in both wt and sting-/ survivors. here we observed enhanced neuronal apoptotic death in the sting-/-cohort, suggesting sting may have a direct or indirect role in neuronal defense in the cns (fig c) . in order to determine if sting is required for neuronal defense against wnv, primary cortical neurons were isolated and cultured, followed by infection with wnv to determine viral growth kinetics under conditions of single and multi-step growth ( fig d) . surprisingly, no difference was detected between in wnv replication in wt and sting-/primary cortical neurons (fig d) . to determine if the actions of sting might be restricted to the cns for wnv protection, we performed an intracranial virus inoculation bypassing the role of the peripheral immune response and physical barriers such as the blood-brain barrier to directly infect the brain with wnv (fig e) . at dpi, there was no difference in cns viral load found in wt vs sting-/-mice nor was viral load different between sting-/-and wt mice. taken together, our observations imply that the role of sting is not limited to mediating viral control in the cns. it is possible that sting is therefore required in the development of a protective immune response in the periphery such that in the absence of sting the immune response is aberrantly programmed, leading to cns immunopathology. given that sting deficiency was associated with enhanced mortality (see fig ) without a significant increase in cns viral burden (fig ) , we considered that sting deficiency could result in defective antiviral innate immune signaling and lead to loss of viral control in the periphery, thereby leading to enhanced morbidity and mortality. we first tested the role of sting in bmdms, as macrophages are a tropic cell and key modulator of peripheral viral control during wnv infection ( fig a) [ ] . as expected, wnv levels were significantly increased by and hours post inoculation (hpi). unexpectedly however, sting-/-bmdm had increased innate immune and inflammatory gene expression, including enhanced level of type i ifn expression during wnv infection ( fig b) . we then examined the spleens of infected mice to determine if there was an overall loss of viral control manifested as increased viral load over wt. as expected, virus was detected at dpi in both wt and sting-/-. surprisingly however, there was no difference in dpi viral titers between wt and sting-/-, nor was there a sustained virologic response in sting-/-mice ( fig c) . these data indicate that peripheral loss of viral control does not occur in the absence of sting (fig c) . similarly, viral rna was detected equally in spleens of infected wt and sting-/-mice at dpi, but the virus was largely cleared from the spleen by dpi (fig d) . in the cns however, we observed a trend toward increased viral rna and innate immune gene expression at dpi in wnv-infected sting-/-mice, similar to that observed in bmdm (fig a and d) . these data were unexpected as we initially predicted that sting deficiency would reduce innate immune activation based on the known role of sting signaling in ifn induction. these data demonstrate that innate immune activation and the inflammatory response are exacerbated in both in vitro and in vivo sting deficient models, possibly culminating in enhanced immunopathology in sting-/-mice. the canonical sting sensing pathway is dependent on upstream recognition of dna dangeror pathogen-associated molecular patterns (damp, pamp) such as dna viruses, cell-free or mitochondrial dna, by cyclic gmp-amp synthase (cgas). in mammals, cgas binding to dsdna activates its synthase activity to produce a cyclic di-nucleotide, cgamp (cyclic guanosine monophosphate-adenosine monophosphate), which binds to sting, initiating downstream activation of sting by phosphorylation, sting relocalization from diffuse cytosolic to punctate pattern, and subsequent induction of innate immune signaling and ifn production [ , , , , ] . during rna virus infections however, the role for sting defense has not been well-characterized. to evaluate the activation of sting during wnv infection, we utilized a recently described telomerase reverse transcriptase human foreskin fibroblasts (hff) model to assess activation of endogenous sting by phosphorylation and relocalization from the cytoplasm to the perinuclear space during wnv infection [ ] . transfection of interferon-stimulated dna (isd; calf-thymus dna) into hffs initiated re-localization of sting as previously reported by hpi [ , ] . intriguingly however, sting was not relocalized in wnv infected cells (fig a) . it is possible that the kinetics of sting activation are different from isd activation of sting as compared to wnv infection, so we performed a time course experiment to detect sting activation by phosphorylation status [ ] , assessing a range of - hpi at moi = ( fig b) . similar to what was observed by ifa, sting phosphorylation was not observed at any time point during wnv infection, although phosphorylated stat and wnv protein was detected at hpi, suggesting virus replication and innate immune signaling were occurring normally ( fig b) . to determine if activation was dependent on viral load, we infected hff with a moi = and moi = of wnv, but also observed no sting activation as measured by phosphorylation ( fig c) . these data suggest that sting is not canonically activated during wnv infection in hff cultures and reveals a potential noncanonical role for sting in host defense during infection with wnv. in order to determine if there was a systemic change in the innate immune profile in sting-/-mice, we examined the cytokine and chemokine profile in the serum of wt and sting-/-mice at the peak of peripheral viremia ( dpi) and cns viral burden ( dpi). we found that mock infected sting-/-mice had an increased basal production of multiple cytokines and chemokines at dpi. we also observed significant increases in il , il , il , il , mcsf, gro-alpha, while at dpi ip- (cxcl ) was decreased in sting-/-compared to wt mice (s fig). while these cytokines have multiple roles in immune modulation, a common role among them is in activation and recruitment of t cells. these data suggest that sting is required for regulation of immune cytokine and chemokines that program immune cell trafficking and actions during wnv, as has been shown for sting in cancer immunity and autoimmune signaling [ ] . to determine if sting is required for proper programming of the t cell response during wnv infection, we examined splenic t cells from wt and sting-/-mice at dpi, a time point when the adaptive immune response is established in wt mice [ ] . we observed a reduction in the frequency of cd + t cells, along with a trend toward decreased numbers of t cells in the spleens of sting-/-mice compared to wt during wnv infection (fig b) . additionally, within the cd + t cell subset (fig c) , there was a significant decrease in frequency of activated (cd +) and cxcr + t cells, and we observed a consistent trend of decrease in the frequency of wnv-specific cd + t cells in the spleens of sting-/-mice compared to wt, suggesting that sting is required for optimal anti-wnv cd + t cell responses. we also observed a significant increase in the frequency of cd + t cells in sting-/-mice (fig b) , with a corresponding trend toward increased absolute cell numbers. while we observed a trend toward differences in the absolute number of most cell populations examined between wt and sting-/-mice, we found that significant differences most typically occurred in cell frequencies, suggesting that the balance of t cells subsets may be skewed in the absence of sting. in particular, we found skewing within the t regulatory cell (foxp +) populations ( fig e- g) , with significant deficits in ki +, cd + and cd + tregs, cd and cd + tregs. these data suggest that sting is required for modulating t cell responses and t cell frequencies during wnv infection that lead to a protective rather than pathogenic outcome. because of the heightened innate immune profile and aberrant programming of the t cell responses in spleens of sting-/-mice, we examined the cns-specific t cell profile across mouse lines. histological analyses revealed trends toward increases in cns immune cellularity, both in the form of perivascular and parenchymal mononuclear infiltrate, suggesting the cns pathology may be immune-mediated (fig a) . we then performed a cd ihc stain in the brains of survivors, we found increased clusters of cd infiltrate in the hind and midbrain regions (fig b) co-localized with robust lesions. in serial slices of the same tissues, we did not observe wnv staining by ihc in sting-/-survivors (fig ) , however we did observe continued gliosis, suggesting that a potential immunopathology may occur in the brain of sting-/-mice infected with wnv. previous studies indicated that cellular infiltrate in the brain is predominantly comprised of cd + t cells during wnv infection [ ] . therefore, we characterized t cell responses of wt and sting-/-mice in the cns on dpi to examine baseline differences at dpi when wnv and leukocytes are both present in the cns (fig j) . lymphocyte and t cell responses in both mock and wnv-infected mice were comparable at dpi, indicating that there was no gross difference in the cns between wt and sting-/-mice ( fig c and d ). by dpi however, we found statistically significant decreases in the frequency and numbers of cd + t cells in sting -/-mice ( fig f) . although there was no difference in the total numbers of cd + t cells, there was a statistically significant increase in the frequency of cd + t cells in the cns of sting-/-mice, likely due to overall trend of decreased numbers of lymphocytes in the brain (fig c- e ). by dpi, these changes resulted in a significantly decreased cd /cd ratio of t cells, indicating an imbalanced t cell response to wnv in the cns of sting-/-mice ( fig i) . of cells that made it to the brain by dpi, no differences were found in the absolute number of activated (cd +) or wnv-specific (ns b tetramer+) cd + t cells (fig g and h ), foxp +cd +cd + t cells (fig k) in the brain. these data suggest that sting is not essential for recruitment of wnv-specific cytotoxic t cells in the cns, however it may be required for balancing the cytotoxic vs immunosuppressive adaptive response. furthermore, it is also possible that the enhanced recruitment of cells to the cns is in response to damage caused by the virus, aberrant immune signaling, or both. this outcome would suggest that sting plays an essential role in modulating the balance between immunopathogenic and immunoprotective response in the cns during wnv infection. the increase in clinical disease and pathological damage observed in the sting-/-versus wt mice, particularly in survivors, could be due to an aberrant immune response resulting in cns damage after initial viral insult. we found that cns pathology in wt mice is largely restricted to the cortex and meninges, while sting-/-mice display increased pathology in the cerebellum and hind/mid brain regions in addition to the cortex and meninges (fig a and c) . these data correlate with the increased cd staining observed by ihc in sting-/-mice (fig b) , also noted as the same brain regions where wnv is often detected by ihc (fig b) . these observations suggest that sting plays a role in directing or maintaining the t cell response to specific loci within the cns or that initial viral infection led to increased sting is required for host defense against neuropathological west nile virus infection recruitment of a localized adaptive immune response that resulted in immunopathology. furthermore, pathology in the spine was more diffuse, suggesting that sting has a widespread protective role in the cns during wnv infection (fig b and d) . these observations led us to investigate if there was a localized polarization of microglia or infiltrating macrophages in cns regions toward an m or m phenotype (fig e) . microglia have the highest levels of sting (tmem ) expression observed in any cell within the adult mouse [ , ] and it is possible that in the absence of sting, microglia are aberrantly polarized, enhancing immunemediated pathology. to examine this possibility, we assessed the expression of m (cxc and il ) and m (pparg, arg , chil and retnla ) associated genes by rt-qpcr in different regions of the cns. in wt mice, we found that cxcl (marking an m phenotype) was present in the brain stem by day post infection, and retnla expression (marking an m phenotype) occurred in both the mock and dpi tissues within the brain stem and sub-cortex (containing the thalamus) regions of the brain (fig e) . this profile suggests that cns homeostasis includes a localized m phenotype that is induced to a m phenotype in wt mice following wnv infection. in sting-/-mice however, we found a widespread increase in the m response gene expression (marked by cxcl and il ) with the highest expression observed in the brain stem and spinal cord. simultaneously, there was also a corresponding increase in pparg and chil (marking the m phenotype), with no clear difference in arg expression and an overall trend toward decreased expression of retnla. these observations reveal a widespread increase in both m associated genes, with altered regulation of the m associated genes in sting-/-mice, potentially resulting in aberrant balance of the m and m polarization in the cns. to determine where in the cns sting is actually localized and if this tissue localization overlaps with the location of the cellular infiltrate noted histopathologically or with expression of innate immune genes, we utilized the allen brain institute database to search for sting (tmem ) localization in the mouse brain [ ] . within the brain, sting expression is found within the olfactory bulb, thalamus/midbrain, brainstem and cerebellum, as well as low levels throughout the cortex, overlapping areas that are affected most severely by wnv infection (s ) [ , ] . these regions of brain affected correlate with the clinical signs we observed including loss of balance, tremors, and loss of motor function (figs e and c- e). furthermore, these areas of sting expression overlap with the brain regions where altered regulation of m or m gene expression were most readily observed, implicating a role for sting in polarization of either or both microglia and macrophages in the cns. cumulatively, these data suggest that sting has an essential role in maintaining immune response homeostasis and immune programming in initial defense against wnv infection. without sting, immunopathology occurs, leading to exacerbated cns disease and clinical sequelae. recent years have seen a marked increase in the global health threat presented by emerging and re-emerging encephalitic viruses, particularly those with increased neurotropism and neuropathology such as wnv [ , , , , ] . previous studies indicated an important role for sting in host survival during wnv infection [ ] , however it is unclear what role sting plays in conferring host defense against rna viruses [ , ] . here, we demonstrate that sting is essential to prevent host morbidity and mortality during wnv infection where it plays a role in immune homeostasis and programming. however, sting is not canonically activated in vitro upon infection with wnv, revealing a novel function for sting during infection with rna viruses. furthermore, we show that sting is essential for host neuropathological defense against wnv through regulation of the innate-adaptive immune interface in vivo. we found that sting deficient mice exhibit increased mortality and morbidity including increased and sustained neurological clinical signs, particularly in mice that survive infection (fig ) . these data were corroborated by pathological analysis, which also revealed distinct differences in cns pathology. intriguingly, there seems to be a stratification in clinical and pathological findings between the sting-/-mice that meet euthanasia criteria and those that go on to survive. survivorship bias has been previously reported in the wnv model, with these data further implicating this bias as a critical factor to consider when performing time course vs. end-point experiments [ ] . unexpectedly, these studies also revealed that there was minimal cns pathology in wt mice that met euthanasia criteria. it is typically assumed that mice meeting euthanasia criteria do so because of neuroinvasion and subsequent encephalitis. our data instead indicates that both wt and sting-/-terminal mice have severe gross gi abnormalities, with corroborating abnormalities by histopathology, which may be the proximate cause of morbundity and meeting euthanasia criteria (s ). gi complications during wnv have been previously described, however further study is necessary to understand the implications of gi pathology on wnv induced morbidity and mortality [ ] [ ] [ ] ] . recently it has been shown that during wnv infection causes delayed gi transit, dependent on infiltrating antiviral cd + t cells [ ] . furthermore, both in this model and in a lung model where sting exhibits a gain-of-function mutation, t cell-dependent chronic tissue damage occurs, supporting our findings that sting may play a broad and significant role in communicating between the innate and adaptive immune responses [ , ] . together, these data demonstrate an essential neuroprotective role for sting during wnv infection, potentially through a cellular mediated mechanism instead of the canonical interferon antiviral function typically attributed to sting. wnv typically is cleared through development of an innate immune response and effective t cell immunity [ ] . to prevent progression to neuroinvasion, both the innate and adaptive immune response are critical to control wnv viremia and prevent viral induced pathology [ - , , , ] . because the known function of sting is to initiate a type i ifn response to both pamps and damps, we anticipated that the type i ifn response would be diminished both in vivo and in vitro explaining the increased viral loads. surprisingly, we actually observed an increased inflammatory and antiviral innate immune response in sting-/-mice in the cns during wnv infection. this same increase in the cytokine-chemokine response was also observed in bmdm (fig ) and in serum of infected mice (fig ) . these outcomes were highly unexpected as the most commonly described role for sting is known as initiating a type i ifn response [ - , , ] . in particular, sting was shown previously to facilitate the actions of the elf transcription factor to promote type i ifn expression from wnv-infected cells wherein loss of sting associated with reduced ifn and isg expression ( ) . while we observed significant increases in ifn and isg expression in bmdm lacking sting, it is likely that sting imparts cell type-specific actions for regulation of innate immune signaling, similar to other pathogen recognition receptors that govern innate immune signaling against wnv, likely explaining this discrepancy between studies [ ] . it is also important to note that our studies employed sting-/-mice produced through classical gene targeting approach [ ] while the previous study used sting gt/gt mutant mice produced from n-ethyl-n-nitrosourea mutagenesis and encoding a t a point mutation of sting [ ] , highlighting that genetic differences between mouse lines might impact findings. importantly, both mouse lines exhibit increased susceptibility to lethal wnv infection, and together reveal expanded roles for sting in immune regulation during wnv infection. our data also suggest that sting has a role in controlling wnv replication and tropism, as we found increased viral loads in bmdm, as well as a trend toward increased viral load in the cns, particularly in the hindbrain regions, but not in the spleens of infected mice lacking sting (figs and ) . the trend toward increased virus in the cns of sting-/-mice could either suggest increased susceptibility of the virus in the cns, delayed clearance of the virus after entering the cns, or possibly a combination of the two. variation observed within strains could be the result of harvesting mice at set time points instead of following them until a determination if they would survive or meet euthanasia criteria, highlighting the potential import of survivorship bias within this model. it does not appear that the requirement for sting in viral control is restricted to neurons or the cns, as no difference was observed in the viral load of sting-/-primary cortical neurons or intracranial infection (fig ) . this outcome suggests that while there is a peripheral requirement for sting in conferring cns protection, it is not due to complete inhibition of viral control in the periphery. intriguingly, base-line expression of type i ifn and isgs were significantly reduced in sting-/-bmdm compared to wt, but not other inflammatory genes (fig ) . it is possible that this reduction in baseline ifn allows wnv to establish an earlier and more robust infection, that is later controlled by the rig-i dependent antiviral response [ , ] . however, we favor that sting plays a role in innate immune homeostasis, as in its absence the control of the inflammatory response is lost (figs and ) , thus leading to immune-mediated pathology. this function for sting may explain why we had a trend but not significant increase in viral load in the cns; it is possible that virus is able to establish a stronger infection in the cns earlier on but is cleared through an exacerbated innate inflammatory and antiviral response in the absence of sting. alternatively, it is possible that in the absence of sting clearance of the virus takes longer due to an ineffective immune response. following either of these events subsequent t cell recruitment is likely, but in a manner that leads to enhanced immunopathology and lack of recovery from clinical illness. in addition to its role in mounting a type i ifn response to pamps and damps, recent studies demonstrated an essential role for sting in developing antitumor t cell responses [ ] . these studies suggested that dead and dying cells are phagocytosed by dendritic cells, which requires sting to present antigen and produce a type i ifn signaling cascade that informs and develops the adaptive immune response. this outcome could also implicate a requirement for sting in microglial-dependent phagocytosis of dead and dying cells, with subsequent sting-dependent polarization and release of soluble factors that effectively recruit and maintain a protective cellular response in the cns. upon examining the cns of infected mice, particularly in sting-/-with ongoing signs, we observed increases in mononuclear cellular infiltrate, implicating possible immunopathology. previous studies have shown that there is an essential requirement for both cd + and cd +foxp + (treg) t cells to control wnv and prevent immunopathology [ , , ] . cd + t cells in particular are essential for wnv clearance, however without an adequate treg response or appropriate balance of cd + and cd + t cells an uncontrolled cytotoxic t cell response could result in immune mediated pathology. examining the programming of the adaptive immune response in spleens (fig ) we found that expression of ki , cd and cd in splenic foxp +cd + tregs were impaired, implicating a role for sting in the proliferation, activation and suppressive potential of tregs. upon examining the brains of mice at baseline ( dpi) and following infection ( dpi), we observed no differences at baseline between wt and sting-/-mice, however the total cd + t cells and the cd /cd ratio was significantly decreased in sting-/-mice, suggesting that there is a defective recruitment or maintenance of t cells in the brain (fig ) . these data in combination with enhanced cns pathology suggest that the cytotoxic effect of cd + t cells may not be controlled adequately in the absence of sting. it is also possible that increases in cellular response within the cns recruit an enhanced protective cellular response as a result of viral damage or aberrant immune signaling. consistent with this either of these options, we found that in sting-/-survivors there were large clusters of cd + cells (fig ) as well as other cellular infiltrate (fig ) in the same vicinity as we observed increased pathology and where sting is localized in the brain (fig ) . recently, a noncanonical sting-dependent signaling pathway was described where multiple cell types initiated an innate immune response following il b release in response to mitochondrial dna release in the cytoplasm [ ] . furthermore, this sting-induced response to il- b was essential for the control of dengue virus infection, a flavivirus related to wnv [ ] and that this response is linked with protection against wnv neurovirulence in vivo [ , ] . thus, it is intriguing to speculate that noncanonical sting activation in response to proinflammatory cytokine signaling serves to direct immune programming that protects against viral neuroinvasion and cns pathology during wnv infection. in summary, our study reveals that that sting is required for immune response programming to restrict wnv infection and neuropathogenesis. all animal experiments were approved by the university of washington institutional animal care and use committee (iacuc) guidelines as per protocol # - and follow the recommendations in the guide for the care and use of laboratory animals of the national institutes of health. invasive infections and manipulations were performed under anesthesia and every effort was made to limit suffering. c bl/ j (wt) and tmem -/-(sting-/-) mice were genotyped and bred under specific pathogen-free conditions in the animal facility at the university of washington. sting-/mice were gifted by the stetson lab, who generated them as previously described [ ] followed by speed congenics to bring them to a . % c bl/ j background. additional c bl/ j (wt) mice were purchased from jackson laboratories, bar harbor, me. both male and female mice, ages - weeks were represented in both the control and infected groups. mice for primary cortical neurons (wt and sting-/-) were set up as timed breeders and embryos were harvested. mice were monitored daily and assigned a clinical score to describe overall well-being and signs of hind-limb dysfunction (paresis). clinical scores (cs) of ( ) without clinical signs, or ( - ) dependent on severity of clinical signs presented. cs = : ruffled fur, lethargic; no paresis; cs = : very mild to mild paresis (in or more hind limbs with minimal gait disturbance or limb-dysfunction); cs = : frank paresis involving at least one hind limb and/or eye conjunctivitis; cs = : severe paresis and/or paresis in both hind-limbs; cs = : true paralysis; cs = : moribund. additionally, mice were observed daily for the presence or absence of various specific signs. each mouse was scored as either exhibiting the clinical sign (yes = ) or not, (no = ). each sign was monitored through the duration of the experiment and the results were graphed as the average daily score/mouse. results of clinical signs monitored represent the entire population until they reached euthanasia criteria, at which point the remaining mice continued to be scored until day post infection or study end point. clinical signs monitored daily include: lethargy (l), ruffled fur/decreased of grooming, hunched, paresis/ paralysis (any degree of severity), tremors, abdominal (ab) distension/gi distress, loss of balance, increased reflex/tone in limbs (fore and/or hind) and tail, decreased reflex/tone in limbs and tail. the clinical scoring system incorporated signs based off of predicted involvement of different anatomical regions within the cns and was created using modifications of various previously described scoring systems for experimental autoimmune encephalomyelitis [ ] [ ] [ ] [ ] . similar neuroanatomic regions were examined pathologically in an attempt to correlate clinical and neurological phenotype of disease. subcutaneously-infected mice were monitored for days post infection (dpi). euthanasia criteria was determined as a clinical score � for or more consecutive days, or % loss in body weight. a clinical score of (moribund) or respiratory distress resulted in immediate euthanasia. mice meeting euthanasia criteria were identified as terminal (t) and were euthanized by co asphyxiation followed by cervical dislocation. mice who did not meet euthanasia criteria were monitored until end point ( dpi) were identified as survivors (s). all remaining s mice were euthanized at the end of study ( dpi) as described above. mice used for morbidity and mortality analysis were necropsied when meeting euthanasia (t) criteria, or study end (s). after euthanasia by co , a complete necropsy was performed and tissues were collected and immersion fixed in % neutral buffered formalin [ ] . the head was removed and skull cap lifted, leaving the brain within the skull cavity during fixation. the spine was fixed in situ in order to preserve the mesenteric ganglia. histological preparation hematoxylin and eosin (h&e) and immunohistochemical (ihc) staining was performed by the uw histology and imaging core (hic) and the vanderbilt university medical center translational pathology shared resource (tpsr). primary pathological analysis was performed on the cns (brain and spine) and gastrointestinal (gi) tract by a board-certified veterinary pathologist (pmt) (supplemental methods table ). in the brain, the following changes were scored on a subjective - scale of increasing severity: perivascular inflammation, parenchymal inflammation, hemorrhage, neuronal necrosis, and meningitis. in the spinal cord the presence ( ) or absence ( ) of mononuclear inflammation was documented from different sections of the spine (c -c , c -t , t -l , l -s , s ) for a maximum score of per mouse. for the enteric nervous system (ens), the degree of mononuclear cells present in the myenteric ganglia, extent to the changes and any secondary gi lesions such as dilation or mucosal change were scores on a on a subjective - scale of increasing severity. ihc staining of wnv (vrl w ) and cd + t cells (mca abd serotec) were performed by the uw histology core. verowho (european collection of authenticated cell cultures; ecacc) cells were cultured in dulbecco's modified eagle medium (dmem) supplemented with % fbs, mm sodium pyruvate, mm l-glutamine, antibiotic/antimycotic solution and non-essential amino acids (complete dmem; cdmem) and split using . % trypsin following pbs wash. hff cells were kindly gifted from stetson lab and were grown in cdmem. cells were split using . % trypsin following pbs wash. bone marrow was collected from sting-/-and wt mice and frozen in % dmso/ % fbs. to generate bone marrow derived macrophages (bmdm), bone marrow stocks were thawed, washed and resuspended in cdmem containing [ μm] bme and [ ng/ml] murine mcsf (mmcsf). cells were cultured for days in non-tc coated plates, then scraped, washed with pbs and seeded at e cells/well in -well tc coated plates with cdmem+bme+mmcsf. cells were infected or transfected the next day. wnv-tx biological isolates ( ) were utilized for in vivo work, while wnv-tx ic (infectious clone) stocks were utilized for cell culture (in vitro) studies. working stocks were propagated in vero-e (american type culture collection; atcc) and titered by standard plaque assay on verowho and bhk (american type culture collection; atcc) cells as previously described [ ] . single-use aliquots from the same viral stock lot were prepared and utilized for all experiments described here. age and sex-matched - week old mice were anesthetized by isofluorane and inoculated subcutaneously (s.c.) in the right rear footpad with pfu wnv-tx (wnv-tx) diluted in ul pbs, administered via ml insulin needle. mice were monitored daily for clinical score and loss of body weight. euthanasia criteria was determined as a clinical score � for or more consecutive days, or % loss in body weight. a clinical score of (moribund) or significant respiratory distress resulted in immediate euthanasia. mice were anesthetized with ketamine/xylazine, the top of the head was cleaned with etoh, and the mouse was then restrained manually on a solid surface. the site of injection was approximately halfway between the eye and ear, and just off the midline, in the medial posterior region of the top of the skull. the injection was done with a g needle using a hamilton syringe into the cerebral cortex. following infection, mice were monitored for revival from anesthesia and monitored daily for clinical score and loss of body weight. euthanasia criteria was determined as a clinical score � for or more consecutive days, or % loss in body weight. a clinical score of (moribund) or significant respiratory distress resulted in immediate euthanasia. to determine the viral load from in vivo tissue samples, mice were terminally anesthetized using ketamine/xylazine mixture followed by cardiac perfusion with - ml pbs. kidney(s) and spleen were collected whole; brains were harvested and macrodissected into four anatomical regions, including the cerebellum, cortex, sub-cortex, and brainstem [ ] ; spinal cords were collected by perfusion with pbs. tissues were harvested into ml pbs on ice in percelly's tubes with ceramic beads. following harvest, tissues were homogenized (percellys ) / x s/ min and centrifuged at ˚c/ min/ k rpm. supernatant was collected and analyzed by plaque assay on vero-who cells ( . % agarose overlay, % neutral red counter stain after five days post inoculation; plaques counted - h post staining). cells were inoculated with wnv in serum-free media and the inoculum left for hr rocking at ˚c. inoculum was removed, cells washed x and media replaced with cdmem. at the indicated time-points, supernatant was collected for virologic and cytokine analysis; cells were treated with ripa buffer for wb analysis (or) with rlt for total cellular rna isolation. primary cerebral cortical neuron cultures were generated from e wt and sting-/embryos as previously described [ ] and maintained in serum free neurobasal-a medium (life technologies - ) with b supplement (gibco - ). neuron cultures were used for virologic experiments after days in vitro. cortical neuron cultures were infected at moi . with wnv-tx [ ] . multistep growth curve experiments were performed as described [ ] and quantified via plaque assay using bhk - cells. mice were euthanized in an isoflurane chamber followed by cardiac perfusion with - ml pbs. tissues were harvested; right kidney and spleen were collected whole; brains were harvested and macrodissected into four anatomical regions, including the cerebellum, cortex, sub-cortex, and brainstem [ ] ; spinal cords were collected via pbs perfusion. tissues were harvested into ml rnalater and stored at ˚c for a minimum of week to stabilize the rna. tissues were removed from rnalater solution and transferred to ml trireagent in percelly's tubes with ceramic beads at rt. following harvest, tissues were homogenized in a percelly's homogenizer ( / x s/ min) followed by centrifugation ( ˚c/ k rpm/ min). rna isolated with the ribopure kit from trireagent using per manufacturer's instructions. cdna was generated from ng rna using iscript kits with random primers per manufacturer's instructions. cellular and viral gene analysis was assessed by sybr green rt-qpcr using an abi viia and analyzed as the linear fold change ( ^-dct) over a housekeeping gene (gapdh) from wt mock infected sample or mouse (table ). cells were harvested in rlt and total cellular rna isolated for rt-qpcr analysis using qishredders and the quiagen rneasy kit per the manufacturer instructions. cdna was generated from ng total rna using the iscript kit per manufacturer instructions using their provided oligo(dt) and random primers. cellular and viral genes were analyzed by sybr green rt-qpcr using an abi viia . primers for bmdm experiments described above. protein extracts from cells were prepared in ripa buffer. - ng protein lysate was analyzed by - % gradient sds-polyacrylamide gel electrophoresis by immunoblotting, using % bsa blocking buffer and nitrocellulose membranes. the following antibodies were utilized: wnv ns (r&d baf ), actin (c ; emd mab ), stat (cst p), sting (cst d pzf), pstat (y ; cst d ), psting (cst d c s). e (or) x ^ hff cells were seeded onto glass coverslips in a -well plate. the following day, cells were infected with wnv at moi = or transfected with calf-thymus dna (ctdna; isd) (thermo fisher, waltham, ma, usa) at ug/ml final concentration using lipofectamine and following the manufacturer's protocol. h after wnv infection or h after ctdna transfection cells were fixed with % paraformaldehyde for min at room temperature (rt). cells were permeabilized with . % triton x- for min at rt. after blocking the cells for min with % bsa in pbs, immunofluorescent staining was performed overnight at ˚c with the following primary antibodies: rabbit-anti-sting ( : , gifted by glen barber), mouse-anti-dsrna (j , : , scicons, budapest, hungary). nuclei were counterstained with ', -diamidino- -phenylindole, dihydrochloride (dapi, thermo fisher). fluorophore coupled secondary antibodies (thermo fisher) were applied for h at rt. after washing with pbs samples were mounted onto glass slides using prolong gold (thermo fisher). images were acquired with a nikon eclipse ti confocal microscope equipped with a x oil immersion objective using the nikon confocal software. insets were captured with x enlargement of x images. images were merged and processed using the nikon confocal analysis software (nikon, melville, ny, usa). mice were euthanized by isoflurane and perfused with - ml pbs to ensure systemic removal of blood and residual intravascular leukocytes. spleens were homogenized and single cell suspensions were treated with ack lysis buffer to clear any remaining red blood cells, washed and resuspended in facs buffer ( x pbs, . % fbs). cells were plated at e cells/ well and stained for surface markers minutes on ice. cells were then fixed, permeabilized (foxp fixation/permeabilization concentrate and diluent, ebioscience) and stained intracellularly with antibodies for minutes on ice. flow cytometry was performed on a bd lsrii machine using bd facsdiva software. analysis was performed using flowjo software. the following directly conjugated antibodies were used: b -foxp , b -cxcr , g -ki , g -cta- , g -cd , g -klrg , r -ns b tet, r -cd , r -cd , uv -cd , uv -cd , v -cd , v -cd , v -cd , v -live/dead. cells were counted by hemocytometer using trypan blue exclusion. brains were harvested into rpmi and mechanically suspended using a um strainer. each brain suspension was added to hypertonic percoll to create a % percoll solution, vortexed then centrifuged at rpm for minutes at ˚c. following centrifugation, the supernatant was aspirated and cell pellet treated with ack lysis buffer to remove any residual red blood cells. cells were then washed and filtered through a um nylon mesh to remove residual debris and resuspended in facs buffer. cells were counted using beads during facs analysis. cells were plated at e cells/ well and stained for surface markers minutes on ice. cells were then fixed and extracellularly stained with antibodies for minutes on ice. flow cytometry was performed on a bd lsrii machine using bd facsdiva software. analysis was performed using flowjo software. the following directly conjugated antibodies were used for fig c- b: gross pathology scores of the gi tract from necropsied mice. mice were visually examined at necropsy and scored. scores were assigned to each mouse ranging from (normal gi tract) to (grossly distended or aberrant morphology). n = - per condition; students t-test (unpaired). p = . � . c: pathological analysis was performed on randomly selected representative mice. sections of the gi were scored including sections from: ) the duodenum and upper jejunum; ) jejunum; ) ileum; ) cecum; ) colon; ) stomach. graphed as the mean sum of all scores. n = - per condition. d: representative hematoxylin and eosin-stained small intestinal sections. mock tissues were unremarkable with readily detectable myenteric ganglia (black ovals) and normal scant intestinal contents (inserts). survivor mice have mild enteritis and minimal changes in the myenteric ganglia (ovals) with normal intestinal contents. in contrast, terminal mice have myenteric ganglia with neuritis, degeneration and neuronal loss. there is bacterial overgrowth and exudative material within the intestinal contents (inserts at lower right of each image). in the sting-/mice, there is readily observable vacuolation of the inner tunica muscularis (arrow), dilation of lacteals (asterisk) and intramucosal hemorrhage and lymphocytic and proliferative enteritis. figure ) : sting localization in the mouse brain. sting localization in the brain is centralized to the hind/mid-brain, hippocampus, primary motor-cortex and olfactory bulb in the brain. square: midbrain/thalamus region. oval: hindbrain (cerebellum and brain-stem). image is from the allen institute for brain science. emerging and reemerging neurologic infections emerging viral infections of the nervous system west nile virus: review of the literature epidemiology of neuroinvasive arboviral disease in the united states division of vector-borne diseases ncfe, zoonotic infectious diseases cdc. west nile virus and other arboviral diseases-united states estimated cumulative incidence of west nile virus infection in us adults neurologic manifestations and outcome of west nile virus infection lessons from the west nile viral encephalitis outbreak in new york city, : implications for bioterrorism preparedness the outbreak of west nile virus infection in the new york city area in emergence and re-emergence of viral diseases of the central nervous system neurocognitive and functional outcomes in persons recovering from west nile virus illness neuroinvasive disease and west nile virus infection west nile fever characteristics among viremic persons identified through blood donor screening immune surveillance of the cns following infection and injury gastrointestinal manifestations of acute west nile virus infection in humans systemic distribution of west nile virus infection: postmortem immunohistochemical study of six cases clinical characteristics and functional outcomes of west nile fever clinical spectrum of muscle weakness in human west nile virus infection west nile virus infection and immunity a mouse model of west nile virus infection a mouse model of chronic west nile virus disease evaluation of a mouse model for the west nile virus group for the purpose of determining viral pathotypes the essential, nonredundant roles of rig-i and mda in detecting and controlling west nile virus infection ips- is essential for the control of west nile virus infection and immunity interferon-lambda restricts west nile virus neuroinvasion by tightening the blood-brain barrier cell-specific irf- responses protect against west nile virus infection by interferon-dependent and -independent mechanisms immune responses to west nile virus infection in the central nervous system the innate immune playbook for restricting west nile virus infection a systems biology approach reveals that tissue tropism to west nile virus is regulated by antiviral genes and innate immune cellular processes nile virus evades activation of interferon regulatory factor through rig-i-dependent and -independent pathways without antagonizing host defense signaling establishment and maintenance of the innate antiviral response to west nile virus involves both rig-i and mda signaling through ips- resistance to alpha/beta interferon is a determinant of west nile virus replication fitness and virulence alpha/beta interferon protects against lethal west nile virus infection by restricting cellular tropism and enhancing neuronal survival distinct rig-i and mda signaling by rna viruses in innate immunity irf- , irf- , and irf- coordinately regulate the type i ifn response in myeloid dendritic cells downstream of mavs signaling pattern recognition receptor mda modulates cd + t cell-dependent clearance of west nile virus from the central nervous system the rig-i-like receptor lgp controls cd (+) t cell survival and fitness deficient ifn signaling by myeloid cells leads to mavs-dependent virus-induced sepsis a temporal role of type i interferon signaling in cd + t cell maturation during acute west nile virus infection role of cd + t cells in control of west nile virus infection cd + t-cell responses are required for clearance of west nile virus from the central nervous system cd + t cells mediate recovery and immunopathology in west nile virus encephalitis tregs control the development of symptomatic west nile virus infection in humans and mice pan-viral specificity of ifn-induced genes reveals new roles for cgas in innate immunity panviral specificity of ifn-induced genes reveals new roles for cgas in innate immunity elf is critical for induction of type i interferon and the host antiviral response sting is an endoplasmic reticulum adaptor that facilitates innate immune signalling sting regulates intracellular dna-mediated, type i interferon-dependent innate immunity recognition of cytosolic dna by cgas and other sting-dependent sensors sting and the innate immune response to nucleic acids in the cytosol the cgas-sting defense pathway and its counteraction by viruses message in a bottle: lessons learned from antagonism of sting signalling during rna virus infection sting: infection, inflammation and cancer cgas and sting: at the intersection of dna and rna virus-sensing networks virus-cell fusion as a trigger of innate immunity dependent on the adaptor sting influenza a virus targets a cgas-independent sting pathway that controls enveloped rna viruses denv inhibits type i ifn production in infected cells by cleaving human sting sting mediates neuronal innate immune response following japanese encephalitis virus infection zika virus infection activates sting-dependent antiviral autophagy in the drosophila brain circulating nucleic acid analysis: diagnostic applications for acute pathologies activation and regulation of cellular inflammasomes: gaps in our knowledge for central nervous system injury the concentration of cell-free dna in focal epilepsy the value of serial plasma nuclear and mitochondrial dna levels in patients with acute ischemic stroke systemic challenge with the tlr agonist poly i:c induces amplified ifnalpha/beta and il- beta responses in the diseased brain and exacerbates chronic neurodegeneration sting-mediated type-i interferons contribute to the neuroinflammatory process and detrimental effects following traumatic brain injury self-dna, sting-dependent signaling and the origins of autoinflammatory disease autoimmunity initiates in nonhematopoietic cells and progresses via lymphocytes in an interferon-dependent autoimmune disease cyclic gmp-amp synthase is a cytosolic dna sensor that activates the type i interferon pathway cyclic gmp-amp is an endogenous second messenger in innate immune signaling by cytosolic dna interleukin- β induces mitochondrial dna release to activate innate immune signaling via cgas-sting phosphorylation of innate immune adaptor proteins mavs, sting, and trif induces irf activation the neuropathology of west nile virus meningoencephalitis. a report of two cases and review of the literature tmem (sting) cellular expression single-cell transcriptomics of mouse organs creates a tabula muris allen mouse brain atlas: allen brain institute zika virus as an emerging global pathogen: neurological complications of zika virus epidemiology and neurological complications of infection by the zika virus: a new emerging neurotropic virus end-point disease investigation for virus strains of intermediate virulence as illustrated by flavivirus infections oral antibiotic treatment of mice exacerbates the disease severity of multiple flavivirus infections sting-associated lung disease in mice relies on t cells but not type i interferon rig-i like receptors and their signaling crosstalk in the regulation of antiviral immunity il- beta signaling promotes cns-intrinsic immune control of west nile virus infection the n-ethyl-n-nitrosourea-induced goldenticket mouse mutant reveals an essential function of sting in the in vivo interferon response to listeria monocytogenes and cyclic dinucleotides dopamine: a parallel pathway for the modulation of spinal locomotor networks a neuropathological analysis of experimental autoimmune encephalomyelitis with predominant brain stem and cerebellar involvement and differences between active and passive induction b cells promote induction of experimental autoimmune encephalomyelitis by facilitating reactivation of t cells in the central nervous system the more the merrier? scoring, statistics and animal welfare in experimental autoimmune encephalomyelitis experimental autoimmune encephalomyelitis in the mouse mouse necropsy ripk restricts viral pathogenesis via cell death-independent neuroinflammation neuronal cxcl directs cd + t-cell recruitment and control of west nile virus encephalitis propagation, quantification, detection, and storage of west nile virus we thank daniel stetson, lauren aarreberg, sunil thomas, aimee sekine and megan de la riva (university of washington) for critical discussion, technical assistance and reagents. we thank glen barber (university of miami) for providing sting antibody for immunostaining. key: cord- - r q authors: dzimianski, john v.; beldon, brianna s.; daczkowski, courtney m.; goodwin, octavia y.; scholte, florine e. m.; bergeron, Éric; pegan, scott d. title: probing the impact of nairovirus genomic diversity on viral ovarian tumor domain protease (votu) structure and deubiquitinase activity date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: r q post-translational modification of host and viral proteins by ubiquitin (ub) and ub-like proteins, such as interferon stimulated gene product (isg ), plays a key role in response to infection. viruses have been increasingly identified that contain proteases possessing deubiquitinase (dub) and/or deisgylase functions. this includes viruses in the nairoviridae family that encode a viral homologue of the ovarian tumor protease (votu). votu activity was recently demonstrated to be critical for replication of the often-fatal crimean-congo hemorrhagic fever virus, with dub activity suppressing the type i interferon responses and deisgylase activity broadly removing isg conjugated proteins. there are currently about known nairoviruses classified into fourteen species. recent genomic characterization has revealed a high degree of diversity, with votus showing less than % amino acids identities within the family. previous investigations have been limited to only a few closely related nairoviruses, leaving it unclear what impact this diversity has on votu function. to probe the effects of votu diversity on enzyme activity and specificity, we assessed representative votus spanning the nairoviridae family towards ub and isg fluorogenic substrates. this revealed great variation in enzymatic activity and specific substrate preferences. a subset of the votus were further assayed against eight biologically relevant di-ub substrates, uncovering both common trends and distinct preferences of poly-ub linkages by votus. four novel x-ray crystal structures were obtained that provide a biochemical rationale for votu substrate preferences and elucidate structural features that distinguish the votus, including a motif in the hughes orthonairovirus species that has not been previously observed in otu domains. additionally, structure-informed mutagenesis provided the first direct evidence of a second site involved in di-ub binding for votus. these results provide new insight into nairovirus evolution and pathogenesis, and further enhances the development of tools for therapeutic purposes. introduction nairoviruses are negative sense single stranded rna [(-) ssrna] viruses within the order bunyavirales. initial classification of nairovirus species relied on antigenic cross-reactivity, leading to the clustering of viruses into seven serogroups; however, with the recent increase in the number of available viral sequences the classifications have shifted to a comparative genomics approach. this not only confirmed the diversity observed based on the serogroup classification, but also further accentuated how these viruses vary across the nairoviridae family. the family nairoviridae now consists of approximately viruses that are currently classified into species (fig ; [ - ] ). most nairoviruses are tick-borne viruses infecting multiple vertebrate host species they parasitize in nature. several have been implicated in human disease, the most notable being crimean-congo hemorrhagic fever virus (cchfv), which has reported case fatality rates in humans that can exceed % [ ] . other nairoviruses associated with human disease include dugbe virus (dugv), nairobi sheep disease virus (nsdv) and the asian variant ganjam virus (ganv), erve virus (ervev), issyk-kul virus (iskv) and kasokero virus (kasv). these viruses have been reported to cause a myriad of symptoms, some of which include fever, headache, and diarrhea [ ] [ ] [ ] [ ] [ ] [ ] . nairoviruses have also been observed to cause fatal animal disease. for example, nsdv has been reported to have a > % mortality rate in sheep and goats making it a significant economic as well as human health concern [ ] . a recently characterized nairovirus, leopards hill virus (lphv), was isolated from bats and causes severe gastroenteric hemorrhaging and hepatic disease in mice [ ] . hazara virus (hazv) was isolated from ticks collected from the royle's mountain vole and has been proposed as a model system to study cchfv based on its ability to cause similar fatal disease in interferon (ifn)-receptor knockout mice [ , ] . beyond these viruses causing disease in mammalian hosts, other nairovirus have been associated with a broad taxonomic diversity of vertebrate hosts such as birds, fish, and reptiles. for example, viruses in the hughes orthonairovirus species, such as farallon virus (farv), have been implicated in infecting birds [ ] . nairoviruses possess a tripartite genome consisting of small (s), medium (m), and large (l) segments that encode the viral nucleoprotein, glycoproteins, and rna-dependent rna the tree was constructed utilizing the jones-thornton-taylor model in the mega program [ ] . current species groupings are indicated by colored ovals, and the assigned species denoted. previous serogroup classification, if applicable, is shown in parentheses. virus votus included in this study are denoted by red lettering. inset is a structure-based phylogenetic tree of votus, with the mammalian cezanne, a , and otulin otus included for comparison. the tree was constructed using pdb ids prp, hxd, jze, dx , dx , dx , dx , lrv, lrx, and znz in the multiseq module of vmd [ ] . sequence accession numbers are included in s table. polymerase, respectively. interestingly, the nairoviral l segment also encodes a viral homologue of the ovarian tumor protease (otu) at the n-terminus. this feature uniquely distinguishes the nairoviridae family and genus tenuivirus from other members of the order bunyavirales. the viral otu (votu) does not appear to play a direct role in genome replication and is dispensable in minigenome replication systems [ ] . instead, the votu's primary function appears to be the reversal of post-translational modifications by ubiquitin (ub) and the ub-like protein interferon stimulated gene product (isg ) . this votu-encoded deubiquitinase (dub) and deisgylase activity has been implicated in evading the innate immune response [ ] [ ] [ ] . ub is an . kda protein that is involved in a wide range of cellular processes, including key regulatory functions in innate immunity. ub is conjugated to target proteins by means of a three step process involving activating (e ), conjugating (e ), and ligating (e ) enzymes, and can either occur as a single ub moiety (mono-ub) or in polymeric and branched forms (poly-ub). these chains can be formed by linkage through either the n-terminus (linear) or one of seven lysine residues in ub (k , k , k , k , k , k , and k ), with different forms often mediating different downstream effects. the most thoroughly studied forms, k and k , play important roles in regulation of the innate immune responses. specifically, k -mediated proteasomal degradation has been associated with feedback control, while k polyubiquitination is required for pathway activation, including retinoic acidinducible protein i (rig-i), mitochondrial antiviral signaling protein (mavs), tumor necrosis factor (tnf) receptor associated factor (traf ), tank binding kinase (tbk ), and ifn regulatory factor (irf ). this signaling cascade leads to the production of ifn-α/β, which ultimately results in the upregulation of numerous ifn-stimulated genes, including isg [ , ] . the role of isg is complex and not well understood but is generally associated with mediating and regulating antiviral responses both as a co-translational modification and as free isg in the cytosol and secreted form inducing the secretion of ifn-γ and il- by binding cell surface receptor lfa- [ ] [ ] [ ] [ ] [ ] [ ] . initial studies on nairoviruses, including cchfv, dugv, and nsdv, established the potential immune modulatory effects of votu activity based on overexpression of the respective isolated otu domain in cell culture [ ] [ ] [ ] . the ability to probe the specific role of the votu during the viral replication cycle remained elusive, however, until the recent development of a reverse genetics system for cchfv. these studies revealed distinct roles for dub versus deisgylating activity during the course of a cchfv infection [ ] . specifically, that cchfv votu dub activity is not as promiscuous towards ubiquitinated host proteins as it first seemed based on the overexpression studies, but appears to be restricted to a targeted subset of cellular substrates associated with suppression of rig-i-mediated early cellular responses to infection. in particular, wildtype cchfv was able to reduce the induction of several immune components, including rig-i, while cchfv with a votu specifically lacking dub activity resulted in enhanced cellular responses to infection and establishment of a cellular antiviral state that reduced viral titers. in contrast, deisgylating activity appears to play a role in later stages of cchfv infection. a recent study demonstrated a similar impact of dub activity in viral immune suppression during the replication cycle of severe acute respiratory study aligned using the t-coffee sequence alignment program [ ] . percentages show the sequence identity relative to cchfv votu. generic votu secondary structure based on define secondary structure of proteins (dssp) algorithm calculations for the votus is shown in reddish orange, with the α and α helices of farv votu shown in teal. the catalytic triad is boxed in black and the selectivity pocket in orange. mutation sites related to the selectivity pocket are shown by yellow stars, sites related to differences in how farv votu engages mono-ub by blue stars, and the dgkv votu catalytic triad mutant by a green star. mutation sites for the second ub binding site in farv votu are denoted by red stars. the region deleted in the farv votu Δ - construct is indicated by a bracket. syndrome coronavirus (sars-cov) [ ] . specifically, when the dub activity of the sars-cov papain-like protease (plpro) was selectively disrupted, the virus showed increased sensitivity to ifn and slower growth kinetics. furthermore, domain exchanges of plpro's between different sars-cov variants supported this observation, establishing dub activity to be a distinguishing virulence trait. these emerging insights into the impact of dub activity in the cchfv votu and sars-cov plpro during viral replication emphasizes the importance of robust dub activity among pathogenic viruses. the demonstrated votu-associated dub/ deisgylase activity of other nairoviruses such as dugv, ervev, and nsdv/ganv, further highlights a potentially substantial role of the votu in viral replication and immune suppression for viruses in the nairoviridae family [ ] [ ] [ ] ] . remarkably, the nairoviral votu domain shows a great degree of sequence diversity, with sequence identities that can drop below % between species (fig ) . a particularly striking case of this diversity is found in members of the hughes orthonairovirus species, such as farv, which possess - additional residues in the middle of the otu domain ( fig b) . these sequence differences between votus suggest a plasticity in the otu domain that could play a role in evolutionary adaptation. currently, exploration into the phenotypical effects of this diversity has been restricted to only a few taxa that include cchfv, dugv, nsdv/ ganv, and ervev [ , ] . these studies revealed that votus possess different enzymatic and structural characteristics. in particular, votus display a wide degree of variation in the efficiency with which they engage ub and isg that is driven by specific sequence and structural features. these substantial differences in viruses within closely related taxa raises questions on the impact of votu diversity across the nairoviridae family. specifically, how votus from viruses in each species vary in structure and activity, and the implications of this for the potential to suppress the innate immune response and affect viral pathogenesis and host tropism. to better understand the impact of votu diversity, we sought to obtain a more complete perspective of the functional and structural features of votus within the nairoviridae family. in vitro assays revealed that votus across diverse taxa possess ub activity, but that activity towards isg appears more restricted. further characterization of votu activity uncovered distinct trends and preferences for specific poly-ub linkages. to better understand the molecular mechanisms driving ub activity and specificity, novel x-ray crystal structures were solved revealing features that distinguish the votus from each other, including a pocket that correlates with ub specificity. additionally, a structure of the farv votu provides details into the structural nature of the additional residues in hughes orthonairovirus votus. structureinformed mutagenesis of farv votu identified residues involved specifically in di-ub binding, representing the first report of the role of a second site involved in di-ub binding in nairovirus votus. this novel enzymatic and structural data not only provides insight into the nature of votu diversity, but also lays a foundation for understanding the impact of the votu interaction with the innate immune response and its connection to viral pathogenesis. to gauge votu diversity across the nairoviridae family, viruses representing the divergent species were selected and the otu domain recombinantly expressed. initially selected based on the traditional serogroups as well as emerging genetic characterization, these viruses include members of the most distantly related taxa and represent of the currently recognized species in the dynamic classification landscape of nairoviruses (fig ) . included were the votus from cchfv, nsdv and ganv, dugv and kupe virus (kupev), hazv, taggert virus (tagv), ervev, farv, dera ghazi khan virus (dgkv), huángpí tick virus (hptv- ), lphv, qalyub virus (qybv), and iskv (fig ) . to better understand the global diversity of nairoviral engagement with ub and isg substrates, these votus were assessed for activity towards ub and human isg fluorogenic substrates. these specific activities were measured by the accumulation of the fluorescent molecule -amino- -methylcoumarin (amc) as a result of cleavage from the c-terminus of ub or isg (fig ) . intriguingly, the votus showed a diverse range of activity towards ub. in general, votus can be divided into groups possessing high (cchfv, hazv, nsdv/ganv, tagv), moderate (dugv, kupev, farv, qybv, iskv), or low activity (ervev, dgkv, lphv, hptv- ) (fig a) . for some of these votus, their deubiquitination activity mirrors that observed in dub-deficient cchfv mutants that impact cellular ubiquitination levels leading to an impaired ability to suppress the ifn response [ , ] . to a large degree, viruses more closely related phylogenetically with cchfv possess the most robust activity (figs a and a ). beyond this, there is not an obvious phylogenetic trend to how well the votus cleave ub-amc, with disparate taxa showing similar low to mid-range activity. overall, engagement with ub is observed to be a feature that can be present in diverse species in the nairoviridae family, with some taxa demonstrating enhanced activity. the patterns of activity for ub are in stark contrast to those of isg -amc, which shows a more dichotomous pattern as a substrate for the votus (fig b) . specifically, there appears to be an abrupt break phylogenetically between groups that contain votus with deisgylating activity, compared to others for which activity is almost negligible. this break appears to exist at the node separating the thiafora, artashat, sakhalin, crimean-congo hemorrhagic fever, nairobi sheep disease, dugbe, and hazara orthonairovirus species from the remaining seven ( fig a) . interestingly, the presence of isg activity does not encompass every votu in these species, suggesting individual factors may have driven the development or retention of isg activity for viruses within this clade. naturally, this also implies that dub activity could be a more broadly utilized mechanism to evade cellular responses. this led us to further explore the dynamics of different nairovirus votu's interactions with ub. while the ub-amc assay provides general information on the ability of votus to engage monomeric ub, cellular substrates are typically modified by poly-ub chains through various linkage types [ ] . additionally, dubs in general and votus in particular have been observed to prefer some linkage types over others [ , [ ] [ ] [ ] [ ] [ ] . to assess the patterns of ub linkage preferences of diverse votus, a subset of the votus were analyzed against di-ub fret-tamra substrates ( fig a) . hazv votu and ganv votu were selected because they have been considered to be a potential model system for cchfv and have significant health and economic impact, respectively. tagv votu represents a more distantly related votu that also has substantial dub activity, while the votus encoded by qybv, farv, and dgkv display diminished activity towards mono-ub. to reduce the influence of interactions with the fret pairs that may disrupt interaction, multiple fret pair configurations were assessed when available and the one displaying the highest activity selected (two positions each for k and k ; s fig) . comparison of votu activities towards different di-ub fret substrates reveals that each species' votu has distinct preferences for specific di-ub linkages. while hazv votu and ganv votu both possess notable activity for k and k di-ub, there appears to be more substantial activity towards k . tagv votu, on the other hand, prefers k and k to a greater extent, while not possessing as much activity towards k . for farv votu, the opposite is observed, with k being preferred. dgkv votu, consistent with its low ub-amc activity, possesses very low activity for the di-ub fret substrates, regardless of the linkage. similar to the pattern observed for hazv and ganv, qybv votu shows the most activity towards k , though at lower overall levels. additionally, qybv votu shows a more pronounced difference in the relative preference for k versus k linkages, with substantially more activity towards k . regrettably, the commercial availability of fret-tamra di-ub substrates is restricted to these tested linkages. additionally, limitations are known to exist due to how the positions of diversity of nairovirus votu structural fold and substrate preference the donor-quencher pairs affects binding of these substrates with the proteases. to gain a more complete and natural perspective of di-ub linkage preferences, these votus were also assessed by sds-page for the ability to cleave unlabeled di-ub substrates of all eight linkage types ( fig b) . as expected, the votus did not show equal preferences for the different linkages. intriguingly, some of the results appeared to differ from the fret analysis. specifically, for both hazv votu and farv votu the gel cleavage assay would suggest the k activity to be the highest with k and k roughly equal, suggesting that the positions of the donorquencher pairs may have hindered binding to some of the substrates. as expected, the hazv, ganv, and tagv votu showed substantial cleavage of several di-ub substrates that is consistent with their high ub-amc activity, while the dgkv and qybv votus showed low level of substrate cleavage over the same time course. intriguingly, farv votu showed substantial cleavage of some of the substrates, despite not possessing high ub-amc activity. this may be reflective of differences in the assays in measuring dub activity. alternatively, it may also suggest the existence of an additional site of interaction with the proximal ub that enhances the efficiency of di-ub cleavage. assimilation with previously reported data reveals interesting trends and points of divergence between the votus ( [ ] ; fig c) . linear and k -linked di-ub does not show any sign of cleavage with any votu. votus demonstrate varying levels of low/detectable activity on k -linked and k -linked di-ub. in contrast, votus show a consistent pattern of higher activity towards k , k , k , and k di-ub. while most of the votus show some level of enhanced activity towards these linkages, the specific linkage most preferred can differ. the cchfv, hazv, tagv, and farv votus all show a distinct preference for k over k linkages, while dugv and qybv votu show more activity towards k . ganv votu shows approximately equal preference for these linkages. interestingly, ganv votu also shows more activity towards both k and k di-ub at approximately equal levels. the high degree of preference towards these substrates extends to the majority of the votus, as even for dgkv votu, which shows minimal or no cleavage of most of the substrates, cleavage of k -linked di-ub can be identified within an hour ( fig b) . the other votus, with the exception of ervev, all show detectable levels of k cleavage, with most also cleaving k . the cchfv, hazv, dugv, and farv votus all showed a greater relative preference for k over k , while tagv votu was the opposite. qybv votu, similar to ganv, showed approximately equivalent activity towards k and k , though overall activity was lower. overall, these patterns of activity suggest that votus do not merely cut any ub moiety, but that they are specific to a subset of linkages that may influence specific aspects of cellular biology. to gain a better understanding of how sequence diversity translates into structural differences, x-ray crystal structures were sought of votus from divergent species. the votus from dgkv, qybv, and tagv readily crystallized and were solved to . Å, . Å, and . Å, respectively (table ) . these votus represent diverse nairovirus species, and possess extensive variation in ub activity with the dgkv, qybv, and tagv votus possessing low, medium, and high activity towards ub-amc, respectively (fig ) . tagv provides a glimpse into the sakhalin orthonairovirus species, a taxon that is more closely related to the ervev and cchf/nsd/dugbe/hazara cluster, while dgkv and qybv are from the dera ghazi khan and qalyub orthonairovirus species, respectively, and are much more distantly related (fig ) . these structures reveal global similarities among the votus (fig and s fig) . each votu possesses a seven-stranded beta sheet as the core feature, with five major alpha helices framing the rest of the structure. the catalytic triads perfectly superimpose over each with the exception of dgkv votu ( fig a) . in dgkv votu, aspartate is replaced by a glutamate that alters the spatial dynamics of the catalytic triad, possibly contributing to a less rigid structure that allows the histidine to adopt the alternate conformation. while atypical for the votus, it does not appear to be the cause of dgkv's low dub activity, as mutating the glutamate to an aspartate only further diminished activity. looking beyond the catalytic triad, a structural overlay of the votus highlights a point of difference in the overall structure that distinguishes the proteases from each other. specifically, there appears to be substantial variability in the region encompassing the α ("selectivity") helix that has been associated with substrate preference, and the loop between the β and β a strands ( fig a; [ , ] ). comparing the root mean square deviation (r.m.s.d.) for the positions of the main chain atoms of these different structures further emphasizes how they deviate from structure to structure (s fig). closer examination of the structures reveals distinct molecular interactions that account for these observed structural differences within the votus. specifically, particular amino acid differences can be identified that form interactions that would promote the observed conformation of each protease, suggesting these differences to not merely be a consequence of dynamics diversity of nairovirus votu structural fold and substrate preference or crystal packing. intriguingly, these residues are not limited to just the selectivity helix and β -β a loop but extend to other secondary structural elements in local proximity, forming an ensemble of interactions that drive the noticeable variability of the α region (fig b and c ). the first area of prominent influence centers around position of cchfv votu (fig c, panel i). this position is strongly conserved in possessing an aromatic residue, consisting of a phenylalanine or a tyrosine in cchfv and more closely related viruses, while consisting of a tryptophan in the rest of the votus studied ( fig b) . while subtle, this change results in distinctly different local interactions that influence the positioning of the selectivity helix. in the cchfv votu, tyr forms hydrogen bonds with the backbone of leu within the α -α loop. in contrast, the tryptophan residues in the other votus fill into a hydrophobic cleft that involves residues within the selectivity helix. in dgkv votu, trp packs with the methylene group of ser on one side and the aliphatic portion of lys 's side chain on the other. lys itself is stabilized in this permissive conformation by hydrogen bond pairing with the carbonyl of gly . in qybv votu, trp packs with pro . additionally, it is in proximity to his , suggesting potential stacking of the rings. such an interaction could have an indirect effect on the positioning of pro at the surface of the votu-substrate interface. in tagv votu, trp fits between leu and thr . the second area centers around helix α and shows a great degree of variation between the votus (fig c, panel ii) . it can encompass interactions that can extend to the α and α helices as well as the β a sheet with the potential to influence the local structural architecture. cchfv votu possesses a number of interactions within this region, including unique lysine pairings consisting of lys , glu , lys , and glu that accommodate hydrophobic packing with tyr and phe . along with hydrogen bond pairing of glu with thr , these work in conjunction in orienting the position of the selectivity helix, a region that has been implicated in substrate preference [ , ] . the other votus, in contrast, possess fewer interactions but still could influence the structure. in dgkv votu, gln within the selectivity helix is central to the interaction, pairing with both ser and tyr by hydrogen bonding. similarly, for tagv votu thr and glu form electrostatic interactions with gln and tyr , respectively. in contrast, for qybv votu there do not appear to be any direct interactions with the selectivity helix. instead, lys and ser form electrostatic interactions with asp and asn , respectively, suggesting a role in manipulating the positioning of the β -β a loop. the third region consists of the selectivity helix and β -β a loop themselves ( fig c, panel iii). specifically, direct interactions, or the lack thereof, work in conjunction with the other interactions to complete the structural features. this is most notable in qybv votu, in which phe appears to pack with pro . in cchfv votu, the corresponding residue is ile , which does not appear to be able to bridge the distance and form an interaction. in tagv votu, asn and thr are in general proximity, but appear to be too distant to form a strong interaction with each other. similarly, dgkv votu appears to lack any direct interactions. overall, these interactions contribute to structural features that influence spatial and chemical presentation of the votu interface, potentially affecting how these votus engage substrates. binding with ub often centralizes around the specific hydrophobic residues leu , ile , and val [ ] . looking at x-ray crystal structures of the cchfv and dugv votus bound to ub reveals that leu in particular has to be spatially accommodated in a pocket deep within the interaction interface (fig a) . to confirm that this interaction with leu is likewise involved in ub binding with these other votus, isothermal titration calorimetry (itc) was performed using the tagv votu to determine the relative binding efficiency of alanine and asparagine ub mutants (ub-l a and ub-l n) compared to wt ub (table , s a fig) . this revealed a stark difference in the affinity. while wt ub bound strongly with a dissociation constant (k d ) of . ± . μm, ub-l a showed no detectable binding under similar conditions. the ub-l n mutant faired only slightly better than the ub-l a mutant with a times weaker dissociation constant, k d of . ± . μm, compared to wt. these results further underscore the importance of votus being able to accommodate ub leu in order to have robust deubiquitinating activity. examining the analogous residues in the other votus reveals a diverse composition for this pocket that could influence how well leu can be accommodated. in tagv votu, this pocket is largely hydrophobic possessing two tyrosines as well as a valine. in contrast, the dgkv and qybv votus possess more polar residues, including asparagine, glutamate, and threonine for dgkv and glutamine and lysine in qybv. when considering the activity towards ub based on the amc assay, a general trend emerges that correlates the degree of an enzyme's ability to engage mono-ub with the hydrophobicity of this pocket. looking more closely at this interface suggests an additional nuance to the ability to accommodate particular substrates. specifically, spatio-chemical characteristics could largely influence what defines a good or acceptable pocket composition for binding a given substrate. in the votus that most effectively engage with ub, such as cchfv, hazv, nsdv/ganv, and tagv, the residue that most directly interfaces with ub's leu is an isoleucine, valine, or threonine that corresponds to position in cchfv votu (figs b, and a ). this correlation is consistent across the nairoviridae family. despite being phylogenetically distant from cchfv and the other robust votu dubs, qybv demonstrates substantial ub activity and possesses ile that could pack with ub's leu . in other votus that have poor ub activity this residue is typically polar, such as ervev's asn (fig b, panel i) . this creates an environment that would discourage binding with ub. mutation of this residue in ervev to the corresponding hydrophobic residues in cchfv has been observed to generate robust ub activity [ ] . the farv and iskv votus appear to have similar characteristics, with both encoding a glutamine at this position. intriguingly, other votus may go even further in discouraging ub binding. these include lphv and htv- , which possess an arginine and lysine, respectively, at their equivalent positions to asn . modeling in an arginine at this position, such as what lphv votu possesses, reveals that this type of change would be prohibitive for ub binding (fig b, panel ii) . to test the central role of this pocket for ub activity, a series of mutants were made in the dgkv, qybv, and tagv votus and tested against ub-amc ( fig c) . as expected, the disruptive mutants i r in qybv and y r and v r in tagv completely knocked out the ability to process ub-amc, in keeping with a previous report demonstrating that the presence of arginine hindered ervev votu ub activity [ ] . further, increasing the hydrophobicity of the pocket in qybv votu was able to enhance ub-amc cleavage, boosting activity by % and % for the q v and q a mutants, respectively. interestingly, changing the pocket in dgkv votu failed to improve activity. this suggests that some votus lacking any outright dub activity may have evolved to a degree that prevents the generation of this activity through simple changes to better accommodate leu in ub. this leaves the presence of a ub leu accommodating pocket in votus as a major marker for deubiquitinating activity and, if present, the ability to dictate variable levels of activity based on the hydrophobicity. table ). inspection of the structure immediately revealed the impact of this additional sequence on the protease's structure ( fig a) . while possessing the familiar core domain and secondary structure features, the protease possesses extended α /α helices that are connected by several intervening residues. thirteen residues could not to be built due to a lack of well-defined electron density in the crystal structure, and those that could be modeled possess high b factors, suggesting this region to have a high degree of flexibility. this contrasts with the votus from cchfv and other nairoviruses that possess relatively small α /α helices connected by a short loop (fig b) . additionally, the α /α helices of farv votu appear to interact with each other in a manner resembling a coiled-coil motif. this is facilitated by hydrophobic packing between ile , val , ala , and the aliphatic portion of the arg sidechain. this relationship between the helices is further promoted by electrostatic interactions, including a salt bridge between arg and asp , as well as a hydrogen bond between tyr and asp . beyond this interaction, tyr is also positioned to hydrophobically interact with tyr , which together create an environment in which trp can insert. trp further promotes the interaction between these helices through a hydrogen bond with asn , as well as through additional hydrophobic packing with lys and leu . the presence of the extra sequence/structural motif in the hughes orthonairovirus species raises the question of whether it could be involved in substrate interaction. a model of how farv votu could interact with ub further accentuates this possibility, suggesting the α /α helices to be in close enough proximity to participate in binding (fig ) . such an interaction could potentially offset other factors in farv votu are not ideal for binding. looking at the coot. (b) the extra space (black arrow) existing in the ervev-mouse isg structure (pdb id jze), with the cchfv votu bound to ub included for comparison (panel i). qybv votu with ub and mouse isg (green) modeled in based on votu secondary structure alignments, with an arginine also modeled into the space opened up by the mouse isg conformation (panel ii). (c) activities for ub-amc for mutant dgkv, qybv, and tagv votus relative to wt. error bars represent the standard deviation of two independent experiments. https://doi.org/ . /journal.ppat. .g selectivity pocket of farv votu reveals it to possess more of a hydrophilic character and contains a relatively bulky gln residue in the equivalent site to position in cchfv (fig a, panel i) . additionally, farv votu possesses a potential steric hindrance to efficient binding with the presence of arg (fig a, panel ii) . this residue may be less accommodating for the leu in ub than other votus, such as cchfv and tagv which contain a histidine at this site. further, farv votu may lack a significant interaction that cchfv votu possesses with arg of ub (fig a, panel iii) . in contrast to gln in cchfv votu that is able to form a hydrogen bond, farv votu possesses a leucine that is unable form this interaction. to test the influence of these sites on dub activity, mutations were made to arg and leu in farv votu to histidine and glutamine, respectively. as anticipated, r h was able to improve ub-amc activity, boosting it by~ %. making the reverse mutation in tagv votu, h r, essentially knocked out this activity suggesting this residue to have a key impact in diminishing farv votu's activity compared to other votus. interestingly, the l q mutation in farv votu led to a large reduction in ub-amc cleavage. looking more closely at this region shows that leu is in the middle of a large hydrophobic region in farv votu (s b fig) . the swap to a large polar residue may impact the structural integrity of the β -β a region, further underscoring the nuances created by the variability of this region. to probe the potential significance of the α /α motif in offsetting these other effects in farv votu, a construct was synthesized lacking residues - ("farv votu Δ - ") and assessed for activity against ub substrates (fig b) . removing this region reduced activity towards ub-amc by almost %, suggesting that this motif could play a significant role in ub binding (fig b, panel i) . interestingly, when tested against k and k fret di-ub substrates a more modest reduction in activity is observed, with only about a % and % reduction in activity, respectively. this is further borne out with unlabeled di-ub, with there being no substantial difference between the wt and Δ - votus over the longer reaction time course (figs b and b, panel ii). although the di-ub cleavage assays are able to differentiate linkage preference, the structural architecture is still relatively simple. to gauge whether this motif can engage with more complex poly-ub structures, the wt and Δ - farv votus were tested with k and k linked tri-ub (fig b, panel iii) . interestingly, both constructs showed a clear preference for k over k tri-ub. this is in contrast to the gel cleavage assay for di-ub, which showed a slight preference for k di-ub (figs b and b, panel ii) . beyond this, both constructs showed similar patterns of activity for these substrates. despite possessing low to moderate activity towards ub-amc, farv votu possesses substantial activity towards some di-ub linkages (figs and ) . this suggests an additional site of interaction with the proximal ub molecule that substantially increases the overall efficiency. to ascertain where this site may be located, a model of how farv votu may bind di-ub was generated (fig ) . examining the potential interface with the proximal ub, two residues in farv votu, arg and lys , immediately stand out as potential contributors. these residues are just beyond the active site, and are part of a region that likely forms the closest contact with the proximal ub. beyond these two residues, thr of farv votu also stands out as being in an area with a higher r.m.s.d. between the votu structures, which in farv votu positions it closer to the general area of the proximal ub (fig b and s fig) . to assess whether these sites could play a role in the farv votu's interaction with di-ub, mutations at these positions were designed in an attempt to alter activity towards k and k fret di-ub substrates. as a control, each mutant was also run with mono-ub substrates. due to the proximity of arg and lys to the space that would be occupied by the fluorogenic molecule, assays were performed with both ub-amc and ub-rhodamine (rh ) to mitigate artifacts. excitingly, these mutations substantially altered the rate of di-ub cleavage, often towards both substrates (fig ) . individually mutating arg and lys to leucine reduces activity towards k di-ub to - % of wildtype and activity towards k to - %. although the mono-ub activity appears to suffer as well in the case of r l, the~ % difference in the ub-amc versus ub-rh mono-ub substrates suggests this to be an artifact of interactions with the amc fluorophore. otherwise, these mutants had little or no effects on mono-ub activity. when a charge flip was introduced at position , activity was reduced to % for k and % for k while not substantially altering the activity for mono-ub. a charge flip at position had the most pronounced effect, driving it down to % for k and % for k . however, there is also a substantive corresponding reduction in both the amc and rh mono-ub substrates, indicating a potentially large disruptive interaction with the hydrophobic fluorophores or possible influence on the local fold. interestingly, while mutating thr to valine did not appreciably change the activity, introducing an arginine at this position increased the activity by % for k and doubled it for k , suggesting the longer sidechain may be able to form a new interaction. ub is among the most conserved and important cellular regulatory components, influencing almost every key aspect of cell biology. ub itself is tightly regulated by an array of endogenous dub enzymes that specifically curb and tailor its effects. the realization that viruses also possessed enzymes with dub activity introduced a paradigm in which these normal regulatory mechanisms could be manipulated to suppress immune responses and enhance viral propagation [ , [ ] [ ] [ ] [ ] . further investigation into these mechanisms continues to uncover how these viral dubs disrupt cellular responses to infection. in particular, the role of robust dub activity in promoting viral replication and conferring virulence in cchfv and sars-cov emphasizes the impact of the respective proteases and highlights the emerging importance of understanding their effects when considering potential pathogenicity and therapeutic strategies. with the almost perfectly conserved sequence of ub, it is not surprising that tick-borne nairoviruses from disparate taxa possess notable dub activity. such a mechanism could provide broad utility in infecting hosts beyond the primary arthropod reservoir by enabling a route of horizontal as well as vertical transmission that amplifies viral replication. the diversity in the observed activity, however, raises questions as to the specific effects relating to arthropod versus vertebrate hosts. in general, the votus from viruses most closely related to cchfv appear to have the most substantial dub activity based on the ub-amc assay (figs and ) . these viruses are known to cause viremia in vertebrate hosts, including mammals. this raises the prospect that increased ub activity may be an adaptive mechanism allowing these nairoviruses to infect a wider host range. while ticks are known to possess rnai and toll sensingmediated antiviral responses, there is little information pertaining to whether ub plays a significant role in arthropod responses to viral infection [ ] [ ] [ ] . further characterization of ub systems in arthropods will be needed to shed light into these questions and would clarify the significance of votu enzymatic diversity in nairovirus adaptation for arthropod versus vertebrate hosts. in contrast to some mammalian dubs, otu proteases generally show poly-ub linkage specificity that ranges from moderate to highly specific [ ] . nairovirus votus reflect this tendency, possessing activity towards poly-ub linkages that is neither highly promiscuous nor completely selective for a single linkage type with each votu possessesing its own respective preferences for the different linkages. while showing individual variation, the votus consistently show the ability to process k , k , k , and k linked di-ub. the fact that nairovirus votus generally show activity towards k and k di-ub is significant. these well-studied forms of di-ub have clearly established roles for cellular processes in general, as well as in antiviral responses specifically. it can be easily envisioned how disruption of k and k -mediated functions could dampen antiviral responses. it is intriguing, however, that votus as a whole also possess substantial activity towards k and k linkages. these forms of ub have been studied much less extensively, with roles that have typically been associated with dna damage responses and cell cycle regulation [ , ] . as part of the l protein, votu activity would be restricted to the cytosol, raising questions as to whether these observed activities of votus are incidental, or if there are important cytosolic functions of k and k linkages that could be manipulated. recent studies have begun to expand knowledge of these linkages. specifically, k poly-ub has been associated with tnf signaling, providing a direct link to the innate immune response [ ] . even more recently, k has emerged as a key component in regulating mitophagy [ , ] . given the key role of mitochondria in innate immunity, this raises an interesting question of how votu activity could impact this process, and whether such manipulation could provide benefits for the virus [ ] . what other functions remain to be identified for these linkages is still an open question, as well as how votus may engage with them to modify cellular responses. the differences in linkage preferences between votus implies potential differences in the degree to which specific viruses may influence these pathways. alternatively, it's possible that the relative importance of the linkages may differ in different hosts, and that different votu preferences reflects virus adaptation to their specific preferred hosts. the new votu structures reveal an array of conserved and divergent features. the conserved elements of nairovirus votu structure distinguishes these from other otu proteases, as highlighted by how they cluster together in a structure-based phylogenetic tree (fig a, inset; [ , , ] ). most notably, this includes the presence of two additional beta sheets and a helix at the n-terminus of votus that are absent from eukaryotic otus. while possessing these characteristic features of the votu fold, the nairoviruses show distinguishable differences from each other that can be traced to specific residue differences. this is particularly noteworthy when looking at the relationship between the selectivity pocket and the observed ub activity by a given protease. it is significant that votus possessing the highest activity for ub all possess highly hydrophobic residues in this region. while many of the votus possessing robust dub activity are closely related phylogenetically, the presence of substantial activity in the more distantly related qybv votu demonstrates that it is not exclusive to this subset of viruses. this suggests the votu fold to be a flexible platform that has allowed dub activity to evolve independently to the benefit of each virus. beyond the central role of this pocket that is deep in the binding interface, the votus also display structural diversity in more peripheral regions. this includes areas that have been observed to influence substrate binding in votus, such as the α selectivity helix, suggesting a potential impact on how votus engage with other proteins. viruses in the hughes orthonairovirus species possess a motif previously unobserved for otu domains. this raises the question of whether this structural feature could have a functional impact. in particular, whether this motif could impact engagement with substrate. the effect of removing this motif from the farv votu on ub-amc activity suggests that it can at least contribute to mono-ub binding. this is consistent with what is observed when comparing farv votu to a ub-bound structure of cchfv votu, where elements of this motif are in proximity for potential interactions with ub ( fig a) . this additional interface provided by the motif likely compensates for the presence of other less optimal factors for ub binding, including an arginine that hinders interaction with the tail of ub. overall, these structural features suggest a mixture of elements that either promote or hinder interaction, with some that may carry a more dominant effect. the involvement of the structural motif in farv votu formed by the two helices and intervening loop, which we also refer to as a substrate interacting bundle (sib), suggests it may form a region that introduces potential to engage with otherwise inaccessible surfaces. interestingly, removing the sib motif from farv votu appears to have a lesser impact on di-ub activity compared to mono-ub (fig b) . this could be accounted for by the presence of an additional site of interaction in farv votu that interacts with the proximal ub. the existence of one or more subsites has been postulated as a mechanism for discriminating different di-ub linkages based on the proximal ub, and has been demonstrated in several mammalian otus [ , ] . while not definitively observed in votus, the ability to distinguish between different linkages implies a similar mechanism. the farv votu mutants provide the first reported direct evidence identifying such a site in a votu, confirming that votus can utilize this mechanism to distinguish various linkages. in addition to supplying potential leads for elucidating such sites in other votus, it also demonstrates a case where this site can have a major impact on activity towards substrate, even when factors hindering binding with the distal ub are present. although di-ub wouldn't directly interact with the sib motif in a manner that would directly influence cleavage, it is possible that a more complex poly-ub structure could engage with it. modeling how tri-ub might bind suggests that a k linkage could place one of the ub molecules in close proximity to the structural motif (fig c) . in contrast, for the other linkages farv votu most readily cleaves-k , k , and k -this ub would likely be too distant to form any interaction. surprisingly, removal of the sib motif has no noticeable impact on k tri-ub cleavage, despite the apparent proximity the tri-ub could have. it's possible that tri-ub may not possess a large enough architecture to be influenced by the sib motif, and that a longer poly-ub may interact with it. additionally, ub is able to form complex chains consisting of multiple linkage types [ ] . it may be that the sib motif can engage more effectively with these "heterotypic" ub chains. alternatively, the primary role of the sib motif may go beyond ub and facilitate interactions with other binding partners. the votu domain exists in the context of the multifunctional l protein. apart from the votu domain, the structural features and dynamics of the nairovirus l protein are currently unknown. this leaves open the possibility that the sib motif could be involved in binding another feature of the l protein to stabilize the overall architecture, or in facilitating interactions with other proteins. in addition, the sib motif could potentially bind to other host factors in the innate immune system. viruses in the hughes orthonairovirus species have been isolated from birds or from ticks that infest them. the immune system of birds, including antiviral responses, possesses considerable differences from mammals in terms of what elements are present and how they are regulated (reviewed in [ ] and [ ] ). this includes the apparent absence of an isg homologue in birds. these differences from mammals raises the possibility that the sib motif could play a role in adaptation to the avian innate immune system, perhaps by facilitating interactions with proteins other than ub. in addition, the lack of isg in birds leaves open the possibility that the motif could engage with other ub-like entities that are involved in regulating the innate immune response. while divergent votus possess the ability to engage with ub, it is possible that this may not be the only, or even predominant function of all votus. in the case of ervev, it has been observed that it possesses poor activity towards ub, while showing potent ability to engage with isg (fig ; [ , ] ). this raises the possibility that other votus that possess poor ub activity may be able to engage with other ub-like entities. while none of the new votus assessed possess notable deisgylase activity, the availability of amc-derived substrates is limited to human isg (hisg ). in contrast to ub, isg shows considerable species-species variances that have been shown to impact binding with viral proteins, including votus from nairoviruses [ , , ] . this leaves open the possibility that votus, while not engaging with hisg , may still possess the ability to interact with isg from species they productively infect. the presence of arginine, lysine, or glutamine in the selectivity pocket of several of the votus, while not ideal for ub, may still allow them to engage with other substrates. the structure of the ervev votu bound to mouse isg (misg ) has a gap in the area that ub's leu would typically occupy (fig b, panel i) . modeling suggests that this feature would also be more permissive of binding with votus possessing a bulky residue such as arginine at position (fig b, panel ii) . this gap is caused by a pairing of glu with lys in misg that pulls the sidechain of glu away from the interface, and suggests a possible mechanism that could allow votus with hydrophilic or bulky residues to effectively engage with non-ub moieties. as highlighted by the lack of isg in birds, however, it's also possible that votus, particularly in the hughes orthonairovirus species, may engage with other ub-like entities that can modulate the immune response. the lack of either ub or isg activity in a number of votus further accentuates this possibility, implying possible biochemical functions that have yet to be characterized among votus. further developments shedding light on these questions could yield key insights into these influential virus-host interactions. the recent increase in genomic characterization of nairoviruses has uncovered a wealth of diversity among them. while our knowledge of nairoviral sequence diversity has expanded, much is still unknown on how this variability affects virus-host relationships. the exact range of vertebrate hosts and their disease state upon infection is not presently known for all members of the nairoviridae family. this novel characterization of nairovirus votus reveals a diversity in the ability to engage mono-and poly-ub that mirrors the genomic diversity. additionally, this study uncovers motifs that appear to play a predominant role in determining these preferences, making it feasible to begin predicting dub activity in uncharacterized or newly discovered nairoviruses. given the presence of robust dub activity in nairoviruses known to infect humans, including cchfv and nsdv, this could serve as an early flag for assessing the risk posed by emerging viruses, and may shed light on the evolutionary trends leading to some viruses to having this capability over others. further, these new structure and activity insights provide a platform to continue the development of robust tools, such as poly-ub specific votus, that can be paired with reverse genetics systems to better understand the role of the votu in the course of a viral infection and how differences in certain activities impact nairoviruses. such knowledge could help propel the field in fully elucidating the detailed functional mechanism of the votu in the viral life cycle, potentially aiding in the development of better disease model systems. in addition, it provides insight that will further gauge the prospects of the votu as a therapeutic target for nairovirus-caused diseases such as cchf, either through the development of specific inhibitors or live attenuated virus vaccines. further, the diversity of the votu suggests a potential relationship with viral host adaptation, and that the role of the votu may extend beyond its well-known function in engaging with ub and/or isg . the votus were constructed and expressed as previously described in published methods [ , ] . purification of qybv, tagv, and dgkv were carried out as previously described. for farv, a slightly different approach altered from the previously described method was used to optimize the expression. e. coli strains with votus from farv were grown at ˚c in l of luria-bertani broth with μg/ml ampicillin. once the optical density reached . - . , . mm isopropyl-β-d-thiogalactopyranoside (iptg) was added to induce gene expression. the temperature was then dropped to ˚c and expression continued overnight. the culture was subsequently centrifuged at xg for minutes and the pelleted cells stored at - ˚c. assays were carried out as described previously [ , ] . briefly, assays were run in mm nacl, mm hepes [ph . ], . mg/ml bsa, mm dithiothreitol (dtt) at ˚c. reactions were run in -well plates with a μl reaction volume using a clariostar plate reader (bmg labtech, inc.). for ub-amc, all votus were assessed at a final enzyme concentration of nm. for isg -amc, votus were assessed at a final enzyme concentration of nm with the exception of nsdv, ganv, and ervev, which were run at a final enzyme concentration of nm due to the high activity towards the substrate. both ub-amc and isg -amc assays were run at a final substrate concentration of μm. assays with ub-rh were run under the same reaction conditions as ub-amc with instrument settings adjusted to optimize detection of the fluorophore. for dgkv votu additional assays were run with the wt and e d mutant using the peptide z-rlrgg-amc (bachem) substrate with protease concentrations of μm and a substrate concentration of μm. assays with fret tamra/qxl pair tagged k , k , and k di-ub substrates were performed as previously described with nm votu and μm substrate [ ] . untagged poly-ub cleavage assays were adapted from the previously published method. briefly, nm of each votu was tested against μm linear (m ), k , k , k , k , k , k , and k linked di-ub (boston biochem, ma). reactions were initiated by the addition of votu and incubated at ˚c in reaction buffer ( mm nacl, mm hepes [ph . ], mm dtt). the reactions were stopped at the time points indicated by mixing μl of each reaction with x laemmli sample buffer and heat killed by boiling at ˚c for minutes. the cleavage over time was visualized using - % mini-protean tgx precast gels (bio-rad) by coomassie staining. assays with k and k linked tri-ub were run in the same manner except that tri-ub was present at μm. all four votus were screened against a series of qiagen nextal suites in a -well hanging drop format with a ttp labtech mosquito (ttp labtech, herfordshire, united kingdom). qybv votu was screened at . mg/ml, tagv votu at . mg/ml, dgkv votu at . mg/ml, and farv votu at . mg/ml. initial hits were optimized along salt, precipitant, and ph gradients as applicable. the tagv and farv votu hits were also optimized with an additive ht screen from hampton research. peg as the cryoprotectant. for selenomethionyl (se-met) derivative qybv votu crystals, bacterial cells were grown in minimal media to od . and induced with . mm iptg at ˚c for hrs. prior to induction, the cultures were supplemented with eight amino acids (leu, ile, val, and trp at . g/l; thr, lys, phe, and cys at . g/l) as well as selenomethionine ( . g/l). cells were harvested and protein purified as previously described. final crystals were grown in . m magnesium acetate, % peg , in drops formed from μl of solution and ul of . mg/ml protein. native datasets of the qybv, dgkv, tagv, and farv votus were collected at a wavelength of Å. a se-met single anomalous dispersion (sad) dataset for qybv votu was collected at the absorption edge of se at . Å. the data sets were indexed, integrated and scaled with hkl- [ ] . experimental phasing of the se-met-sad dataset was performed using the phenix suite of programs [ ] . hyss was utilized to locate the se-met sites, with phaser solving the experimental phases [ ] [ ] [ ] . initial model building was performed using autobuild, with subsequent cycles of refinement and model building carried out in phenix and coot ( [ , , ] . this structure was then used as a search model to solve the qybv votu native dataset by molecular replacement in phaser [ ] . the other votus were solved by molecular replacement. a qybv votu-based homology model was used to solve dgkv votu, while homology models based on dugv votu (pdb entry hxd) were used to solve tagv votu and farv votu. all the structures were built with autobuild, followed by alternating manual building and refinement in coot and phenix. structures were validated using the molprobity server [ ] . mutations were made using the quikchange lightning kit according to the manufacturer's protocol (agilent technologies, inc.). the pcr-generated plasmids were transformed into escherichia coli neb- α cells by heat shock. the mutant plasmids were confirmed by sequencing and transformed into t express cells (new england biolabs). t express cells expressing ub, ub-l a, and ub-l n in pet- b were grown to od . - . at ˚c. expression was induced with . mm iptg and continued at ˚c overnight. the cells were pelleted and stored as described above. the pellet was resuspended in mm nacl, mm tris [ph . ] supplemented with lysozyme at ˚c for minutes. the cells were sonicated on ice at % power with a % duty cycle for a total of minutes, followed centrifugation at , xg for minutes. the supernatant was filtered through a . μm and applied to a gravity flow ni-nta column (goldbio) pre-equilibrated with mm nacl, mm tris [ph . ]. the column was washed with the same buffer containing mm imidazole, followed by elution with mm imidazole. thrombin was added to cleave the x his-tag and the elution dialyzed overnight in mm nacl, mm hepes [ph . ], mm dtt at ˚c. after dialysis the protein was filtered through a . μm membrane and run over a superdex column (ge healthcare) equilibrated with mm nacl, mm hepes [ph . ], mm dtt. the fractions were pooled based on the chromatogram and concentrated to~ - . mm, supplemented with % glycerol, and flash frozen in liquid nitrogen followed by storage at - ˚c until further use. tagv votu was purified as previously described and dialyzed alongside ub and ub-l n in mm nacl, mm hepes [ph . ], mm tcep overnight at ˚c. itc was performed with a microcal peaq-itc (malvern, worcestershire, uk). ub or ub-l n were titrated into the cell in series of injections, μl each with a spacing of seconds. the temperature was kept constant at ˚c with a reference power ranging from - μcal/s. final protein structures were deposited in the protein data bank with ids dwx, dx , dx , dx , and dx for se-met qybv votu, native qybv votu, dgkv votu, tagv votu, and farv votu respectively. complete genome coding sequences of artashat genomic characterization of the genus nairovirus (family bunyaviridae) taxonomy of the family arenaviridae and the order bunyavirales: update genomic characterization of yogue a global genomic characterization of nairoviruses identifies nine discrete genogroups with distinctive structural characteristics and host-vector associations crimean-congo hemorrhagic fever: history, epidemiology, pathogenesis, clinical syndrome and genetic diversity investigation of tick-borne viruses as pathogens of humans in south africa and evidence of dugbe virus infection in a patient with prolonged thrombocytopenia isolation of ganjam virus from a human case of febrile illness: a report of a laboratory infection and serological survey of human sera from three different states of india outbreak of arbovirus infection in the tadzhik ssr due to the issyk-kul virus laboratory infections with ganjam virus thunderclap headache caused by erve virus? kasokero virus: a new human pathogen from bats (rousettus aegyptiacus) in uganda nairobi sheep disease a nairovirus isolated from african bats causes haemorrhagic gastroenteritis and severe hepatic disease in mice tick-borne viruses of west pakistan. ii. hazara virus, a new agent isolated from ixodes redikorzevi ticks from the kaghan valley, w. pakistan hazara virus infection is lethal for adult type i interferon receptor-knockout mice and may act as a surrogate for infection with the diversity of nairovirus votu structural fold and substrate preference soldado virus (hughes group) from ornithodoros (alectorobius) capensis (ixodoidea: argasidae) infesting sooty tern colonies in the seychelles, indian ocean crimean-congo hemorrhagic fever virus-encoded ovarian tumor protease activity is dispensable for virus rna polymerase function epub / / ovarian tumor domain-containing viral proteases evade ubiquitin-and isg -dependent innate immune responses inhibition of interferon induction and action by the nairovirus nairobi sheep disease virus/ganjam virus dugbe virus ovarian tumour domain interferes with ubiquitin/isg -regulated innate immune cell signalling viral evasion and subversion of pattern-recognition receptor signalling interferon-inducible antiviral effectors isg functions as an interferon-mediated antiviral effector early in the murine norovirus life cycle isg is critical in the control of chikungunya virus infection independent of ube l mediated conjugation human intracellular isg prevents interferon-alpha/beta over-amplification and auto-inflammation human isg conjugation targets both ifninduced and constitutively expressed proteins functioning in diverse cellular pathways isg conjugation system targets the viral ns protein in influenza a virus-infected cells extracellular isg signals cytokine secretion through the lfa- integrin receptor crimean-congo hemorrhagic fever virus suppresses innate immune responses via a ubiquitin and isg specific protease the papain-like protease determines a virulence trait that varies among members of the sars-coronavirus species diversity of ubiquitin and isg specificity among nairoviruses' viral ovarian tumor domain proteases biochemical and structural insights into the preference of nairoviral deisgylases for interferon-stimulated gene product originating from certain species molecular basis for ubiquitin and isg cross-reactivity in viral ovarian tumor domains the ubiquitin code lys -linked ubiquitin chains adopt compact conformations and are preferentially hydrolyzed by the deubiquitinase cezanne an ankyrin-repeat ubiquitin-binding domain determines trabid's specificity for atypical ubiquitin chains otu deubiquitinases reveal mechanisms of linkage specificity and enable ubiquitin chain restriction analysis molecular basis of lys -polyubiquitin specificity in the deubiquitinase cezanne evidence for bidentate substrate binding as the basis for the k linkage specificity of otubain ubiquitin-binding domains-from structures to functions deubiquitinating function of adenovirus proteinase the papain-like protease of severe acute respiratory syndrome coronavirus has deubiquitinating activity the papain-like protease from the severe acute respiratory syndrome coronavirus is a deubiquitinating enzyme deubiquitination, a new function of the severe acute respiratory syndrome coronavirus papain-like protease? tick-virus interactions: toll sensing antiviral responses of arthropod vectors: an update on recent advances induction and suppression of tick cell antiviral rnai responses by tick-borne flaviviruses brca : bard induces the formation of conjugated ubiquitin structures, dependent on k of ubiquitin, in cells during dna replication and repair k -linked ubiquitin chains as novel regulators of cell division c-iap and ubch promote k -linked polyubiquitination of rip in tnf signalling usp and park /parkin-mediated mitophagy usp regulates mitophagy by removing k -linked ubiquitin conjugates from parkin the essential role of mitochondrial dynamics in antiviral immunity. mitochondrion structural analysis of a viral ovarian tumor domain protease from the crimean-congo hemorrhagic fever virus in complex with covalently bonded ubiquitin structural basis for the removal of ubiquitin and interferon-stimulated gene by a viral ovarian tumor domain-containing protease the increasing complexity of the ubiquitin code defense genes missing from the flight division structural insights into the interaction of coronavirus papain-like proteases and interferon-stimulated gene product from different species species specificity of the ns protein of influenza b virus: ns binds only human and non-human primate ubiquitin-like isg proteins processing of x-ray diffraction data collected in oscillation mode phenix: a comprehensive python-based system for macromolecular structure solution substructure search procedures for macromolecular structures simple algorithm for a maximum-likelihood sad function phaser crystallographic software iterative model building, structure refinement and density modification with the phenix autobuild wizard coot: model-building tools for molecular graphics molprobity: allatom structure validation for macromolecular crystallography mega : molecular evolutionary genetics analysis version . for bigger datasets multiseq: unifying sequence and structure data for evolutionary analysis a novel method for fast and accurate multiple sequence alignment data were collected at southeast regional collaborative access team (ser-cat) -id beamline at the advanced photon source, argonne national laboratory and the university of georgia x-ray diffraction core facility (xrdc). the findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the centers for disease control and prevention. key: cord- - hnal ad authors: agbeci, maxime; grangeon, romain; nelson, richard s.; zheng, huanquan; laliberté, jean-françois title: contribution of host intracellular transport machineries to intercellular movement of turnip mosaic virus date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: hnal ad the contribution of different host cell transport systems in the intercellular movement of turnip mosaic virus (tumv) was investigated. to discriminate between primary infections and secondary infections associated with the virus intercellular movement, a gene cassette expressing gfp-hdel was inserted adjacent to a tumv infectious cassette expressing k( ):mcherry, both within the t-dna borders of the binary vector pcambia. in this system, both gene cassettes were delivered to the same cell by a single binary vector and primary infection foci emitted green and red fluorescence while secondarily infected cells emitted only red fluorescence. intercellular movement was measured at hours post infiltration and was estimated to proceed at an average rate of one cell being infected every three hours over an observation period of hours. to determine if the secretory pathway were important for tumv intercellular movement, chemical and protein inhibitors that blocked both early and late secretory pathways were used. treatment with brefeldin a or concanamycin a or expression of arf or rab-e d dominant negative mutants, all of which inhibit pre- or post-golgi transport, reduced intercellular movement by the virus. these treatments, however, did not inhibit virus replication in primary infected cells. pharmacological interference assays using tyrphostin a or wortmannin showed that endocytosis was not important for tumv intercellular movement. lack of co-localization by endocytosed fm - and ara (atrabf b) with tumv-induced k( )-tagged vesicles further supported this conclusion. microfilament depolymerizing drugs and silencing expression of myosin xi- gene, but not myosin viii genes, also inhibited tumv intercellular movement. expression of dominant negative myosin mutants confirmed the role played by myosin xi- as well as by myosin xi-k in tumv intercellular movement. using this dual gene cassette expression system and transport inhibitors, components of the secretory and actomyosin machinery were shown to be important for tumv intercellular spread. plant viruses move from the initially infected cell to neighboring cells during local spread and then over long distances through vascular tissues to establish a systemic infection in the plant. transport of viruses between cells first involves the intracellular movement of the viral rna from the site of replication to plasmodesmata (pds) and then its delivery into neighboring cells through pds. pds are tunnels in the cell wall that connect the cytoplasm, the endoplasmic reticulum (er) and the plasma membrane between adjoining cells (reviewed in [ ] ). the size exclusion limit (sel) of pd is normally too small to allow passive transport of large molecular complexes, but plant viruses encode movement proteins (mps) that increase the sel of pds to allow passage of the viral rna (reviewed in [ , ] ). intracellular movement likely involves a membrane-associated viral rna-host and viral protein complex, but the exact configuration of the viral entity that enters the neighboring cells has not yet been determined (reviewed in [ , ] ). in the case of tobacco mosaic virus (tmv), the viral rna appears to spread between cells as membrane mp-associated viral replication complexes (vrcs) [ ] . for members of the comovirus and caulimovirus genera, viral particles transit through mp-induced tubules that go through pds for their delivery into non-infected cells [ ] [ ] [ ] [ ] [ ] . although mps and other viral protein components are important for viral rna intra-and intercellular movement, it is clear that host factors also are required for these activities. the cytoskeleton is an essential component of organelle trafficking in plant cells (reviewed in [ , ] ) and it has been shown to be involved in vertebrate virus intracellular movement (reviewed in [ ] ). in the case of tmv, several studies have shown that microtubules and microfilaments are necessary to anchor and release, or aid the movement of the vrc or mp granules often associated with er (reviewed in [ , , ] ). microfilaments influence the intracellular or intercellular transport of other mps or viruses [ ] [ ] [ ] [ ] [ ] [ ] . myosin motors are also required for mp or viral trafficking [ , [ ] [ ] [ ] [ ] . the secretory pathway is further involved in intra-and intercellular trafficking by several viruses [ , , , , , ] . finally, recent studies suggest that the endocytic transport pathway may be involved in viral movement [ , ] . however, not all viruses or their components use the cytoskeleton or the secretory pathway for movement. for example, pd targeting of the tubule-forming mp of cowpea mosaic virus (cpmv) is not affected by either the disruption of er-golgi transport or by cytoskeleton disruption [ ] . similarly, the targeting of the triple gene block protein (tgbp ) of poa semilatent virus to pd does not require a functional cytoskeleton or the secretory pathway for its intracellular transport [ ] . the genome of potyviruses is a single , kb rna molecule that codes for a polyprotein, which is processed into ten mature proteins. in addition to polyprotein-derived polypeptides, an , kda protein termed pipo is produced in infected cells [ ] and is also found as a trans-frame protein consisting of the amino-terminal half of p fused to pipo (p n-pipo) [ ] . potyviruses have no designated mp but many viral proteins have been reported to have mp-related functions. for instance, hcpro and the coat protein (cp) can increase pd sel [ ] . in addition, cp and cylindrical inclusion (ci) protein are required for virus intercellular movement [ ] [ ] [ ] and are associated with pd [ , ] . recently, the targeting of ci to pd was shown to be mediated by p n-pipo [ ] , which itself is targeted to the plasma membrane through an interaction with the host protein pcap [ ] . one last protein involved in viral movement is the kda membrane-associated k protein. it induces the production of motile vesicles that contain viral rna and have been proposed to be the vehicle for intracellular trafficking of potyviral rna [ , ] . these findings, while providing a partial understanding of the mechanism of tumv intracellular and intercellular movement, do not sufficiently explain the involve-ment of the host secretory pathway or the cytoskeleton in potyvirus movement. in this study, we used a novel dual gene cassette construct that differentiated primary infected cells from cells infected after virus intercellular movement to show that the early as well as the late secretory pathway, but not endocytosis, was important for tumv transport. we also determined that myosin xi- and xi-k, but not myosin xi-f and viiis, influenced tumv intercellular movement. although these cellular components were required for intercellular movement of tumv, they did not appear to be involved in the virus protein production in primary infected cells. an in vivo quantitative assay for tumv intercellular movement a recombinant tobacco etch virus (tev) (genus potyvirus) engineered to express the reporter protein b-glucuronidase (gus) allowed direct observation of virus spread in leaves [ ] . virus spread is influenced by the rates of virus rna replication and virus intercellular movement. hence, the use of the above tev-gus construct to fully interpret results from virus spread studies is limited since virus replication in live tissue cannot be quantitated through gus staining. in order to discriminate initially infected cells from later infected cells in live tissue, we introduced within the t-dna borders of a binary vector a gene cassette expressing the er-localized gfp-hdel adjacent to a tumv infectious genome cassette expressing k :mcherry (fig. a) . since both gene cassettes are delivered to the same cells and gfp-hdel does not move between cells [ ] , primary infected cells should display concomitant green and red fluorescence while secondary infected cells should display red-only fluorescence. this system also allows differentiation between virus rna replication and virus intercellular movement. a single infiltration with an a. tumefaciens suspension containing the above plasmid was performed on leaves of three-week old n. benthamiana plants, resulting in an agroinfiltrated area of - mm in diameter (fig. b) . fluorescence emitted by gfp-hdel was generally observed at approximately hrs post infiltration (hpinf) and mcherry fluorescence resulting from tumv replication was detected at approximately hpinf. systemic tumv infection was observed at - days post infiltration (dpinf) in leaves above the infiltrated one by western blot analysis using a rabbit serum against the cp of tumv (data not shown). a similar systemic movement was obtained when more dilute agrobacterium suspensions (e.g. . - . ) were infiltrated, indicating the bacterial load did not elicit a plant defense response that might have affected virus infection rate. n. benthamiana cells displayed the expected green polygonal er pattern and virus-induced k tagged red vesicles (fig. c) . virus intercellular movement was assayed by observing red and green fluorescence at the perimeter of the infiltrated area. at hpinf, a majority of cells in the infiltrated area emitted both green and red fluorescence and just a few red-only cells were observed (fig. d) , indicating that viral movement was just starting. viral movement was followed in the same agroinfiltrated region at , , and dpinf ( fig. e-g) . at the end of the observation period, the surface area of green fluorescence did not changed, indicating that gfp-hdel did not moved into neighboring cells. intercellular movement of fluorescent signal was also followed by observing spread of green and red fluorescence for consecutive hours starting at hpinf in order to evaluate the rate of cell-to-cell movement. this was achieved by securing an infiltrated leaf still attached to the plant on the confocal plant viruses move from the initially infected cell to neighboring cells during local movement and then over long distances through vascular tissue to establish a systemic infection in the plant. virus intercellular transport requires viral and host factors to move viral rna-protein complexes through plasmodesmata (pds). virus intercellular movement is normally assessed by assays that cannot always differentiate between reduced viral rna replication and intercellular movement. by using a dual cassette of genes encoding fluorescent proteins that can differentiate between primary infected cells and cells infected after intercellular transport, we provide evidence that turnip mosaic virus (tumv) needs a functional secretory pathway where pre-and post-golgi trafficking and the actomyosin network are important for its movement. interestingly, disruption of these host transport machineries had no impact on tumv accumulation in initially infected cells. these results support the idea that virus replication activities can be influenced separately from those involved in other virus activities such as movement, although aspects of both are likely coordinated. benthamiana leaf agroinfiltrated hrs before with a. tumefaciens strain agl containing the above plasmid. the confocal image tiles was formed using the objective by assembly images in xy and the three-dimensional rendering was created by z stacks of mm thick confocal images that overlap by mm. left panel, red microscope stage for observation. when the perimeter of the agroinfiltrated area was initially imaged, intercellular movement have already begun as indicated by the presence of red-only fluorescent patches (movie s ). to calculate the rate of cell-to-cell movement in cells/hour, we first measured the number of leaf epidermal cells for every linear mm in three-week-old n. benthamiana plants and we calculated that mm corresponded to . cells (n = ). we then measured the distance from the agroinfiltrated front to the limit of expansion of the red fluorescence at the end of the observation period. in this experiment, the red-only fluorescence, indicative of tumv secondary infection, had spread an average distance of mm (n = ) in the xy plane in h. this expansion corresponded to a rate of one new infected cell every hours. we repeated this experiment five times and we observed the same rate of cell-to-cell movement. this rate of intercellular movement was similar to that observed for tev expressing gus and for tmv [ , ] . the increase in red fluorescence surface area was not due to the diffusion of k :mcherry from agroinfected cells in the absence of virus spread because replacement of the tumv cassette with a cassette expressing only k -mcherry did not produce red-only fluorescent foci (fig. s a ). these findings validated the use of the double cassette, gfp-hdel and tumv k :mcherry to follow intercellular movement by tumv. chemical and protein inhibitors were used to evaluate the role of the early and late secretory pathways in the intercellular movement of tumv using the dual gene cassette system. the plant secretory pathway consists of the er, the golgi apparatus, various post-golgi intermediate compartments (e.g. trans-golgi network, endosomes), the vacuoles/lysosomes and the small vesicular transport carriers that shuttle between these compartments. the early secretory pathway embraces the er-golgi interface while the golgi apparatus and the various post-golgi organelles that control plasma membrane or vacuolar sorting is categorized as the late secretory pathway (reviewed in [ ] ). brefeldin a (bfa) is an inhibitor that interferes with protein transport between the er-golgi interface [ ] . concanamycin a (cma) inhibits protein transport at the trans-golgi network (tgn) [ ] by inhibiting the function of tgn-localized proton-atpases, which leads to the acidification of the tgn lumen [ ] . to evaluate the influence of these secretory inhibitors on tumv intercellular movement, n. benthamiana leaves were treated with dmso, mg/ml bfa or . mm cma h before pcambia-tumv/ k :mcherry//gfp-hdel agroinfiltration. bfa at this concentration effectively blocked the secretory pathway ( fig. s b ). at dpinf, dmso alone had no inhibitory effect on tumv movement ( fig. a) . on the other hand, bfa and cma treatment reduced cell-to-cell movement of tumv ( fig. b-c) . the surface area for mcherry-only expressing foci for each treatment was measured and the statistical analysis confirmed the inhibitory effect of bfa and cma on tumv intercellular movement (fig. d ). this experiment was repeated two more times and the statistical data are presented in fig. s a . to assess whether or not bfa or cma inhibited tumv replication, we quantified mcherry fluorescence intensity over gfp fluorescence intensity in primary infection foci for all treatments. fig. e shows that that there was no significant difference in the ratio of red over green fluorescence during bfa and cma treatments compared with the no inhibitor treatment (tumv alone) at dpinf, indicating that viral protein production in the primary infected cells was not affected by the drug treatments. green fluorescence levels were also similar between dmso-and bfa-or cma-treated primary cells indicating that steady state level of gfp, which was not associated with virus replication, was not affected by the treatments (data not shown). protein inhibitors were used to further support the role of the secretory pathway in tumv intercellular transport. the adpribosylation factor (arf ) is a small gtpase regulating the recruitment of copi coatomer proteins. a dominant negative mutant of arf [arf (ni)] impaired in gtp/gdp binding inhibits the transport of soluble markers from the er to golgi, and causes a re-absorbance of golgi membrane proteins into the er [ ] . rab-e d is a small rab gtpase acting at a post-golgi trafficking pathway and the dominant negative mutant rab-e d(ni) inhibits trafficking from the golgi apparatus to the plasma membrane [ ] . these two dominant-negative mutants were coinfiltrated with pcambiatumv/ k :mcherry//gfp-hdel. four days post-agroinfiltration, red-only foci were reduced in the tissue co-infiltrated with the two mutant protein cassettes compared with those not infiltrated with these constructs, indicating reduced intercellular movement of tumv in the presence of these secretory pathway inhibitors (fig. b-c) . surface area measurements for mcherry-only expressing patches confirmed the inhibitory effect of both arf (ni) and rab-e d(ni) on tumv intercellular movement (fig. d ). this experiment was repeated two more times and the statistical data are presented in fig. s b . expression of these two mutant proteins did not hamper virus protein production in primary infected cells as measured by red over green fluorescence ratios in the dual expressing regions of the infected leaves (fig. e) . we therefore concluded that inhibition of both early and late secretory pathways inhibited tumv intercellular movement but did not affect virus replication in primary infected cells. the last assertion is in line with the prior observation that bfa treatment did not affect the production of tumv-induced k -tagged perinuclear structures and peripheral vesicles [ ] . we examined if endocytosis was involved in tumv intercellular movement. to this end, we first used a pharmacological interference assay with tyrphostin a , tyrphostin a and wortmannin. in mammalian cells, tyrphostin a inhibits the recruitment of endocytic cargo into clathrin-coated vesicles formed at the plasma membrane by preventing the interaction between the clathrin-binding ap- adaptor complex m subunit and the sorting motif within the cytoplasmic domain of plasma membrane proteins [ ] . tyrphostin a is a structural analog of tyrphostin a but has no inhibitory effect and is routinely used as negative control [ ] . tyrphostin a is active in plant cells [ ] and it has been shown that the drug inhibits endocytosis of some plasma membrane proteins [ ] . wortmannin is a phosphatidylinositol kinase inhibitor that inhibits in mammalian cells receptor sorting and/or vesicle budding required for delivery of endocytosed material to ''mixing'' endosomes [ ] . in plant cells, it has been shown that the drug inhibits endocytosis of fm - (an fluorescence channel imaging tumv producing k :mcherry; middle panel, green fluorescence channel imaging gfp-hdel; and right panel, merged images. (e-g) same infiltrated area as in d but confocal images were taken at , and dpinf. scale bar = . mm. doi: . /journal.ppat. .g amphiphilic styryl dye used to monitor endocytosis) [ ] and morphogenesis of mvb/pvcs [ , ] but it does not affect protein transport from the trans-golgi network (tgn) to the plasma membrane [ , ] . n. benthamiana leaves were infiltrated either with tyrphostin a , tyrphostin a , wortmannin or dmso hrs prior to agroinfiltration with a. tumefaciens agl containing pcambiatumv/ k :mcherry//gfp-hdel. the drug concentrations used previously were shown to block endocytotic pathway in plants [ , ] and inhibition of endocytosis of fm - in the presence of wortmannin was confirmed in our system (fig. s c) . four days post agroinfiltration, tumv movement was examined (fig. b-e) . tumv intercellular spread was not significantly inhibited in the presence of the different drugs that modify cellular endocytotic activity. this experiment was repeated two more times and data are presented in fig. s c . thus maintenance of tumv intercellular movement does not require endocytic activity within the four day period of observation. we also investigated whether tumv-induced k -tagged vesicles were associated with ara (atrab-f b) and internalized fm - . intracellular trafficking of k -tagged vesicles, which contain viral rna [ , ] , has been shown to be dependent on the secretory pathway and microfilaments [ , , ] . ara is a plant rab protein similar to rab of mammals and to ypt /ypt / ypt of yeast, and is associated with prevacuolar compartments and involved in endocytic and vacuolar trafficking in plant cells [ , ] . co-expression of ara fused to gfp and pcambiatumv/ k :mcherry in n. benthamiana leaves cells showed that there was no colocalization between ara motile dots and k -tagged vesicles (fig. a) . also, k -tagged vesicles were never associated with fm - -labeled vesicles (fig. b) . lack of co-localization of fm - and ara with tumv-induced k -tagged vesicles further indicates that endocytic pathways associated with these markers were not important for tumv cellular spread. many viruses and individual virus proteins require the actomyosin system for their intracellular and/or intercellular movement [ , , [ ] [ ] [ ] , ] . however, recent studies showed that rna viruses might have evolved differently in their requirements for actin and the associated myosin motors [ , ] . we first tested the effect of latrunculin b (latb), and cytochalasin d (cytd), which inhibit maintenance of microfilaments [ ] , on the intercellular spread of tumv. leaves were infiltrated with mm latb, mm cytd, or dmso h before agroinfiltration with pcambiatumv/ k :mcherry//gfp-hdel. the disruption of actin by latb or cytd was confirmed by confocal microscopy observation of microfilaments labeled with the actin-binding domain of a. thaliana fimbrin fused to gfp (gfp-abd -gfp) [ ] (fig. s d) . tumv intercellular movement was assessed by imaging n. benthamiana leaves at dpinf. inhibition of tumv intercellular movement was observed after latb or cytd treatment (fig. b-d) . this experiment was repeated two more times and the statistical data are presented in fig. s a . the ratio of red to green fluorescence in dual expressing cells was unchanged between treatments indicating that virus replication was unaffected by these microfilament antagonists (fig. e) . these results indicate that an intact microfilament network was important for tumv intercellular movement, but not for replication. the last assertion is in line with the prior observation that latb treatment did not affect the production of tumvinduced k -tagged perinuclear structures and peripheral vesicles [ ] . it was previously shown that overexpression of the myosin xi-k tail, a dominant negative mutant of this myosin species, inhibited the intracellular trafficking of tumv k vesicles and reduced tumv infection [ ] , indicating the involvement of this myosin in virus movement. we were also interested to see if other myosins could be involved. tobacco rattle virus-mediated virus-induced silencing (trv-vigs) was adopted to determine the role of myosins on intercellular movement of tmv, potato virus x (pvx), tomato bushy stunt virus, and turnip vein-clearing virus (tvcv) [ ] . we thus used trv-vigs to silence individual myosin genes prior to tumv infection. n. benthamiana leaves were first infected with trv constructs and days later upper leaves were infiltrated with agrobacterium strain containing pcambia-tumv/ k :mcherry//gfp-hdel. tumv intercellular movement was quantified at dpinf. quantitative rt-pcr confirmed that the transcriptional level of the target myosin genes was decreased in plants infected by the trv silencing construct containing the corresponding genes (fig. a) . we then monitored tumv intercellular movement by measuring areas of foci expressing mcherry-only. quantification indicated that there was no significant difference in tumv intercellular movement in mock-and trv-infected plants (fig. b and d) . virus movement was not affected in myosin viii- and viii- -silenced plants. however, silencing of myosin xi- reduced tumv intercellular movement by a factor of compared to the control ( fig. b and d ). reduced tumv movement was observed in myosin xi-f silenced plants, but was not found to be statistically significant. this experiment was repeated two more times and the data are presented in fig. s b . to be sure this effect on virus movement was specific to myosin xi- silencing, we analyzed the effect of silencing myosin xi- on the other myosins (fig. c) . silencing myosin xi- had no significant effect on the transcript level of the other tested myosins. studying the role of different myosins in virus movement has also been carried out with transient expression of dominant negative myosin mutants [ , ] . a significant decrease of movement was observed when we expressed n. benthamiana dominant negative myosin mutants for the myosin xi- and xi-k, but no significant effect was observed for myosin viii- and xi-f ( fig. a and c) . this experiment was repeated two more times and the data are presented in fig. s c . intensity ratio of red over green foci was calculated to determine if replication was affected by the expression of the dominant-negative myosin mutants. the replication was not affected by any dominantnegative myosin mutants assessed in this experiment (fig. b) . results presented here indicate that myosin xi- and xi-k are required for intercellular movement of tumv and that the other myosins analyzed did not appear to play a role in cell-to-cell movement of tumv. studies on intercellular movement have shown that plant viruses may use different trafficking pathways to move from one cell to another (reviewed in [ , ] ). in this study, by discriminating infiltrated and primary-infected cells from cells infected following intercellular virus movement, we were able to evaluate the contribution of the secretory pathway and the cytoskeleton for tumv intercellular movement. using our dual gene cassette construct (fig. ) , we first observed the green fluorescence at hpinf, and mcherry fluorescence at hpinf. this is the time frame normally observed when virus infection is initiated through agroinfiltration [ ] [ ] [ ] . tumv intercellular movement was observed between and hpinf and progressed at a rate of one new infected cell per , hours and systemic infection of the plant occurred at - dpinf. the intercellular rate of spread of tumv was very close to that observed for tmv and tev [ , ] . infections caused by mechanical inoculation with virus suspensions as opposed to agroinfiltration often result in measurable virus replication at hpinf and systemic infection days later [ ] . the delay in observable infection in our agroinfiltration system may be explained by several factors. the first factor is that a t-dna copy of the viral rna genome is delivered in the cell. this t-dna molecule must be transported to the nucleus and transcribed into rna, which is then transported back in the cytoplasm. in the case of potyviruses, there may be an additional delay because the rna transcribed from the t-dna is not linked to vpg. there is consequently a first round of translation that needs to take place before bona fide infection begins. lastly, monitoring fluorescence as opposed to viral rna through an amplification system (e.g. rt-pcr) is likely less sensitive and requires maturation of the fluorescent marker [ ] . importantly, however, after the initial delay in infection, tumv intercellular movement and systemic infection proceeded at the same rate as infections using purified viral particles. this indicates that the infiltrated agrobacterium did not cause an additional defense response by the plant that significantly impeded spread of tumv beyond what is normally observed during virus infections. this latter finding further supports the use of our dual cassette construct as a valid tool to study virus intercellular movement. it was previously shown that intracellular motility of individual potyviral proteins was dependent on the early secretory pathway [ , , , ] . in addition to er, copi, and copii coatomers, the golgi apparatus can be recruited into virus factories [ , , ] but the role of late secretory pathway in plant viral movement was not investigated. although escrt (endosomal sorting complexes required for transport) proteins, which have a major role in the sorting of cargo proteins, are recruited for tomato bushy stunt virus replication [ , ] , it is not known if they have any involvement in virus movement. in the present study, we determined that in addition to the early secretory pathway, post-golgi transport components were also required for tumv intercellular movement ( fig. - ) . likely, this late secretory pathway is required for sorting the membrane-associated viral rna-protein complex to pds. intercellular movement of tumv also depended on microfilaments (fig. ) and myosin motor proteins ( fig. and ) . in plant cells, myosins are classified into class viii or class xi [ ] . among the myosins tested in this study, myosin xi- and xi-k were required for tumv intercellular movement, but not myosin xi-f, viii- or viii- [ ] . class xi myosins are also required for normal sustained movement of tmv [ ] and gflv [ ] . in the case of gflv, it may be that myosin is required to transport a host factor to the pd that then supports gflv movement. for tmv, it has been suggested that the influence of myosin xi- on its sustained intercellular spread may be through metabolism of virus products after virus movement, since the related tobamovirus tvcv does not require actomyosin for intercellular spread and tmv spreads normally for h post treatment with a microfilament antagonist [ , , ] . previously, myosin xi-k was shown to be involved in the intracellular movement of tumv k vesicles [ ] . myosin xi-k and myosin xi- , but not other myosins, have been shown to be major facilitators for cellular motility between actin filaments and the er [ ] . it has also been shown that myosin xi-k localizes to the motile endomembrane vesicles associated with f-actin [ ] . we suggest that there may be more than one myosin-dependent activity necessary for a single virus and its expressed proteins to spread in plants and that xi- , together with xi-k, may be important for the movement of tumv rna complexes. disruption of the secretory pathway had no impact on tumv accumulation in the initially infected cells. we showed previously that bfa treatment had no effect on the formation of tumvinduced k -tagged structures, although motile k vesicles showed a higher incidence of localization with the copii marker sec [ ] . similarly, disruption of the early secretory trafficking by bfa inhibited intercellular virus movement of melon necrotic spot virus but did not modify its accumulation in infected cells [ ] . coronavirus-induced remodeling of the er and viral replication equally took place in the presence of bfa [ ] . breakdown of actin filaments also did not affect the formation of tumv k -tagged vesicles [ ] . these results suggest that replication activities, despite their requirement for membranes, are influenced separately from those involved in movement, although aspects of both are likely coordinated [ ] . in conclusion, we show in this study that the secretory pathway and the actomyosin system are both important for the intercellular movement of tumv. these host components are likely required by the virus to aid its movement out of the initially infected cell. further work is necessary to identify host proteins within the secretory pathway and the actomyosin network that interact with the virus proteins and influence virus movement. tumv infectious clone pcambiatumv/ k :mcherry was as described [ , ] . ara /rabf b was as described [ ] . the binary vectors designed to express n. benthamiana myosin tails viii- , xi-k, xi-f, and xi- were as described [ ] . the introduction of the s-gfp-hdel gene cassette into pcambiatumv/ k :mcherry was done as follows: pbin/ -er-gk [ ] was digested with asei and ligated with similarly digested pcambiatumv/ k :mcherry. kanamycin-resistant escherichia coli colonies were screened for pcambiatumv/ k :mcherry//gfp-hdel. transient expression studies were performed by agroinfiltration on three-week-old n. benthamiana plants. plasmids were introduced by electroporation into agrobacterium tumefaciens agl and selected on lb ampicillin-kanamycin plates. the pellet of an overnight culture was gently suspended in water supplemented with mm mgcl and mm acetosyringone and left at room temperature for h. the solution was then diluted to an od of . for pcambia-tumv/ k :mcherry//gfp-hdel; . for parf (ni), prab-e d(ni) and pyfp-rab-f b ; . for ptrv and ptrv ; . - . for myosin dominant negative mutant. for co-expression, : mixture of the two agl bacteria containing the plasmids of interest were agroinfiltrated. all dominant negative mutants were agroinfiltrated h before pcambiatumv/ k :mcherry//gfp-hdel agroinfiltration. plants were kept for - dpinf in a growth chamber until observation. small pieces of n. benthamiana leaves were cut and dipped in mg/ml of fm - (molecular probes). leaves were incubated at room temperature for - minutes and observed by confocal laser microscopy. stock solutions of latrunculin b (latb: . mm calbiochem) and cytochalasin d (cytd; mm calbiochem) were prepared in dimethyl sulfoxide (dmso) and diluted to the desired concentration in water prior to their infiltration into -week-old n. benthamiana leaves. the final concentration of brefeldin a (bfa), cma, tyrphostin a , tyrphostin a and wortmannin were mg/ml, . mm, mm, mm, and mm, respectively. the maximal surface of n. benthamiana leaves were agroinfiltrated with the inhibitors hours before pcambiatumv/ k :mcherry//gfp-hdel agroinfiltration. pcambiatumv/ k :mcherry//gfp-hdel agroinfiltration was restricted to a small region in the leaf, to be sure that this region received the inhibitors treatment, and in order to be able to follow the cell to cell movement. ptrv with myosin fragments was as described [ ] . virusinduced gene silencing (vigs) studies were conducted as described previously [ , ] . vtrv infections were established in n. benthamiana by co-agroinfiltration of ptrv and ptrv . to confirm silencing of specific myosin transcripts, rna was isolated from day-old trv-infected systemic leaves (two plants/ construct) using the rneasy plant mini kit (qiagen). dnasetreated rna ( mg) was used to generate cdna with iscript cdna synthesis kit (bio-rad). after a -fold dilution of the cdna, ml of solution was used for quantitative rt-pcr through a rotor gene real-time dna detection system (corbett research). the following primers were used to detect n. agroinfiltrated leaf sections were mounted on a depression microscope slide, aligning the leaf tissue in the well. cells were observed using a objective, , and oil immersion objective on a lsm metaconfocal microscope (zeiss) or on a lsm metaconfocal microscope (zeiss). for lsm metaconfocal microscope experiments, argon and hene lasers were used to excite fluorescent proteins and for a lsm metaconfocal multiline argon and dpss were used. data from both green and red channels were collected at the same time. after acquisition, images were processed using metamorph and/or imagej to quantify the average intensity of fluorescence, carl zeiss lsm image browser, and/or adobe photoshop software for post-capture imaging processes. statistical analysis was performed from a total of - patches from leaves and different plants. graphpad prism one-way analysis of variance ( way anova) was used to assess the overall statistical differences between the means of different groups. following way anova, tukey's multiple comparison test was also used to assess whether the mean of two particular groups were different from each other. p value summary (p, . ) shows statistically significant differences between different treatments. figure s (a-c) repeated experiment as described for fig. d , fig. d and fig. e , respectively. one-way analysis of variance calculation followed by tukey's multiple comparison test allowed analysis of differences between means: = ns, not significant, ***, . ,p value, . , **, . ,p value, . , *, p, . . (tif) figure s (a-c) repeated experiment as described for fig. d , fig. b and fig. a , respectively. one-way analysis of variance calculation followed by tukey's multiple comparison test allowed analysis of differences between means: = ns, not significant, ***, . ,p value, . , **, . ,p value, . , *, p, . . movie s time lapse series of three-dimensional rendering tile confocal image of n. benthamiana leaf agroinfiltrated hrs before with a. tumefaciens strain agl containing pcambiatumv/ k :mcherry//gfp-hdel. the confocal image tiles was formed using the objective by assembly images in xy and the three-dimensional rendering was created by z stacks of mm thick confocal images that overlap by mm. images were taken every hour for consecutive hours, indicated at upper left. scale bar = mm (avi) plasmodesmata -membrane tunnels with attitude cellular pathways for viral transport through plasmodesmata plasmodesmata: gateways to local and systemic virus infection intracellular transport of plant viruses: finding the door out of the cell tobacco mosaic virus infection spreads cell to cell as intact replication complexes tubule-guided cell-to-cell movement of a plant virus requires class xi myosin motors tubular structure induced by a plant virus facilitates viral spread in its vector insect identification of distinct steps during tubule formation by the movement protein of cowpea mosaic virus tubular structures involved in movement of cowpea mosaic-virus are also formed in infected cowpea protoplasts the movement proteins of cowpea mosaic virus and cauliflower mosaic virus induce tubular structures in plant and insect cells plant organelle positioning cytoskeleton-dependent endomembrane organization in plant cells: an emerging role for microtubules subversion of the actin cytoskeleton during viral infection rna transport during tmv cell-to-cell movement role of rice stripe virus nsvc in cell-to-cell movement and symptom development in nicotiana benthamiana the intra-and intercellular movement of melon necrotic spot virus (mnsv) depends on an active secretory pathway the tobacco etch virus p protein forms mobile inclusions via the early secretory pathway and traffics along actin microfilaments the cauliflower mosaic virus protein p forms motile inclusions that traffic along actin microfilaments and stabilize microtubules actin cytoskeleton is involved in targeting of a viral hsp homolog to the cell periphery involvement of the secretory pathway and the cytoskeleton in intracellular targeting and tubule assembly of grapevine fanleaf virus movement protein in tobacco by- cells the early secretory pathway and an actin-myosin viii motility system are required for plasmodesmatal localization of the nsvc protein of rice stripe virus differing requirements for actin and myosin by plant viruses for sustained intercellular movement class viii myosins are required for plasmodesmatal localization of a closterovirus hsp homolog endomembranes and myosin mediate assembly into tubules of pns of rice dwarf virus and intercellular spreading of the virus in cultured insect vector cells intracellular trafficking of potato leafroll virus movement protein in transgenic arabidopsis endoplasmic reticulum export and vesicle formation of the movement protein of chinese wheat mosaic virus are regulated by two transmembrane domains and depend on the secretory pathway two plant-viral movement proteins traffic in the endocytic recycling pathway arabidopsis synaptotagmin syta regulates endocytosis and virus movement protein cell-to-cell transport the cytoskeleton and the secretory pathway are not involved in targeting the cowpea mosaic virus movement protein to the cell periphery intracellular targeting of a hordeiviral membrane-spanning movement protein: sequence requirements and involvement of an unconventional mechanism an overlapping essential gene in the potyviridae interaction of the trans-frame potyvirus protein p n-pipo with host protein pcap facilitates potyvirus movement capsid protein and helper component-proteinase function as potyvirus cell-to-cell movement proteins distinct functions of capsid protein in assembly and movement of tobacco etch potyvirus in plants capsid protein determinants involved in cell-to-cell and long distance movement of tobacco etch potyvirus genetic evidence for an essential role for potyvirus ci protein in cell-to-cell movement the coat and cylindrical inclusion proteins of a potyvirus are associated with connections between plant cells ultrastructural and temporal observations of the potyvirus cylindrical inclusions (cls) show that the cl protein acts transiently in aiding virus movement formation of complexes at plasmodesmata for potyvirus intercellular movement is mediated by the viral protein p n-pipo turnip mosaic virus rna replication complex vesicles are mobile, align with microfilaments, and are each derived from a single viral genome sequential recruitment of the endoplasmic reticulum and chloroplasts for plant potyvirus replication hijack it, change it: how do plant viruses utilize the host secretory pathway for efficient viral replication and spread? enigmatic brefeldin a conserved arabidopsis echidna protein mediates trans-golgi-network trafficking and cell elongation vacuolar h+-atpase activity is required for endocytic and secretory trafficking in arabidopsis in tobacco leaf epidermal cells, the integrity of protein export from the endoplasmic reticulum and of er export sites depends on active copi machinery a rab-e gtpase mutant acts downstream of the rab-d subclass in biosynthetic membrane traffic to the plasma membrane in tobacco leaf epidermis impact on the endoplasmic reticulum and golgi apparatus of turnip mosaic virus infection tyrphostin a inhibits internalization of the transferrin receptor by perturbing the interaction between tyrosine motifs and the medium chain subunit of the ap- adaptor complex testing for endocytosis in plants endocytosis restricts arabidopsis knolle syntaxin to the cell division plane during late cytokinesis wortmannin alters the transferrin receptor endocytic pathway in vivo and in vitro fm-dyes as experimental probes for dissecting vesicle trafficking in living plant cells uptake of a fluorescent marker in plant cells is sensitive to brefeldin a and wortmannin a specific role for arabidopsis trappii in post-golgi trafficking that is crucial for cytokinesis and cell polarity rab-a c gtpase defines a population of the trans-golgi network that is sensitive to endosidin during cytokinesis in arabidopsis biogenesis of cytoplasmic membranous vesicles for plant potyvirus replication occurs at endoplasmic reticulum exit sites in a copi-and copii-dependent manner atrabf b (ara ) acts on the vacuolar trafficking pathway in tobacco leaf epidermal cells the arabidopsis aaa atpase skd is involved in multivesicular endosome function and interacts with its positive regulator lyst-interacting protein the tobacco mosaic virus -kilodalton protein, a constituent of the virus replication complex, alone or within the complex aligns with and traffics along microfilaments inhibition of tobacco mosaic virus movement by expression of an actin-binding protein hypersensitivity to cytoskeletal antagonists demonstrates microtubule-microfilament cross-talk in the control of root elongation in arabidopsis thaliana improved imaging of actin filaments in transgenic arabidopsis plants expressing a green fluorescent protein fusion to the c-and n-termini of the fimbrin actin-binding domain renilla luciferase-based quantitation of potato virus a infection initiated with agrobacterium infiltration of n. benthamiana leaves potato virus x and tobacco mosaic virus-based vectors compatible with the gateway cloning system high-efficiency protein expression in plants from agroinfection-compatible tobacco mosaic virus expression vectors improved monomeric red, orange and yellow fluorescent proteins derived from discosoma sp. red fluorescent protein the tgb movement protein of potato virus x reorganizes actin and endomembranes into the x-body, a viral replication factory a unique role for the host escrt proteins in replication of tomato bushy stunt virus ubiquitination of tombusvirus p replication protein plays a role in virus replication and binding to the host vps p escrt protein arabidopsis myosin xi-k localizes to the motile endomembrane vesicles associated with f-actin the cell biology of tobacco mosaic virus replication and movement myosindependent endoplasmic reticulum motility and f-actin organization in plant cells integrity of the early secretory pathway promotes, but is not required for, severe acute respiratory syndrome coronavirus rna synthesis and virus-induced remodeling of endoplasmic reticulum membranes missing links? -the connection between replication and movement of plant rna viruses eukaryotic elongation factor a interacts with turnip mosaic virus rnadependent rna polymerase and vpg-pro in virus-induced vesicles a multicolored set of in vivo organelle markers for co-localization studies in arabidopsis and other plants virus-induced gene silencing in plants virus-induced gene silencing as a tool for functional analyses in the emerging model plant aquilegia (columbine, ranunculaceae) we are grateful to v. dolja (oregon state university) for providing the dominant negative myosin mutants. we thank m. desrosiers, j. tremblay (inrs-institut armand-frappier) and j. lacoste (the cell imaging and analysis network, mcgill university) for helping with confocal microscopy. key: cord- -c elsnag authors: fusco, marnie l.; hashiguchi, takao; cassan, robyn; biggins, julia e.; murin, charles d.; warfield, kelly l.; li, sheng; holtsberg, frederick w.; shulenin, sergey; vu, hong; olinger, gene g.; kim, do h.; whaley, kevin j.; zeitlin, larry; ward, andrew b.; nykiforuk, cory; aman, m. javad; berry, jody; saphire, erica ollmann title: protective mabs and cross-reactive mabs raised by immunization with engineered marburg virus gps date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: c elsnag the filoviruses, which include the marburg- and ebolaviruses, have caused multiple outbreaks among humans this decade. antibodies against the filovirus surface glycoprotein (gp) have been shown to provide life-saving therapy in nonhuman primates, but such antibodies are generally virus-specific. many monoclonal antibodies (mabs) have been described against ebola virus. in contrast, relatively few have been described against marburg virus. here we present ten mabs elicited by immunization of mice using recombinant mucin-deleted gps from different marburg virus (marv) strains. surprisingly, two of the mabs raised against marv gp also cross-react with the mucin-deleted gp cores of all tested ebolaviruses (ebola, sudan, bundibugyo, reston), but these epitopes are masked differently by the mucin-like domains themselves. the most efficacious mabs in this panel were found to recognize a novel “wing” feature on the gp subunit that is unique to marburg and does not exist in ebola. two of these anti-wing antibodies confer and % protection, respectively, one hour post-exposure in mice challenged with marv. filoviruses are filamentous, enveloped viruses that can cause highly lethal hemorrhagic fever in both humans and non-human primates. the filovirus family includes the major genera ebolavirus and marburgvirus and the newly discovered cuevavirus. in the ebolavirus genus are five known species: ebola virus (ebov), sudan virus (sudv), bundibugyo virus (bdbv), reston virus (restv), and taï forest virus (tafv). in the marburgvirus genus, there is one species, the eponymously named marburg virus (marv) [ ] . marv is further subdivided into different strains, including ci , musoke, ravn and angola. ravn is the most divergent strain of marv, differing by % in genomic sequence from other marburg strains [ ] , and is sometimes referenced as a separate filovirus species. marburg virus was the first filovirus to be identified when it sickened laboratory workers handling infected animals originating from uganda in [ ] [ ] [ ] . marburg virus has since reemerged at least times, and has been imported to the united states and europe by travelers who became infected in africa [ ] [ ] [ ] [ ] . angola, the most lethal strain of marburg virus [ ] , emerged in and caused the largest marv outbreak known to date with an extremely high case fatality rate of % [ ] . the emergence of ebola virus in west africa in has caused an outbreak unprecedented in magnitude, and is a grim reminder of the devastation that can be caused by filoviruses. the filoviruses present a single viral protein on their envelope surface, the glycoprotein (gp), which is responsible for attachment and entry of viruses into target cells. gp is expressed as a precursor that is cleaved by furin in the producer cell to yield two subunits: gp and gp , which remain linked by a disulfide bond [ , ] . gp contains the putative receptor-binding region [ ] , as well as two heavily glycosylated domains: a glycan cap which sits immediately atop the putative receptor-binding site and a larger, largely unstructured mucin-like domain [ , ] . the mucin-like domains contain a dense clustering of n-and o-linked glycans and likely mask the gp from immune surveillance [ , ] . the second subunit of gp, termed gp , possesses the transmembrane domain that anchors gp into the viral surface and the hydrophobic fusion peptide required for fusion. in ebolaviruses, the furin cleavage site lies at residue and the entire mucin-like domain is attached to the gp subunit. in marburg virus, however, the furin cleavage site lies at residue , splitting the mucin-like domain so that a portion of it remains attached to the gp subunit [ ] . we have termed this amino-acid n-terminal gp extension the "gp wing". after cell entry by macropinocytosis [ , ] filovirus gp undergoes additional cleavage by host cathepsin proteases in the endosome [ , ] . this cleavage event removes the glycan cap and the mucin-like domain, resulting in a loss of over % of the molecular mass of gp [ ] [ ] [ ] . endosomal cleavage renders gp competent for receptor binding [ , , ] , allowing the exposed gp head to bind a shared filovirus receptor, niemann-pick c (npc ) [ , ] . although antibodies that broadly cross-react among ebola-and marburgvirus gps would be highly desirable, only one such antibody, mr , has been described [ ] . recent work in non-human primates has demonstrated that passive administration of monoclonal antibody (mab) cocktails against gp can provide highly effective post-exposure therapy for ebov infection [ ] [ ] [ ] [ ] [ ] . polyclonal sera against marburg virus has shown similar efficacy, suggesting that antibodies could also be a viable treatment option for marv infection [ ] . however, fewer monoclonal antibodies, from which such cocktails could be developed, are currently available for marv. one human survivor panel has recently been described; most of these mabs compete for the same site on the gp core [ , ] . antibodies targeting other epitopes on marburg gp would be desirable in order to form a treatment cocktail. in general, monoclonal antibody cocktails are most effective when the component antibodies display synergistic effects. combining mabs with non-overlapping epitopes can significantly increase the overall potency of the cocktail over the individual mabs alone [ ] , and can mitigate antigenic escape by the virus [ , ] . anti-viral antibodies are often selected based on neutralization, or the ability of the mabs to prevent viral entry into target cells in vitro. however, for filoviruses as well as other viruses, neutralization in vitro does not necessarily correlate with protection in vivo [ , ] . non-neutralizing antibodies are known to confer protection by antibody-dependent cellular cytotoxicity (adcc), phagocytosis, prevention of virus budding, and other mechanisms [ , ] . indeed, one successful anti-ebov oligoclonal cocktail is composed entirely of antibodies that are not potent neutralizers [ , ] . in this study we produced a diverse panel of antibodies against marburg virus by immunization of mice with different strains of the surface gp antigen. immunogens included gp -mucin-deleted ectodomains (gpΔmuc) from marburg strains ci , musoke, angola, and ravn. mucin-deleted immunogens were used to direct the immune response away from the highly variable mucin-like domains. ten antibodies were chosen and analyzed for in vitro neutralization, in vivo efficacy, and biochemical recognition of marv and ebov gps. antibodies against multiple epitopes were found. four antibodies target a novel marv-specific "wing" epitope on gp ( g , g , g and g ), and confer - % protection in mice challenged with marv. a separate marv-specific antibody, a , directed against an epitope in gp , confers % protection. another mab directed against gp , g , confers % protection and was found to be broadly cross-reactive among the core of filovirus gps, including both marburg-and ebolaviruses. to generate marv gp-specific mabs, balb/c mice were immunized with gpΔmuc antigens from either marv strain ci , musoke, angola, or ravn ( fig a) . mice for each subset were immunized and boosted with the same antigen with the exception of the series ( g , g , g ). eight of the ten mabs in the panel are mouse igg . the remaining two mabs, a and d , are igg a ( fig a) . to characterize the binding of mabs, we performed enzyme-linked immunosorbent assays (elisas) with recombinant gps from four marv strains, and determined ec values for binding with different forms of marv ravn gp: gp, gpΔmuc, gpcl (fig a) . all ten mabs exhibit medium binding (ec between ng/ml and ng/ml, colored dark yellow) to high binding (ec < ng/ml, colored orange) against ravn gp and gpΔmuc ( fig b) , but only seven of the mabs cross-react with gp from other marv strains ( fig c) . all mabs bind the protease-cleaved ravn gp core, termed gpcl, as well as gpΔmuc, with the exception of a . antibody a exhibits an -fold decrease in binding to gpcl as compared to gpΔmuc ( fig b) . additionally, to evaluate whether the mabs have the capacity to bind cell-surface gp, eli-sas were performed with virus-like particles (vlps) bearing full-length wild-type marv ravn gp. eight mabs bind as well (or nearly as well) to vlps as purified recombinant ravn gp. in contrast, a exhibits nearly -fold weaker binding to vlps than to gp ectodomain, and g binding to vlps is lost at the highest concentration tested (fig b) . to determine antibody epitopes, we performed western blotting with ravn gp and pepscan analysis with overlapping -mer pins of peptides from ravn or musoke gp. five of the mabs bind gp , and five bind gp by western blot (s fig panel a) . pepscan identified linear epitopes for only four mabs, g , g , g and g , all of which overlap within residues - ( fig b) . this shared region lies in an extension of gp that is specific to marv (as a result of the furin cleavage site shift from in ebov to in marv), which we have termed the gp wing (fig a) . in order to confirm the pepscan results, we engineered a gpΔmuc with an additional deletion of residues - , termed gpΔmucΔw (fig a) . indeed, binding to gpΔmucΔw is lost for only the four anti-wing mabs, whilst the remaining six mabs against different epitopes do bind gpΔmucΔw (s fig). no definitive epitope information could be identified by pepscan for the remaining antibodies, suggesting that these mabs bind conformational epitopes. sequence alignment of marv gp residues - reveals that while ci , musoke and angola are completely conserved in this region, ravn has unique residues. the most notable change is residue , which is a glu (e) in ravn but a lys (k) in the other strains ( fig b) . wing mabs g and g are specific for marv ravn. correspondingly, elisa data comparing binding of wild-type ravn gpΔmuc to e k ravn gpΔmuc confirm that the presence of lys at position (as exists in other strains of marv) likely hinders binding of g and g . g , however, still retains some binding to e k, while g is unaffected by this mutation, retaining binding at and . ug/ml ( fig c) . these results agree with pepscan results (based on -mer peptides overlapping by amino acids) which define the epitope for g as slightly shifted away from position , towards the n-terminus of gp ( fig b) . this shift may explain why g is the most cross-reactive of the anti-wing mabs ( fig c) . antibodies were screened for in vitro neutralization using a vsv-pseudovirus containing marv ravn gp on the surface. six of the ten mabs exhibit partial neutralization at the highest concentration tested ( ug/ml), reducing entry by - %. the remaining four mabs do not neutralize (fig ) . notably, all five gp -directed mabs produced in this study exhibit some neutralization, while only one gp -directed mab, a , inhibits entry of ravn gp pseudovirions. polyclonal sera from mice that yielded the series mabs ( g , g and g ) reduces entry by only about %, suggesting that mabs g , g , and g represent the maximum potency of the polyclonal population (fig ) . human survivor mab mr was used as a positive control and reduces pseudovirion entry by almost %. all mabs were evaluated for in vivo protection using balb/c mice challenged with a lethal dose of marv virus [ ] . one hour after challenge with , pfu mouse-adapted marv ravn, mice were treated ip with μg purified mab. two separate studies were performed, with half of the mabs repeated in both studies. control animals in study # , treated with pbs, exhibited / survival (fig a) . both control groups in study # , treated with pbs or anti-ha mab, exhibited / survival (fig b) . marv mab treatment groups varied widely in efficacy, ranging from - % protection. all four mabs against the gp wing were found to be moderately or highly protective: mab g conferred % survival ( / ) , mab g % survival ( / ), mab g % survival ( / ) , and mab g % survival ( / ) . monoclonal antibody a , against gp , conferred % survival ( / ) (fig a and b) . other mabs against the gp core exhibited - % survival; of these, only g offered strongly significant protection (p value . ). the only mab against a gp epitope other than the wing, mab g , exhibited zero protection ( / ) (fig a) . in both studies, mice in all treatment groups displayed an elevation of disease score by day (fig a and b) , and there were no significant differences in weight loss between treatment groups and control groups. in study # , g -treated mice faired only modestly better than the other groups, reaching a disease score maximum of and fully recovering by day (fig a) . two of the highly cross-reactive marv antibodies, mabs g and d , also exhibit binding to ebola, sudan, bundibugyo and reston virus mucin-deleted gps by elisa (fig a) . binding curves show that the affinity of g and d for mucin-containing ebov gp is weak, affinity for gpΔmuc is stronger, and binding to ebov gpcl (the receptor-binding competent core) is strongest and equal to that of marv gpcl ( fig b) . hence, the g and d epitopes are conserved across the filovirus family, exposed on all versions of marburg virus gp, but masked on ebolavirus gp by the mucin-like domain and the glycan cap. single particle electron microscopy of the most protective anti-gp ( a ) and anti-gp ( g ) antibodies was performed in complex with purified antigen. negative stain d class averages of a fabs in complex with marv ravn gpΔmuc show one, two, or three fabs bound to the dense trimeric gp core (fig a) . in contrast, d class averages of the anti-gp wing mab g in complex with marv ravn gpΔmuc show a single fab bound to gp, at a distance further away from the high density gp trimer (fig b) . deuterium exchange mass spectrometry (dxms) studies suggest this gp wing region is unstructured and likely flexible (s fig). to ensure that the wing epitope is not artificially positioned in gpΔmuc as compared to the biologically relevant mucin-containing gp, we also performed em with g fab in complex with the complete ectodomain of marv ravn gp. images obtained were similar to those with gpΔmuc, with only one fab binding per trimer (fig c) . likely footprints of fabs a and g are drawn onto the marv ravn gpcl crystal structure (fig d) . in this study, a small panel of mabs targeting marv gp were isolated from immunized mice. those that conferred the greatest in vivo protection are directed against a novel "wing" domain on marv gp . this wing region is a marv-specific portion of the mucin-like domain attached to gp . such an epitope does not exist in ebolaviruses because the entire mucin-like domain is attached to gp . although this study size was small, we note that gp wing-directed mabs were only obtained when mice were immunized with mucin-deleted ravn gp. it may be tempting to assume that this epitope is masked by the mucin-like domain; however, anti-wing mabs are able to access their epitope on mucin-containing gp, neutralize pseuodviruses bearing mucin-containing gp and provide in vivo efficacy when challenged with marburg virus. we believe that the elicitation of anti-wing antibodies when using ravn gpΔmuc may instead result from the greater homogeneity and stability of ravn gpΔmuc over other marv antigens. a seven-year protein engineering effort in our laboratory to identify crystallizable versions of marv gp indeed found that gps produced from strain ravn are the most homogenous, and have a lesser tendency to aggregate than those from other strains of marv [ ] . the homogeneity may have lead to improved presentation of this protective epitope within this study. it is interesting to note that among this panel of murine mabs and the recently published panel of human survivor mabs [ ] , no antibodies that bind the gp -and gp -containing base of marv gp were identified. the "base" of gp is a common site of neutralization for the ebolaviruses and is the epitope target of anti-ebov neutralizing antibodies kz [ ] , g and g [ ] , as well as the anti-sudv mab f [ ] . perhaps the presence of the flexible gp -wing in marv blocks access to this site on the gp core. nonetheless, antibodies directed against the gp wing itself do have the potential to be fully protective, and represent a novel epitope in marv for therapeutic cocktail design. the most protective of these mabs, g , is promising but only binds with high affinity to the gp from ravn, and hence, protection by g against other marv strains may be limited. in contrast, monoclonal antibody g only confers % efficacy, yet cross-reacts with mucin-containing gps from four strains of marv. however, g is a murine igg , an isotype that typically exhibits weaker immune effector activity than murine igg a [ ] . replacement of the constant domain framework may improve its in vivo efficacy. in this panel, two mabs against gp were identified which also bind the gp cores of ebolaviruses. these antibodies, g and d , bind all marv gps, but only bind ebola, sudan, bundibugyo and reston gp from which the mucin-like domain is deleted (fig ) . hence, these highly conserved epitopes are exposed on marburgvirus gps, but masked on ebolavirus gps. these observations parallel those obtained from a panel of anti-marv gp antibodies isolated from a human survivor [ ] , and support structural observations that the orientation of the mucin-like domains differs between ebov and marv [ ] . indeed, no cross-filovirus anti-gp antibody (reactive to both ebola and marburg) has yet been elicited by an ebolavirus gp immunogen, nor has any such antibody yet been isolated from an ebolavirus survivor. although the filovirus cross-reactive mab g confers only % survival, g or another antibody like it [ ] may be useful in an immunotherapeutic cocktail because a highly conserved epitope would likely be less subject to antigenic escape. antibody a is also directed against gp but its pattern of binding is distinct from g and d . a is the only mab in this panel that has a lower affinity to gpcl than gp or gpΔmuc. this suggests that the epitope of a is partially lost upon cleavage and that a could be similar to a glycan cap binder like c or h for ebov gp [ ] . unfortunately, due to the single preferred orientation of gpΔmuc + a fab particles on negative stain em grids, a high-resolution reconstruction could not be determined, and better understanding of the a epitope awaits further study. a affords % protection in vivo and is highly crossreactive among marv ectodomain gps. for ebola virus, in vitro neutralization is not necessarily an effective predictor of in vivo protection. one anti-ebov cocktail is composed entirely of non-neutralizing or weakly neutralizing mabs, yet still confers in vivo protection, presumably by recruiting immune effector function [ , ] . more recent cocktail formulations have included a mix of neutralizing and non-neutralizing antibodies [ ] . in this study of mabs against marv, none of the mabs offered significant in vitro neutralization, yet several did confer partial to complete in vivo protection against marv one hour after challenge. although this study is limited in scope, we note that among this set of antibodies, those that exhibited in vitro neutralization also conferred the best in vivo protection. (there was only one mab that weakly neutralized but offered no protection, mab g ). future studies, performed at longer time periods after challenge and with lower treatment doses, will test the limits of efficacy of the individual mabs. promising mabs could then be evaluated in non-human primates (nhps) to predict therapeutic potential in humans. in this work, we provide biochemical and structural mapping of antibody epitopes on marv gp, and analyze the conservation of these epitopes among different strains of marv. we find antibodies against a novel gp "wing" epitope that confer - % protection in vivo, and two mabs against different sites in gp that confer % and % protection. mab cocktails are thought to be most effective when the component antibodies display synergistic effects. combining mabs with non-overlapping epitopes can significantly increase the overall potency of the cocktail over the individual mabs alone [ , ] , and can mitigate antigenic escape by the virus [ ] . the panel of antibodies described here, although limited in number, provides three possible components of an anti-marv immunotherapeutic cocktail: an anti-gp core mab such as g (or a neutralizing mr mab), the anti-gp mab a , and an anti-gp wing mab such as g or g . future studies will determine the limits of protection and therapeutic potential of these antibodies when delivered in combination. this study was approved and carried out in accordance with protocols provided by the institutional animal care and use committee (iacuc) at tsri, emergent biosolutions, niaid, and usamriid. research at usamriid was conducted in compliance with the animal welfare act and other federal statutes and regulations relating to animals, and adhered to principles stated in the guide for the care and use of laboratory animals, national research council, . marburg angola, ravn and ci gpΔmuc (tsri). marburg virus gp immunogens used to raise antibodies at emergent were designed and produced at tsri. dna encoding the marv gpΔmuc ectodomain (residues - with a mucin deletion of residues - ) was cloned into a derivative of the invitrogen pdisplay vector. in this derivative vector, the pgdfr sequence is replaced by a c-terminal purification tag (either an ha or strep tag). large-scale production was performed by pei transfection (polysciences, inc mw , ) of plasmid into % confluent hek gnti-/-cells (atcc) in corning -layer cellstacks. supernatants were harvested four days post-transfection, concentrated with a centramate tangential flow system, and affinity purified using streptactin (qiagen) or anti-ha f (roche) affinity resin. trimeric gpΔmuc was then isolated by s size exclusion chromatography (sec) in mm tris, mm nacl, ph . ( x tbs). in order to improve furin cleavage processing during expression, which decreased aggregation and improved yield of purified trimers, bulky hydrophobic residues near the furin cleavage site were mutated in the gpΔmuc constructs. ravn gpΔmuc mutations included f l, w a, f g, f n and angola gpΔmuc mutations included w v, m a, f g. marburg musoke and angola gpΔmuc (ibt). marburg virus gpΔmuc antigens used to raise antibodies at ibt were produced at ibt. musoke gpΔmuc ( - Δ - ) was produced by pei transfection of hek t cells with the derivative pdisplay plasmid containing a c-terminal ha-tag. protein was purified by anti-ha f affinity (roche) followed by lectin affinity and superose size exclusion chromatography (ge healthcare). angola gpΔmuc ( - Δ - ) was produced by baculovirus infection (bac-to-bac, invitrogen) of sf cells (invitrogen) according to the manufacturer's protocol, and purified via a c-terminal hisx tag on ni +-nta sepharose resin (ge healthcare). production of g , g , g , g , g , g , and g . six week-old balb/c mice were injected subcutaneously (sc) with μg (in μl volume pbs) of purified marv gps in freund's complete adjuvant (cfa; brenntag biosector). additional boosts were injected intraperitoneally (ip) on day and with μg of the same gp in incomplete freund's adjuvant (ifa; brenntag biosector). thereafter mice received a final push of μg purified gp (in pbs by ip) before conducting fusions. standard protocols were used to produce hybridoma cell lines [ ] , and monoclonal antibodies specific to gpΔmuc antigen were purified on protein g resin. mice immunized with ravn gpΔmuc raised antibodies g , g and g . mice immunized with ravn gpΔmuc, then boosted two times with a complex of ravn gpΔmuc bound to g fab, raised antibodies g , g and g . mice immunized with angola gpΔmuc yielded antibody g . immunization of mice at emergent was performed according to animal use protocols (aup) approved by the protocol management and review committee (pmrc), university of manitoba. production of a and d . six week-old balb/c mice were immunized intramuscularly (im) three times with μg purified marv gps in glucopyranosyl lipid adjuvant (gla) adjuvant at week intervals, and boosted intravenously (iv) with μg antigen days before harvest of spleen/lymph nodes for fusions. standard protocols were used to produce hybridoma cell lines [ ] , and monoclonal antibodies specific to gpΔmuc antigen were purified on protein g resin. mice immunized with musoke gpΔmuc yielded antibody a . mice immunized with angola gpΔmuc yielded antibody d . immunization of mice at ibt was performed according to aup approved by noble life sciences iacuc. production of a . immunization of mice was performed by bio-quant inc (san diego, ca). six week-old balb/c mice were injected subcutaneously (sc) with μg of purified ci gpΔmuc in cfa followed by additional boosts at week intervals with μg antigen ( μg in ifa by ip and μg in ifa by sc) before conducting fusions. standard protocols were used to produce hybridoma cell lines, and mab a was purified on protein g resin. purified filovirus gp antigen preparation. all filovirus ectodomains for elisa, western blot, em, or dxms were produced at tsri in drosophila s cells [ ] , with the exception of musoke gp, which was produced by ibt in sf cells (as described above for musoke gpΔmuc). briefly, effectene reagent (qiagen) was used to transfect s cells with pmtpuro plasmids containing a strep-tagged filovirus gp gene of interest, followed by stable selection of transfected cells with μg/ml puromycin in insect xpress protein free medium (lonza). secreted gp ectodomain expression was induced with . mm cuso and supernatants harvested after days. proteins were affinity purified using streptactin resin (qiagen), followed by purification via superdex sec in x tbs. the cleaved "core" ectodomain for marv (marv gpcl) was produced by incubating mg ravn gpeΔmuc with . mg trypsin (sigma) at °c for hour in tbs ph . , followed by s sec purification. the cleaved "core" ectodomain of ebov (ebov gpcl) was produced by incubating mg ebov gpeΔmuc with . mg thermolysin (sigma) overnight at room temperature (rt) in tbs buffer plus mm cacl , followed by s sec purification. sds-page gels comparing purity and molecular weight of several antigens in shown in s panel b. vlp preparation. virus-like particles were produced by co-transfection of hek t cells with pcaggs plasmids expressing full-length marv ravn gp or marv vp . supernatants were harvested after sixty hours, vlps pelleted down at , xg for minutes, washed with pbs, and re-pelleted. vlp pellets were then gently resuspended in pbs containing . % np and . % triton x , diluted : with pbs, and used as coating antigen for elisa. western blotting. purified marv ravn gp reduced and non-reduced samples were run on - % sds-page gels and transferred onto pvdf immobilon membranes (millipore). membranes were blocked overnight in % milk (biorad blotting grade) pbs- . % tween (pbs-t), incubated for hour at room rt with anti-marv mabs at a concentration of ug/ml in % milk pbs-t, washed with pbs-t, then incubated with goat anti-mouse (or anti-human for mr ) alkaline phosphatase (ap) conjugated antibody at a : dilution. ap activity was detected with sigmafast bcip/nbt substrate. recognition of various forms of gp and cross-reactivity by elisa. to determine half maximal effective concentrations, or ec s, mabs were tested for binding to gp ( - ), gpΔmuc ( - Δ - ), and gpcl of marv ravn at a concentration range of μg/ml to . ng/ml using -fold serial dilutions. data was analyzed using graphpad prism . software. to determine cross-reactivity, mabs were tested for binding to marv angola, musoke, ci , and ebov gp ( - ) at , , . and . μg/ml. because mucin-containing musoke and ci gps readily aggregate, binding in fig c is reported as high, medium, or low rather than as a quantitative ec value. antibodies d and g were further analyzed for binding to the gpΔmuc antigens of ebov ( - Δ - ), sudv ( - Δ - ), bdbv ( - Δ - ) and restv ( - Δ - ), as well as to ebov gpcl. binding curves for d and g were determined at a concentration range of μg/ml to . ng/ml using -fold serial dilutions. elisas were performed as follows: corning -well high-binding microtiter plates were coated with filovirus gp antigens, blocked with % bsa in pbs hour at rt, and incubated with anti-marv mabs in . % bsa hour at rt. plates were then incubated with : goat anti-mouse igg (h+l) hrp conjugated secondary (thermo scientific) in . % bsa hour at rt. (plates were washed between each step with pbs containing . % tween ). color development was produced with tmb substrate (thermo scientific), stopped with n sulfuric acid, and quantified by measuring absorbance at nm. pepscan and gp wing analysis. in an attempt to map linear epitopes, all mabs were tested by elisa pepscan against synthetic -mer peptides designed from ravn gp sequences, overlapping by amino acids. pepscan was repeated for mabs a , a , d and g against peptides designed from musoke gp sequences. as a control for gp wing pepscan defined epitopes, additional elisas were performed with a marv gp lacking both the gp mld (Δ - ) and the gp wing (Δ - ), termed gpΔmucΔw. gp wing-directed antibodies were further evaluated for binding to both wild-type ravn gpΔmuc and ravn gpΔmuc containing a point mutant (e k). coating antigens for the point mutant elisas were produced in hek t cells to represent a mammalian glyco-profile in and around the gp wing. pseudovirus neutralization assays. vesicular stomatitis virus (vsv) pseudovirions containing a gfp gene in place of the vsv envelope glycoprotein gene (vsvΔg) and bearing the full-length glycoprotein of marv ravn were generated as previously described [ ] . pseudovirions were incubated with anti-vsv g mab (a gift from a. takada) for hour at rt, then incubated with μg/ml of each anti-marv gp mab in dmem- % fbs (gibco) for an additional hour at rt. pseudovirion/mab complexes were added to vero cell (atcc) monolayers in -well plates at a multiplicity of infection (moi) between . and . . after hours, infection was evaluated by counting gfp-expressing cells. experiments were performed in triplicate and standard deviations displayed. all mammalian cell lines used in this study tested negative for mycoplasma contamination at tsri. animal work. all procedures with infectious marburg viruses were performed in a biosafety level (bsl ) facility at usamriid. male and female balb/c mice between and weeks of age were challenged intraperitoneally (ip) with plaque-forming units of mouseadapted marv ravn [ ] in two separate studies. one hour post-exposure, the mice were treated ip with μg of purified monoclonal anti-marv gp antibody in pbs ( . mg/ml) or pbs alone. study two included an additional negative control group treated with μg of anti-ha igg in pbs ( . mg/ml). each test group consisted of animals for a total of mice. all antibodies were blinded by ibt before submission to usamriid researchers. animals were weighed and monitored daily over a day period post-challenge, at which point mice were euthanized in accordance with an iacuc-approved protocol. once animals were symptomatic, they were examined twice per day. health was scored using the following parameters: = normal, = reduced grooming/ruffled fur, = subdued, = lethargic/hunched posture (provide dietgel for hydration), = unresponsive; euthanize. health scores for the g and a treatment groups in fig are shown for the one or two animals that survived, respectively. no animals were excluded from analysis and the experiments were not randomized. all balb/c mice used in these experiments were obtained from the frederick cancer research and development center, national cancer institute (frederick, md). statistical analysis. graphpad prism . software was used to calculate p values using the log-rank mantel cox test. each treatment group was compared to the corresponding pbs control for either study # or # . p values > . are considered non-significant (ns). purified ravn gpcl was evaluated by deuterium exchange mass spectrometry (dxms) as previously described [ ] . for negative stain em analysis, marv ravn ectodomains were produced in drosophila s cells as described above. fab g and a fragments were generated by standard papain digestion (sigma) of igg and purified by mono q (ge healthcare) ion-exchange chromatography. five molar excess fab was added to trimeric gpΔmuc or gp and allowed to bind overnight at °c. complexes were diluted to . mg/ml in tbs buffer and deposited onto to carbon-coated copper mesh grids which had been plasma cleaned for sec (gatan) and stained for sec with μl of % uranyl formate. the stain was blotted off the edge and the grid was allowed to dry. data were automatically collected with leginon [ ] using a fei tecnai f electron microscope operating at kev with an electron dose of e -/Å and a magnification of , x that resulted in a pixel size of . Å at the specimen plane when collected with a spirit k x k ccd camera (for g ) and . Å at the specimen plane when collected with a tietz k x k ccd camera (for a ). images were acquired at a constant defocus value of - . μm at various tilt angles from to °. particles were picked automatically using dog picker [ ] and placed into a particle stack using the appion software [ ] . reference-free d class averages were calculated by using particles binned by with the xmipp clustering d alignment software [ ] and sorted into~ - particles per class. supporting information s fig. (a) western blot of anti-gp marv mabs incubated at μg/ml, against reduced (+ dtt) and non-reduced (-dtt) purified ravn gp. (b) non-reducing - % sds-page gels of several purified marv gp and ebola antigens from s cells. note ravn gpcl runs larger than ebola gpcl due to extra mass of the gp wing. proposal for a revised taxonomy of the family filoviridae: classification, names of taxa and viruses, and virus abbreviations phylogenetic assessment of filoviruses: how many lineages of marburg virus? on the etiology of an unknown human infection originating from monkeys fatal human disease from vervet monkeys agent of disease contracted from green monkeys forty-five years of marburg virus research response to imported case of marburg hemorrhagic fever, the netherland imported case of marburg hemorrhagic fever-colorado outbreak of marburg virus disease in johannesburg marburg virus angola infection of rhesus macaques: pathogenesis and treatment with recombinant nematode anticoagulant protein c marburgvirus genomics and association with a large hemorrhagic fever outbreak in angola biochemical analysis of the secreted and virion glycoproteins of ebola virus the glycoproteins of marburg and ebola virus and their potential roles in pathogenesis conserved receptor-binding domains of lake victoria marburgvirus and zaire ebolavirus bind a common receptor structure of the ebola virus glycoprotein bound to an antibody from a human survivor structural basis for marburg virus neutralization by a cross-reactive human antibody ebolavirus glycoprotein gp masks both its own epitopes and the presence of cellular surface proteins steric shielding of surface epitopes and impaired immune recognition induced by the ebola virus glycoprotein proteolytic processing of marburg virus glycoprotein cellular entry of ebola virus involves uptake by a macropinocytosis-like mechanism and subsequent trafficking through early and late endosomes ebolavirus is internalized into host cells via macropinocytosis in a viral glycoprotein-dependent manner endosomal proteolysis of the ebola virus glycoprotein is necessary for infection role of endosomal cathepsins in entry mediated by the ebola virus glycoprotein ebola virus glycoprotein needs an additional trigger, beyond proteolytic priming for membrane fusion biochemical and structural characterization of cathepsin l-processed ebola virus glycoprotein: implications for viral entry and immunogenicity cathepsin cleavage potentiates the ebola virus glycoprotein to undergo a subsequent fusion-relevant conformational change proteolysis of the ebola virus glycoproteins enhances virus binding and infectivity ebola virus entry requires the cholesterol transporter niemann-pick c small molecule inhibitors reveal niemann-pick c is essential for ebola virus infection mechanism of human antibody-mediated neutralization of marburg virus sustained protection against ebola virus infection following treatment of infected nonhuman primates with therapeutic intervention of ebola virus infection in rhesus macaques with the mb- monoclonal antibody cocktail protective efficacy of neutralizing monoclonal antibodies in a nonhuman primate model of ebola hemorrhagic fever delayed treatment of ebola virus infection with plant-derived monoclonal antibodies provides protection in rhesus macaques reversion of advanced ebola virus disease in nonhuman primates with zmapp postexposure antibody prophylaxis protects nonhuman primates from filovirus disease potent neutralization of botulinum neurotoxin by recombinant oligoclonal antibody antibodies in infectious diseases: polyclonals, monoclonals and niche biotechnology development of a highly protective combination monoclonal antibody therapy against chikungunya virus an update on the use of antibodies against the filoviruses protective antiviral antibodies that lack neutralizing activity: precedents and evolution of concepts preventing infectious disease with passive immunization inhibition of marburg virus budding by nonneutralizing antibodies to the envelope glycoprotein epitopes involved in antibody-mediated protection from ebola virus development and characterization of a mouse model for marburg hemorrhagic fever structures of protective antibodies reveal sites of vulnerability on ebola virus a shared structural solution for neutralizing ebolaviruses divergent immunoglobulin g subclass activity through selective fc receptor binding delayed treatment of ebola virus infection with plant-derived monoclonal antibodies provides protection in rhesus macaques reversion of advanced ebola virus disease in nonhuman primates with zmapp human monoclonal antibody combination against sars coronavirus: synergy and coverage of escape mutants production of monoclonal antibodies a system for functional analysis of ebola virus glycoprotein automated molecular microscopy: the new leginon system dog picker and tiltpicker: software tools to facilitate particle selection in single particle electron microscopy appion: an integrated, databasedriven pipeline to facilitate em image processing a new generation of the imagic image processing system key: cord- -f l ty j authors: jaworski, elizabeth; routh, andrew title: parallel clickseq and nanopore sequencing elucidates the rapid evolution of defective-interfering rnas in flock house virus date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: f l ty j defective-interfering rnas (di-rnas) have long been known to play an important role in virus replication and transmission. di-rnas emerge during virus passaging in both cell-culture and their hosts as a result of non-homologous rna recombination. however, the principles of di-rna emergence and their subsequent evolution have remained elusive. using a combination of long- and short-read next-generation sequencing, we have characterized the formation of di-rnas during serial passaging of flock house virus (fhv) in cell-culture over a period of days in order to elucidate the pathways and potential mechanisms of di-rna emergence and evolution. for short-read rnaseq, we employed ‘clickseq’ due to its ability to sensitively and confidently detect rna recombination events with nucleotide resolution. in parallel, we used the oxford nanopore technologies’s (ont) minion to resolve full-length defective and wild-type viral genomes. together, these accurately resolve both rare and common rna recombination events, determine the correlation between recombination events, and quantifies the relative abundance of different di-rnas throughout passaging. we observe the formation of a diverse pool of defective rnas at each stage of viral passaging. however, many of these ‘intermediate’ species, while present in early stages of passaging, do not accumulate. after approximately days of passaging we observe the rapid accumulation of di-rnas with a correlated reduction in specific infectivity and with the nanopore data find that di-rnas are characterized by multiple rna recombination events. this suggests that intermediate di-rna species are not competitive and that multiple recombination events interact epistatically to confer ‘mature’ di-rnas with their selective advantage allowing for their rapid accumulation. alternatively, it is possible that mature di-rna species are generated in a single event involving multiple rna rearrangements. these insights have important consequences for our understanding of the mechanisms, determinants and limitations in the emergence and evolution of di-rnas. introduction rna viruses are extremely diverse and rapidly evolving. their rna-dependent rna polymerases (rdrps) readily generate single-nucleotide variants whilst lacking proof-reading capabilities [ ] . rdrps are also highly prone to rna recombination [ ] ; either through template-switching [ ] or through non-replicative end-joining [ ] . rna recombination has been demonstrated to be responsible for the emergence of new strains or species of viruses such as rhinoviruses [ ] and dengue virus [ ] , and the formation of vaccine-derived poliovirus [ ] . non-homologous rna recombination is also responsible for the generation of defective rnas [ , ] . these are versions of the parental viral genome that can arise naturally during the course of viral passaging but have been truncated and rearranged by rna recombination. while not encoding for functional viruses themselves, they can be amplified and co-passaged with the help of the wild-type 'helper' virus that provides the necessary machinery for replication, encapsidation and transmission. a defective rna that accumulates to such an extent as to compete with or otherwise attenuate the replication of the parental virus is known as a defective-interfering rna (di-rna) [ ] . di-rnas can attenuate the viral infection via a variety of proposed mechanisms such as the saturation of the viral replicative machinery, sequestration of essential cellular cofactors, and/ or induction of innate immune responses [ ] [ ] [ ] [ ] [ ] . di-rnas have been well characterized for a number of rna viruses as they provide valuable tools to molecular virologists by revealing conserved regions and functional domains in the rna genome such as binding sites for viral or host factors. moreover, characterizing recombination loci reveal the mechanisms of recombination, impacting our understanding of viral evolution [ , , ] . until recently [ ] , due to difficulties in capturing and characterizing di-rnas in vivo, di-rnas were considered to be a curious epiphenomenon of cell-culturing practices. as a result, our appreciation of the diversity of di-rnas and the range of situations in which they could play a role was greatly limited. increasingly, due to the use and sensitivity of next-generation sequencing (ngs) technologies, di-rnas have been observed in a multitude of viral systems under laboratory conditions (e.g. sars coronavirus [ ] , hiv [ ] ), in clinical settings (e.g. measles [ ] , dengue [ ] and chronic hepatitis c [ ] ) and in metagenomic or 'wild' samples (e.g. west nile virus [ ] , influenza virus [ ] ). despite this burgeoning range of hosts for di-rnas, limitations in ngs technologies including high artifactual recombination rates, short reads and a limited range of bioinformatics tools tailored to viral rna recombination discovery has hindered our ability to detect and characterize di-rnas in complex or clinical samples. flock house virus (fhv) is a positive-sense single-stranded rna (+ssrna) virus originally isolated from grass grubs in new zealand [ ] and is perhaps the best-studied alphanodavirus from the nodaviridae family. fhv infects drosophila flies and cells in culture as well as medically important genera of insects including mosquitos, (anopheles gambiae), the tsetse fly (glossina morsitans morsitans westwood), and the chagas vector (rhodnius prolixus stal) [ ] . infection of these organisms by fhv has been demonstrated to have similar characteristics in terms of viral titer, virus dissemination and mortality as has been shown for fruit fly infections. fhv provides an excellent model system to study +ssrna virus evolution by virtue of having one of the smallest known eukaryotic virus genomes [ ] . moreover, the viral life-cycle and details of the molecular biology of virus particle assembly, cell entry and particle disassembly are highly-characterized. fhv contains two genomic rnas. rna ( nts) encodes the viral rdrp and rna ( nts) encodes the viral capsid protein. rna also expresses a small sub-genomic rna, called rna , that encodes the b protein responsible for inhibition of the anti-viral rnai machinery [ ] . fhv has been demonstrated to form di-rnas in multiple independent studies spanning three decades both in cell-culture [ , , [ ] [ ] [ ] [ ] and in drosophila melanogaster [ ] . many of these studies characterized individual di-rna genomes through sub-cloning and sanger sequencing. intriguingly, many of these di-rnas are highly similar. this indicates that either the di-rnas have emerged due to a common mechanism of formation or the presence of a common selectivity filter, or both. our recent ngs studies of rna recombination in fhv revealed a diverse array of rna recombination events, suggesting that the genomic landscape of di-rnas is highly dynamic and likely contributes significantly to the diversity of viral genomes that form the viral quasi-species [ ] . despite these findings, studies to-date present only a single snap-shot of the di-rna genome landscape and do not capture the pathways of their emergence and evolution nor characterize any intermediate di-rna species that might arise during these processes. in order to resolve the potential mechanisms of di-rna emergence and elucidate the evolutionary pathways that lead to the formation of 'mature' di-rnas, we performed high-titer serial passaging of fhv in cell culture and characterized the encapsidated rna using rnaseq. we used illumina hiseq sequencing of clickseq generated libraries to provide a high-resolution and high-confidence quantification of individual recombination events. we combined this information with long-read oxford nanopore technologies's (ont) minion sequencing to resolve the topology of full-length and defective rna genomes. by combining these data, we aimed to determine the correlation of recombination events within single rna virus genomes, characterize the distribution of defective rna genomes, and determine the exact make-up of di-rnas during serial passaging of fhv in cell culture. we recently developed the 'clickseq' method for rnaseq that uses copper-catalyzed alkyne-azide cycloaddition (cuaac), a click-chemistry reaction, for rnaseq library synthesis [ ] . clickseq provides a robust platform on which to study rna recombination in rna viruses. artifactual recombination is a common contaminant in ngs library generation and can easily obscure rare or non-canonical recombinant species. clickseq does not require template fragmentation and replaces enzymatic ligation steps commonly required in ngs library generation with click-chemistry. clickseq works by introducing small amounts of azido-nucleotides (azntps) into rt-pcr reactions to generate azido-terminated cdna transcripts. these cdna fragments are subsequently mixed with alkyne-labelled dna adaptors. the addition of a copper catalyst results in the 'click-ligation' of the two chemically-functionalized dna substrates to produce an unnatural triazole-linked single-stranded dna molecule [ ] . clickseq prevents template switching during rt-pcr as well as non-specific ligation of rna fragments. we demonstrated that clickseq reduces artifactual recombination to fewer than events per million mapped reads [ ] . as a result, clickseq provides a superior method for the detection of di-rnas and rna recombination events. the oxford nanopore technologies's (ont) minion is a small handheld sequencing device [ ] poised to revolutionize the next-generation sequencing field by providing realtime, high-throughput and long-range (over kbp [ ] ) sequences of dna samples with minimal sample prep. ont nanopore sequencing has been used to rapidly characterize virus genomes from metagenomic samples [ ] , in the midst of ebola virus outbreaks [ ] , and in targeted studies aimed at characterizing sequence variations within influenza virus samples [ ] . highly parallel direct rna sequencing using nanopore technology was also recently reported [ ] . due to the higher error-rate [ ] of the nanopore sequencing technology compared to other rnaseq platforms, the exact identity of recombination events within singlemolecule genomes may be inaccurate. however, long-read nanopore reads provide the distinct advantage of being able to sequence full-length cdna copies of rna virus genomes and thus can resolve multiple recombination events within a single rna virus genome. this study provides a comprehensive analysis of the steps and pathways governing di-rna emergence and evolution starting from a plasmid-driven inoculum through to a highly-passaged sample. by combining short-read and long-read sequencing technologies, we determine both the exact identity of rna recombination sites and their correlation within the viral quasispecies. we find little evidence for the accumulation of intermediate defective rna species that contain either only one, or smaller, deletions during the course of passaging. rather, fully formed 'mature' di-rnas that are characterized by two to three deletions between a limited number of positions in each of the fhv genomic rnas appear after approximately days of viral passaging and accumulate rapidly. the accumulation of di-rnas corresponds with a reduction in the specific infectivity of the viral samples in each passage. this implies that partially formed di-rna species are not competitive and cannot accumulate in the manner that mature di-rna species do, perhaps due to the epistatic interaction of multiple recombination events. alternatively, the formation of mature di-rnas may occur in a single step involving multiple simultaneous genome rearrangements. cell culture and virus passaging d. melanogaster (s ) cells were grown at ˚c in schneider's drosophila media supplemented with % fetal bovine serum and x penicillin-streptomycin using standard laboratory procedures. to generate the initial flock house virus inoculum, s cells were plated at - % confluency in a six well plate and were transfected with . μg of pmt plasmid containing fhv rna (nc_ ) and . μg of pmt plasmid containing fhv rna (nc_ ) using lipofectamine transfection reagent as per the manufacturer's protocol. plasmid transcription was induced hours post transfection with the addition of mm cuso . virus was then allowed to propagate for days post induction. for successive passages (passages - ), s cells were grown in t- flasks to - % confluency (~ x cells), then infected with ml of viral inoculum from the previous passage. virus was grown for days, then fractions were harvested for viral purification or inoculation of the next passage. to purify virus from each consecutive serial passage, cells and supernatant were subjected to a freeze-thaw cycle in the presence of % triton x- to release viral particles from infected cells. virus particles were then purified on a % sucrose cushion by spinning the cell lysate at , rpm for . hours. the viral pellet was resuspended in mm tris (ph . ). virus was further purified by applying resuspended virus atop a - % sucrose gradient and spun at , rpm for . hours. the viral band was collected and subsequently treated with unit dnase and unit rnase and incubated at room temperature for at least one hour to remove any cellular nucleic acids not protected by the viral capsid. the virus sample was concentrated on a , nmwl centrifugal filter column and washed with at least volumes of mm tris ph . . finally, encapsidated viral rna was extracted using a qiagen rneasy mini kit as per the manufacturer's protocol. next generation sequencing (ngs) libraries were generated using ng of rna using the 'clickseq' protocol as previously described by routh et al. [ , , ] . briefly, cdna is synthesized through rt-pcr initiated from semi-random ( n) primers containing a partial illumina p adapter (gtgactggagttcagacgtgtgctcttccgatctnnnnnn) and stochastically terminated by the addition of azido-ntps (azntp) at a ratio of : azntp:dntps. subsequently, the p click-adapter ( '-hexynyl-nnnnagatcggaagagcgtcgtgta gggaaagagtgtagatctcggtggtcgccgtatcatt, idt) was click-ligated onto the azido-terminated cdna fragment using copper-catalysed azide-alkyne cycloaddition (cuaac) in the presence of tbta ligand (lumiprobe) and vitamin c catalyst in % dmso. after purifying the click-linked cdna with a zymo dna clean column, cycles of onetaq (neb) pcr amplification adds the remainder of the p adapter along with the desired truseq index sequence. pcr product was cleaned again with a zymo dna clean column to remove excess primers and then ran on a - % precast agarose e-gel (invitrogen, e-gel electrophoresis system). cdna libraries between to bp were excised corresponding to insert sizes of - bp and cleaned using the zymo research gel dna recovery kit. final cdna libraries were quantified using a qubit fluorimeter (life tech) and loaded on a hiseq single read rapid run flowcell for x reads and nucleotides of the index. fhv libraries used for the triplicate study shown in s fig were sequenced on a miseq platform with v chemistry for cycles ( x ). reads were trimmed to nts prior to analysis to emulate the libraries sequenced on the hiseq. raw reads were processed by first removing the illumina truseq adaptor using cutadapt [ ] with default parameters. next, the first nucleotides (corresponding to the random nucleotides and triazole-linkage included in the click-adaptor) were trimmed and any reads that contained nucleotides with a phred score < were removed using the fastx toolkit (http://hannonlab.cshl.edu/fastx_toolkit/). the remaining reads were aligned end-to-end with bowtie (v . . ) [ ] (command line parameters: -v --best) first to the fhv genome (nc_ and nc_ ) and next to host d. melanogaster genome (fb _ ). the remaining unmapped reads were processed to identify recombination events using the python script 'virema' (viral recombination mapper) [ ] (command line parameters:--n --x --seed --host_seed --defuzz --microindel ). the frequency of a specific recombination event is approximated by dividing the number of reads mapping to this recombination (n) by n plus the average of the number of reads mapping to the wild-type genome at each of the recombination coordinates. the oxford nanopore technologies's (ont) minion and flowcells were acquired as part of the ont early-access program. to prepare sequencing libraries for the minion, ng of rna was reverse transcribed using rna specific primers that were complimentary to the ' end of the respective genome (rna _rp: acctctgccctttcgggcta or rna _rp: acctt agtctgttgacttaa). cdna was then amplified using the standard phusion (neb) pcr protocol using genome specific primers (rna _fp: gttttcgaaacaaataaaac or rna _fp: gtaaacaattccaagttcca) for cycles. excess primers were removed from the pcr product using ampure xp beads (beckman coulter) at a ratio of : ampure bead:pcr product. samples were then barcoded and prepared following the manufacture's protocol (r native barcoding kit i and nanopore sequencing kit) with adjustments to tailor input cdna quantities. a target of ug of fragmented dna at approximately ' nts is considered optimal for library generation using this kit. the input amounts for rna ( bp) and rna ( bp) were thus adjusted to ng and ng respectively and combined in ul water to maintain optimal dna end molarity. after ligation of barcodes, equal amount of each dna library ( samples in total) were pooled and loaded onto a minion mkib device equipped with an r flow cell. the minknow control software was used to select a -hour sequencing protocol and was allowed to proceed for at least hours, until high-quality data accumulation ceased. raw data was uploaded automatically by metrichor software for cloudbased base-calling using default settings and quality filtering for -dimensional reads. reads were extracted from hdf format files (fast ) using poretools [ ] . ont nanopore data processing and alignment full-length ont reads were mapped to the flock house virus genome using the pacbio wrapper from the bbmap v suite (command line parameters: fastareadlen = vslow = t maxindel = minid = . local = f ignorebadquality = t usequality = f). alignment sam files were visualized using the tablet sequence viewer [ ] . sam files were filtered to ensure that minion reads mapped from the first nts to the final nts of the reference genome (accounting for deletions and insertions), due to the presence of truncated nanopore reads and mis-priming during the cdna pcr amplification steps. errors including substitutions, insertions and deletions were counted using the samtools [ ] mpileup command and error rates at each position were calculated by dividing this value by the read depth at this position (s fig). insertion and deletion events longer that nts were extracted using the cigar string of the sam files using simple in-house scripts. for recombination sites containing 'fuzz', where nucleotides surrounding the putative recombination events are the same for both the acceptor and donor sites, the recombination event was reported as occurring in the middle of the 'fuzzy' region, or at the ' side of the middle two nucleotides in the orientation of the reference if the fuzzy site contained an even number of residues. this is the same methodology as employed in the virema script [ ] used to map recombination event in the clickseq data. insertion events and soft-pads longer than nts were extracted and their nucleotide sequence was analyzed using an online blastn search to determine their identity. to annotate the defective genomes detected by minion nanopore sequencing or recombination events detected by clickseq, we use underscores '_' to denote continued mapping, and carets '^' to denote a recombination events. for example, " _ ^ _ ^ _ " indicates an authentic mapping from nt to , then a deletion event removing nts through , then another authentic mapping from to , followed by another deletion removing nts through , and finally an authentic mapping from nt to . the shannon entropy index is given by: hðxÞ ¼ À p nÀ i¼ p i ln p i for the clickseq data, each recombination event is treated as independent with its probability determined by dividing the number of reads mapping to the present recombination event divided by the average coverage over the whole viral rna. for the minion data, each individual read mapping is treated as an individual event with the frequency determined by dividing the number of identically mapping reads divided by total mapped reads. tissue culture infective dose (tcid ) analyses of the supernatants from each passage were performed using standard protocols [ ] . for purified particles of each passage tcid was calculated with slight modifications. specifically, x cells (s ) per well were plated in well format. virus samples were quantified by measuring the od nm . an od of . corresponds to mg virus [ ] , which in turn corresponds to . x virus particles assuming a virion mass of . mda [ ] . purified virus samples were diluted to a starting concentration of ng/μl, which corresponds to x virus particles per μl. these quantities were chosen as particle-to-pfu ratios for rescued fhv has previously been reported to be - particles [ , ] . therefore x particles per cells corresponds to approximately pfus per cell. eight serial -fold dilutions were subsequently made and added to each column of the -well plate ( replicate wells per dilution), as per standard tcid protocols. virus was allowed to grow for days after which the number of positive wells exhibiting cytopathic effect (cpe) were counted. the tcid values and effective moi were calculated using the reed and muench calculator [ ] . we further counted the total number of cells that were present in each well after infection using a guava easycyte ht (millipore) flow cytometer to provide us with quantifiable amount of cell death. μl from each well was diluted in μl pbs and injected using manufactures' protocols. the incyt v . software was used to collect data with the following parameters: collection time: sec; flow rate: . μl/sec; fsc: ; ssc: ; threshold: . the region used to determine live cells was based on scattering features of the negative controls (wells with no viral infection) (s fig). all raw illumina data and demultiplexed minion nanopore data passing quality filters (comprising d, template and complement strands) associated with this manuscript are available on the sra ncbi archive with study number srp and bioproject number prjna . d. melanogaster (s ) cells in culture were transfected with cdna plasmids containing each of the flock house virus genomic rnas followed by a hepatitis d virus (hdv) ribozyme sequence. after induction, the hdv ribozyme regenerates the authentic ' end of the positive sense viral rna, which is thus successfully recognized by the fhv rdrp allowing the initiation of viral replication [ ] . we choose to initiate replication with this method to ensure that the starting viral population would be homogeneous containing only the full-length rnas derived from the plasmid cdna. after transfection, the viral inoculum was allowed to amplify for days (passage number = p ), after which most cells exhibited cytopathic effect. subsequently, the supernatant from infected cells was collected and a ml fraction ( % of the total volume) was used to infect ml of fresh s cells in triplicate (replicates r , r , and r ). again, after three days, ml of the supernatant was harvested and used to infect fresh s cells in series for a total of nine -day passages (passage numbers = p -p ). therefore, one single inoculum was used to generate three distinct lineages as shown in fig . for each passage and replicate, including the original inoculum, viral particles were purified over a sucrose cushion and non-encapsidated genetic material was degraded to ensure that the genetic material subsequently analyzed was packaged within the viral capsid. rna was extracted from the purified viral particles using standard silica-based spin columns. clickseq libraries [ ] were synthesized from the purified viral genomic rna and sequenced on an illumina hiseq for x single-end reads. the inoculum sample was sequenced on a separate flowcell to all other samples to prevent any cross-contamination from incorrect demultiplexing. we obtained . - . million reads after trimming and quality filtering for each passaged sample, and . million reads for the original inoculum ( table and s table) . million reads corresponds to an average coverage of greater than ' x across the fhv genome. reads were aligned to the fhv genome and the host genome (d. melanogaster, fb _ ) using bowtie end-to-end mapping [ ] . as expected, the majority of the reads aligned to fhv rna ( nts) and fhv rna ( nts) in a ratio reflecting the longer length of rna . as we have observed previously [ , ] , . - . % of reads correspond to host rnas that are encapsidated within the viral particles including mrnas, ribosomal rnas and retrotransposons. interestingly, the amount of encapsidated host rna increases modestly through later passages (s fig) . subsequently, we further characterized the unmapped reads with the python script virema ("virus recombination mapper") [ ] . virema is a computational pipeline optimized for mapping virus recombination junctions in ngs data with nucleotide resolution by dynamically generating moving read segments. virema is sensitive to many types of rna recombination events. this includes micro-insertions and deletions (indels comprising or fewer nucleotides), duplications, deletions, inter-rna recombination (denoting recombination between fhv rna and rna ) and virus-to-host recombination events, and reports both the identity and frequency of recombination events. recombination events that cannot be unambiguously identified due to unmapped read segments, mismatches occurring near to putative recombination events, or reads containing fragments of sequencing adaptors are flagged as 'other' (table ) [ ] . in each genomic rna we found hundreds of unique recombination events, reflecting a diverse and complex mutational landscape (s datafile). broadly, we see an increase in the total number of recombination events during serial passaging of fhv and a corresponding increase in the shannon diversity index (fig a and b) . following these mapping procedures, few reads ( - . %, table and s table) remained uncharacterized. as in previous studies, these were found to be derived from incorrect demultiplexing of neighboring samples on the hiseq flow-cell [ ] or from contaminants in the rnaseq library generation [ ] . having accounted for almost all of the reads present in each dataset, we can be confident that we are capturing the full range of recombination events and/ or other rearrangements present within each sample and thus are not missing important or significant events due to computational limitations. to demonstrate the reproducibility of the clickseq approach and to assess the limit in terms of our ability to successfully detect rare recombination events, we generated three virus was allowed to propagate for three days after which cells and the supernatant were collected. a fraction ( : ) was used to further infect a new population of s cells in a series of passages. the remaining fraction was collected for analysis. in total, three replicates of nine passages were collected. replicate clickseq libraries from the rna sample p r , obtained x bp reads and performed the same computational analyses as described above. we mapped . m, . m and . m reads per library, giving an approximate coverage of~ , - , x coverage over fhv rna and~ , - , x over fhv rna (calculated from the average coverage over the conserved ' and ' ends). when comparing the frequency of unique recombination events in either rna or rna between any pair of the three replicates, we find excellent correlation (pearson > . ) even for very infrequent recombination events, as illustrated in the scatter plots in s fig. events that were found in two replicates but not a third, never exceeded more than mapped reads for rna and reads for rna . if we take these values as a cut-offs, below which we begin to fail to detect events, then we can conservatively estimate that we are reproducibly sensitive to recombinant species that are present at approximately . % ( / , ) of rna population and . % ( / , ) of the total rna population when obtaining~ m sequence reads. in the inoculum (p ), less than . % of the all the reads mapped to recombination events ( table , fig a) . inspection of these events reveals that they are dispersed throughout each of the genomic rnas. rna recombination events are the least frequent, with only unique events detected represented by reads and without an apparent bias toward any specific location. the three most common rna events in the inoculum are ^ , ^ , and ^ with , , and mapped reads respectively (s datafile). read depth for the wild-type genome at these loci ranges from k to . m reads, therefore these recombination events make-up at less than . % of the total viral population. these are not the events that have been previously reported as forming fhv di-rnas, moreover none of these events are observed again in subsequent passages, perhaps due to approaching the sensitivity of discovery limit as described above. however, due to the low-rate of artifactual recombination of the clickseq approach (> events per millions reads [ ] ), we can be confident that these are not sequencing artifacts [ ] . therefore, these events likely represent non-viable or transient recombination events that arose due to stochastic non-homologous recombination. for rna in the inoculum, the three most frequently observed events were ^ , ^ and ^ , with , , and mapped reads respectively (s datafile). fig a) . again, these occur throughout each of the genomic rnas. however, we do begin to see events that have previously been characterized as forming di-rnas, such as ^ and ^ , although these events are present at low levels ( and reads in replicate from a total of . m reads mapped to the fhv genome) (s datafile). in subsequent passages there was a rapid increase in the total proportion of mapped recombination events (peaking at . % in p r ) (table , fig a) . in these later passages for each replicate, it can be seen that the most common recombination events are deletions that span two regions in each genomic rna including: nts - and nts - of rna ; and nts - and nts - of rna , consistent with previous observations of fhv di-rnas [ ] (s datafile). however, the exact sites of the recombination events, while repeatedly observed over time in each replicate, varied between replicates and each had distinct 'most popular' species in the final passages (table ). in some instances we are able to find that a specific event is predominant in one replicate while at low levels in another. overall, these trends in the frequency of recombination events throughout passaging reveal that once defective rna species emerge during viral passaging they rapidly accumulate. despite the predominance of certain recombination events in the later passages, there still remained a large number of infrequent events scattered throughout the viral genome. many of these are observed only in one passage and not in subsequent passages. again, these events likely correspond to stochastically generated rna recombination events that form non-viable defective rnas. we reasoned that analyzing these events would more accurately reveal the nucleotide preference of the fhv rdrp for rna recombination as they were not subject to replicative selection (although they must be packaged by fhv particles), unlike the di-rnas. therefore we extracted all recombination events occurring with fewer than mapped reads throughout all fhv passages and replicates ( ' unique events from a total of . m possible permutations [ ] ) and counted the frequency of nucleotides found both up and down-stream of ' and ' recombination sites in the reference genome. as shown in fig c, this revealed a preference for a's - nts downstream of ' sites, a preference for u's - nts upstream of ' sites, a weaker preference for a c nt up stream of ' sites, and an aversion to g's nts both upstream and downstream of ' sites. interestingly, an almost identical trend was observed for the ' sites. this trend was maintained also when analyzing only recombination events without ambiguity pathways of fhv defective rna evolution by clickseq and the minion in the site of recombination (i.e. sites that lacked 'fuzziness' as reported by the virema pipeline [ ] ). this result is similar to what we have previously reported [ ] . however, here we provide a much larger dataset and analyze events that we can determine are not amplified in subsequent passages, providing greater confidence that these sites reflect the preference for rna recombination at these sites rather than the selection of replicatively viable defective rna species. open reading frames are maintained in most di-rnas since many recombination events resulted in deletions of the viral genome, we were curious to see if the open reading frame (orf) was conserved, as conservation of an orf has frequently been observed to be a property of defective and defective-interfering rnas [ ] . moreover, it has previously been shown that cloned di-rnas vectors containing egpf in their putative orfs do indeed express fluorescent protein [ ] although it is not clear whether a functional orf is essential for di-rna formation or propagation. in the earliest passages, only~ % of deletions removed a multiple of nucleotides (i.e. they thus conserved the orf), as would be expected if deletion events occurred randomly throughout the genome. however, with continued passaging, there was a general trend toward conservation of the orf for both rna and rna (fig d) , although this was not the case for all replicates. specifically, while initially showing an increase in orf conservation, replicate of rna showed a decrease in the conservation of the orf after passage , in contrast to the other two replicates, and in fact dips below %. closer inspection of individual recombination events shows that this trend is driven by three of the four most common recombination events in replicate passages to : ^ , ^ , ^ and ^ (the latter three events in bold do not maintain the orf). these events are observed in the other replicates, but at a much reduced frequency (no more than % of the total rna recombination events for reps and ) (s datafile). using virema, we were able to calculate the frequency with which each nucleotide was deleted, revealing areas of the viral genome that are conserved during serial passaging and required for di-rna replication. we plotted these data to generate recombination profiling maps for each rna of fhv throughout passaging (fig and s fig). in the first passage, there is a relatively even distribution of nucleotide deletions along the whole length of the genome with the exception of two frequently excised regions in the ' end in rna due to two common recombination events in each of the replicates: ^ and ^ . by passage the deletions along the genomic landscape begin to be 'sculpted' whereby certain regions are deleted with a greater frequency than others. passages , , and were sculpted further revealing three major regions that were deleted in rna and two in rna . interestingly, for both rna segments, while a range of deletions and rearrangements are generated during early passages, only the deletions that maintain regulatory and control elements are amplified during continued passaging, as previously observed in fhv [ ] . these include the '/ ' utrs and internal response elements (intre) of both genomes, as well as the proximal-and distal-subgenomic control elements (psce and dsce) in rna (fig ) , which correlates to the findings that these regions are important and required for rna replication and encapsidation [ , , , [ ] [ ] [ ] [ ] [ ] . the short-read clickseq data provide in-depth and high resolution details of individual recombination events. however, in order to determine the correlation of these events over time, we used long-read ont nanopore sequencing, which can characterize full-length wildtype and defective genomes (fig and s fig) . we reverse transcribed and amplified both rna genes using primers specific to the ' and ' utrs of rna and rna from all passages of replicate to obtain cdna that could be barcoded and analyzed using protocols for d sequencing on the ont minion. the clickseq data shows that these regions were highly conserved during passaging (fig ) , therefore we were confident using template-specific primers to these regions would capture both the full-length wild-type virus genomes as well as any defective rnas. after pcr amplification, we analyzed the minion cdna libraries using agarose gel electrophoresis to observe the distribution of cdna fragments and ratios of full-length to defective rna genomes (fig a) . while the cdnas from the early passages are predominantly of the expected size for full-length rna genomes, later passages contain an array of band sizes. this shows that in the early passages the full-length genome is the predominant species while in later passages the truncated version becomes predominant. we also observe species appearing to be larger than rna . it is possible that some of these species correspond to rna homodimers or other complex rearrangements that have previously been observed [ ] and would result in an increased molecular weight. evidence of rna homodimers ( ^ ), rna homodimers ( ^ ), and rna to rna heterodimers ( ^ ) can also be found in the clickseq data (s datafile). we pooled the amplified cdnas from each sample at equimolar ratios and loaded the pooled, barcoded library onto a minion mkib device using an r flowcell as per the manufacture's protocol. we ran the standard protocol for obtaining -dimensional reads using the minknow control software and collected nanopore reads for approximately hours, upon which the quality and yield of reads dropped substantially. we obtained a total of ' reads, of which ' passed the default ont filter and were successfully demultiplexed. this yielded between and full-length reads per passage and corresponds to approximately . gigabases of sequence information. this would correspond to~ ' x bp illumina reads per sample, assuming even coverage. with this depth, we can build up a comprehensive picture of the full-length genomic landscape of the viral samples, allowing us to resolve di-rna species even if they were present at less than % of the total viral genomic population. the long-read nanopore sequencing data were aligned to the full-length fhv genome using the bbmap suite (https://sourceforge.net/projects/bbmap/). this pipeline tolerates large insertions and deletions in the long-reads, thus allowing us to characterize the overall topology of the defective rnas. we mapped between and . % of the minion reads from each passage to the fhv reference genome ( table ). an example of reads aligned to fhv rna is shown in s fig. the error rate of aligned reads, including single nucleotide mismatches and small indels, was determined from alignment pileup files. consistent with recent reports [ ] , we found the overall modal and mean error rates for all mapped position was . % and . % respectively, with % of the sites having an error rate better than . % (n value = . ). example of how reads generated from the illumina hiseq would map to a reference genome. the bowtie alignment is able to map bp reads along the reference with a relatively even coverage distribution. unmapped reads are then aligned with virema to accurately identify recombination events. the low error rate of highthroughput sequencing allows us to precisely define the boundaries of the junctions. below the reference genome is an example of how reads generated from oxford nanopore technologies's minion sequencing would map to a reference genome. the minion is able to generate full length reads at the expense of a high error rate, which is~ %. full length analysis allows us to determine what recombination events a genome contains. due to the error rate the exact boundaries of the recombination event are imprecise. (s b and s c fig) . this shows that small indels are frequent, but fall quickly in abundance with increasing length. . % and . % of all minion deletions and insertions respectively were shorter than nts. therefore, we considered only deletions and insertions of at least nts to be likely to be bona fide indels present in the original viral rna and corresponding to recombination events comprising defective rna species rather than a sequencing error. the nanopore data reveals the presence and frequency of large deletions and insertions within defective rna genomes. from these, we can reconstruct the population of either full-length or defective rna genomes present in each of the viral passages (annotated as described in the methods section). the full table of characterized defective rnas and their frequencies in each passage is detailed in s datafile. in total, we found and unique defective rnas of rna and rna respectively throughout all passages. the frequency of individual recombination events found in both the clickseq data and the minion data were compared and correlation coefficients calculated ( table ). the correlation in the earliest passages was poor, due to the low abundance of events in both datasets. however, later passages correlate well with pearson coefficients reaching . . this is important as it demonstrates that the frequency of recombination events was not biased during cdna amplification of the full-length or defective viral genomes. similarly, the shannon entropy indices increase during passaging (fig c) , consistent with those from the clickseq data. the number of deletions in each passage in rna and rna are given in table and illustrated in fig b. the earliest passage contains very few deletions. in passage , . % of the reads map to the full-length genome in its entirety. with subsequent passages, the number of reads containing deletions increases, reaching a plateau at passage with . % of the reads containing two deletions and . % containing three deletions. di-rnas (e.g. _ ^ _ ^ _ ) are easily identifiable as early as passage (s datafile) and match well with the number of demultiplexed nanopore reads passing quality filters are shown for each sample. these were mapped end-to-end to the fhv genome using the bbmap suite, allowing for large deletions and insertions, which were counted using the cigar string from the output alignment sam file. the pearson correlation coefficients of these events to those found using clickseq were also calculated. the number of reads mapping to fhv rna and fhv rna are indicated along with the number that contain - or more deletions of at least nts. only a small number (< %) remained unmapped. https://doi.org/ . /journal.ppat. .t pathways of fhv defective rna evolution by clickseq and the minion the expected identities based on our clickseq results and previous studies [ , ] . by the final passages these species predominate, leaving only a small percentage of full-length wild-type viral rnas. while we can readily identify mature di-rnas containing two or more deletions, few single-reads contain just one deletion (< %) in all of the passages. moreover, individual species are rarely observed again in subsequent passages (s fig and s datafile) . importantly, most of these single events do not delete the expected regions common to fhv di-rnas. therefore, they may either correspond to sequencing artifacts, or transient defective rna species generated due to stochastic rna recombination, similar to the low-frequency events observed in the clickseq datasets. in later passages (beginning at passage ), we do begin to see the presumptive intermediates (e.g. _ ^ _ or _ ^ _ ) of mature di-rnas (e.g. _ ^ _ ^ _ ). however, this is after the mature di-rnas were first observed, and after the point at which di-rnas have begun to accumulate. indeed, in passages and respectively, mature di-rnas make up % and % of the viral population while the singly-deleted intermediates make up % (unobserved) and %. together with the observation of rare di-rnas in the inoculum with the clickseq recombination analysis, these data indicate that single-deletion species do occur early during passaging, but remain poorly abundant and do not accumulate. in contrast, mature di-rnas are observed to rapidly accumulate between passages, indicating that they possess a replicative advantage above both wild-type viral genomes and intermediate defective rna species. in addition to deletions, a small number of defective rnas first appearing at passage contained insertions. interestingly, the majority of these comprised short insertions of~ nucleotides that were found in first nts of the minion reads and were mapped between nts and of rna . in each case, these inserts corresponded to nts - of rna . this region corresponds to an internal response element (intre) of the proximal sub-genomic rna control element (psce) previously identified as being essential for fhv rna replication and conserved in di-rnas species [ ] . the most common deletion in the di-rnas in this region of rna for the final passages are from to , which retains the intre. however, there are also a large number of deletions ranging from to , which would delete the essential intre. closer inspection of the minion data reveals that the majority of these reads (> %) that contain the nt intre insertion concomitantly contained deletions from - , indicating that these two events are correlated. the clickseq data also shows a frequent recombination event, ^ , which appears first in passage and is among the most common events in the final passages (s datafile). this matches precisely the ' junction site of the insertion event detected in the min-ion data. however, the event ^ corresponding to the ' junction site was not detected in our initial virema analysis of the clickseq data as this would have required a search seed length of less than nts. repeating the virema analysis using a seed length of does indeed reveal the presence of the ^ recombination event. this event is rarely observed in either of the other two replicate clickseq data ( and total reads across all passages of replicates and respectively). these data indicate that in a number of defective rna genomes, the intre element has been deleted and subsequently re-inserted at the ' end of the defective rna genome. as the intre element is required for regulation of rna replication, presumably this maintains the ability for this highly-rearranged defective rna to replicate. these data provide a comprehensive overview of the different species of defective rnas that are present during viral passaging. illustrating such a complex set of data is a challenge as each sample contains a large number of genome arrangements ( and for rna and rna respectively) and frequencies of these species vary substantially over time. we found that illustrating these data as a stacked area plot gave the most informative summary of the changes of the many different type of di-rna species over time. due to the moderate errorrate of the nanopore read data, the exact identification of a recombination event and thus annotation of that genome may be incorrect. this would result in an over-estimation in the potential number of unique structural variants. therefore we filtered datasets by requiring genomes to be represented by three or more reads. while removing a lot of noise, this has the drawback that we might be losing rare defective rnas. stacked area plots for genomes represented by three or more reads are shown in the stacked area plot for rna (fig a) shows that the composition of di-rnas in the viral population changes over time and new species appear at each passage. for example, the most abundant defective rna species in passage is ' _ ^ _ ^ _ ' but reduces in relative frequency in later passages. the most abundant species in the final passage is ' _ ^ _ ^ _ ', which appears at low levels as early as passage , but does not begin to accumulate until passage (s datafile). why this di-rna only begins to accumulate at later passages despite being present in the early passages is not clear. the 'complex di-rna' that deletes the psce in rna referred to in the previous section (' _ ^ _ ^ _ ' ) is also observed (annotated in fig a) first appearing at passage . as can be seen in the stacked area plot for rna (fig b) , the general composition of di-rna species is established at passages - . subsequently, the relative frequencies of the di-rna fluctuate but the overall diversity changes little with few new species appearing after passage . this is also observed when calculating the shannon diversity index (fig b) whereby entropy reaches a maximum at passage and decreases thereafter. interestingly, this range of fluctuations resemble the sinusoidal patterns of di-rna abundance that have been observed in other studies of rna viruses where the ratio of the frequency of di-rnas to wildtype genome has been measured through longitudinal studies [ ] . the reduction in full-length infectious viral genomes and the accumulation of defective rnas during passaging is likely to correspond to a decrease in the specific infectivity of the virus samples. to determine the effect of defective rnas characterized by combined clickseq and nanopore sequencing of replicate upon specific infectivity, we performed % tissue culture infectious dose (tcid ) assays for each passage - for both the original inoculum used to infect each sample and for the particles purified from each passage [ ] . the tcid assay is used to determine the dose required to give a % chance that cells in culture will be successfully infected as determined by cpe and is typically used to determine viral titer and the effective moi of the inocula transferred from passage to passage. the results from the tcid assay for each passage are shown in fig a and b . we found that the tcid value (and thus pfus(plaque forming units)/ml) drops considerably during passaging by over four orders of magnitude. the corresponding effective moi (pfus per cell) also drops from . to . during passaging (fig b) . and (b) rna . the passage number is indicated on the x-axis and the stacked frequencies of each detected defective rna is shown in the y-axis. each non-contiguous color represents a specific genome characterized by minion nanopore sequencing. wild-type genomes are colored green, genomes with one deletion are colored in shades of blue, and genomes with two or more deletions are colored in shades of orange (using the same color scheme as in fig b) . raw data and annotations are in s datafile. to determine whether the drop in effective moi was driven by reduced total particle yield or from reduced specific infectivity (i.e. virus particles per pfu), we also performed tcid analysis of our purified and quantified virus stocks (described in methods). this allowed us to pathways of fhv defective rna evolution by clickseq and the minion determine and normalize the number of virus particles delivered per cell between each passage. as the particle-per-pfu ratio has previously been estimated at - particles-per-pfu [ , ] we setup our assay beginning with ' particles per cell in well format and performed -fold serial dilutions. in this assay, we found that the number of viral particles required to induce cpe decreased by over -fold during passaging (fig a) with a trend very similar to that for the unpurified inocula. together, these data indicate virus specific infectivity drops with a corresponding increase in the defective rna population. there was an exception at passage where tcid actually increased~ fold from the previous passage. this could be correlated to our observation of a decrease in the amount of defective rna species in the minion analysis (figs b and b) . we further characterized each well of the tcid assay of our purified particles using flowcytometry to give a quantitative assessment of cell survival and death in response to virus dose. we calculated the number of live cells that remained after infection at each dilution and for each passage (fig c and s fig). we observed a reduced overall cpe in later passages at the highest virus dose as well as an increase in the number of viral particles require to induce the same amount of cpe (fig c) . together these trends reflect a reduced specific infectivity during viral passaging, in agreement with our tcid assays. interestingly however, for the highest particle concentrations in passages and , we saw less cell death at the highest doses ( , and , particles per cell) than for cells infected with the same inoculum (and therefore same ratio of full-length to defective rnas) but at a lower dose ( , - particles per cell). this observation indicates the protection of cells from infection and/or cpe when supplied with a large dose of viral particles that contain a large proportion of di-rnas. in this manuscript, we sought to provide a thorough and comprehensive analysis of the frequency and identity of recombination events present during the serial passaging of flock house virus in cell culture in order to elucidate the pathways and mechanism of di-rna emergence and evolution. we began with a homogenous inoculum derived from plasmid cdnas of each of the fhv genomes. in the inoculum and in the early passages, we find a wide range of low-frequency recombination events corresponding to deletions and duplications that are dispersed through-out the viral genomic rnas. we can be confident that these species do not constitute sequencing artifacts as we made our rnaseq libraries using 'clickseq' [ ] that has previously been demonstrated to reduce artifactual recombination in rnaseq data to fewer than events per million reads. further confidence in the low rates of artifactual recombination in our study is provided internally by inspecting the numbers of inter-rna recombination events (rna to rna and vice versa), which are always low. furthermore, the majority of the detected inter-rna recombination events correspond to genomic rna hetero-and homo-dimers, which have previously been characterized as replication intermediates [ ] . within only - passages, however, deletion events similar to those previously observed in di-rnas appear in all three passaging replicates. in subsequent passages, these recombination events begin to accumulate rapidly so as to predominate over full-length viral rnas. this observation of the emergence of di-rna species, followed by their rapid accumulation is consistent with existing theories on the evolution of di-rnas that postulate that a wide range of potential di-rna species are generated by non-programmed rna recombination and that only a handful are successfully replicated and thus accumulate [ ] . while the short-read data provide high-resolution characterization of individual recombination events, it is through the use of the oxford nanopore technologies (ont) minion that we are able reconstitute the complex full-length genomic landscape of fhv during passaging and determine the relative abundances of the genomic rnas in each passage. as a result, we were able to determine that by the final passage only~ % of the mapped reads are full-length viral rna , which corresponded with a large reduction in specific infectivity. additionally, the nanopore data revealed complex rearrangements of rna genomes, including the excision of an entire functional rna motif and its reinsertion in the 'utr of rna . the variation in recombination boundaries of the di-rnas suggests that a range of deletions can be tolerated. however, it is important to note that while each replicate is its own distinct lineage, each replicate passaging experiment was derived from the same initial inoculum. we chose to design the experiment this way to determine if the same di-rnas would be generated independently or if completely different deletions would arise and be selected for even though the environmental conditions are practically the same. here, we observe the latter ( table ) . few of the rna recombination events observed in the inoculum are observed again in subsequent passages. additionally, even though we found the event ' ^ ' in rna the inoculum, this was not the final predominant species in any replicate. therefore, the evolution of di-rnas was not pre-determined by the presence of rare di-rna species in the common inoculum (a founder-effect), but rather by the selection of well-replicating di-r-nas that arose later during serial passaging. nonetheless, the final recombination events are highly similar between replicates and to previous reports from different laboratories. therefore, this indicates that either the di-rnas have emerged due to a common mechanism of formation, the presence of a common selectivity filter, or both. in addition to providing a thorough analysis of the pathways of defective rna formation and evolution, there are two unexpected and critical observations made through this study. first: while we observe a wide range of recombination events early on during passaging, only a limited number of events are subsequently amplified and later define di-rnas. moreover, these limited sets of events are similar between replicates, and to previous studies. this suggests that while a large pool of potential defective rnas are generated, only a small number are capable of accumulating. secondly: we do not observe the amplification of di-rnas with only one deletion. in contrast, 'mature' di-rnas accumulate rapidly. nonetheless, we do find evidence for intermediate di-rnas as early as the inoculum sample. this indicates that intermediate defective-species are either non-competitive and do not accumulate or are not formed as a pre-cursor to mature di-rnas. these two observations provide important insights into the potential mechanisms of di-rna emergence and evolution. while there is a strong selection pressure for di-rnas to retain essential functional genomic elements, it is also postulated that a shorter defective rna would be replicated more quickly and thus more competitively [ ] . in our analysis, while the~ - deletion in rna (for example) is very common we do not observe the accumulation of deletion events that are smaller than this (e.g. - ). this is despite the fact that we can detect and observe such species in low frequencies both in early and late passages, suggesting that they are indeed generated but are not selectively amplified. this may in part be due to selecting for di-rna genomes that are as small as possible, while retaining the minimal amount of genetic material to form functional genetic elements. however, it may also be the result of a negative selection pressure or restrictive barrier that is released only after excising specific portions of the viral genome. an example of this scenario has been demonstrated for tomato bushy stunt virus (tbsv) associated di-rnas whereby deletion of a translation enhancer functional element removes the competition between translation and replication, thus favoring replication of the smaller di-rna [ ] . therefore, the final structure of di-rnas may depend both on the retention of essential functional rna elements, as well as the removal of restrictive barriers that attenuate rna replication. one model for the evolution of di-rnas is through the step-wise accumulation of deletion events through a series of individual recombination events [ ] . the minion data reveals that the defective rnas that accumulate (rapidly over the course - passages) contain multiple deletion events. however, we do not see the rapid accumulation of the intermediate di-rnas, despite evidence for their presence early in viral passaging. this suggests that the mature di-r-nas have a competitive advantage over their presumed intermediate precursors. if this is the case, the multiple deletions may function epistatically either through an undefined cooperative/additive mechanism or through the release of multiple restriction barriers, as proposed above. if multiple restriction barriers are required to be excised for the formation of di-rnas, small or multipartite rna viruses, such as fhv or influenza [ ] , may therefore generate di-rnas more readily by requiring fewer intermediate steps than long, monopartite rna viruses. moreover, if intermediate defective rnas fail to accumulate, this reduces the likelihood that mature di-rnas can subsequently be generated and may place substantial limitations on the ability of some viruses to generate di-rnas altogether. an alternative reason for the rarity of precursor/intermediate defective rnas is that the mature di-rnas are generated in one single event. we are yet to determine the molecular mechanism of recombination that leads to di-rna formation. both template-switching, secondary-structure jumping, and non-replicative mechanisms have been proposed, and indeed these mechanisms need not be mutually exclusive. our observation of nucleotide preferences at recombination junctions (fig c) may arise through any of these potential mechanisms. alternatively, it is possible that multiple reassembly/deletion events occur in a single step, in a manner reminiscent of chromosome shattering (chromothripsis) [ ] ; or 'virothripsis'. within the confined invaginations of the mitochondrial membranes that form the replication factories of rna viruses such as fhv [ ] , the fragmentation the rna virus genome followed by incorrect re-stitching of these genome pieces, either through forced-copy choice template switching or a non-replicative mechanism, could create the di-rnas observed here including the complex rearrangements observed for rna . a defective-interfering rna is a defective rna that has the ability to compete with or otherwise attenuate the replication and proliferation of the wild-type helper virus. in our study we demonstrate that the viral swarm, even after only a few passages, is replete with many varieties of defective rnas. with a single sequencing experiment, we would not be able to determine whether these defective rnas are accumulating, diminishing or make-up a static component of the viral quasi-species. however, as we perform serial passaging with sequential sequencing experiments, we can determine which defective rnas are accumulating (for example the 'mature' di-rnas) and which are not (e.g. the putative intermediate, or 'immature' defective rnas). it would be impractical to validate each of the many hundreds of detected defective rna species with molecular virological experiments to determine whether they truly can attenuate or interfere with wild-type virus replication and to therefore categorize that species as a defective-interfering rna. indeed, we cannot exclude the possibility that multiple di-r-nas act co-operatively within the viral quasi-species and are mutually dependent upon one another. however, the demonstration here of an accumulation during serial passaging is strong evidence that these species are interfering, as their accumulation essentially dilutes the pool of wild-type functional virus. with this in mind, we believe it would be suitable to describe the mature defective rnas as defective-interfering rnas (di-rnas), and the 'immature' only as defective rnas. it is remarkable that fhv is able to maintain a viable infection despite being burdened with such a gross excess of di-rnas in the final passages presented here. by performing tcid assays of the original inocula used between each of the passages and of the particles purified from each passage, we show that there is a dramatic reduction in specific infectivity during passaging corresponding with the increase in the di-rna content. di-rnas have generally been demonstrated to arise at high mois, as was the scenario with our first passages. however, our calculated moi drops rapidly after di-rnas have formed to levels that might be expected during typical in vivo viral passaging scenarios. however, in this experiment we were actually passaging a large number of virus particles between cells, but with a low specific infectivity. it is also interesting to observe that for the final passages there appears to be a protective property of the di-rnas as determined by flow-cytometry, but only when administered at the highest doses corresponding to over~ , particles per cell. however, the role that di-rnas might play in vivo is not clear, as these very high doses may not be physiologically relevant. di-rnas for fhv similar to the ones described here have been observed to arise in experimental fruit fly infections [ ] and so the mechanism of formation and/or selection is likely to be similar in cell culture and in vivo. however, whether rna viruses such as fhv have evolved to favor the spontaneous formation of di-rnas and if so whether these di-rnas play an important role in modifying the life-cycle of the virus, is yet to be determined. and the stacked frequencies of each detected defective rna is shown in the y-axis. each noncontiguous color represents a specific genome characterized by minion nanopore sequencing. wild-type genomes are colored green, genomes with one deletion are colored in shades of blue, and genomes with two or more deletions are colored in shades of oranges (using the same color scheme as in fig c) . each passage in each replicate as well as the inoculum is shown. output format, as described in routh et al. [ ] , is given as "donorcoord_to_acceptorcoord_#_counts". this is the raw data used to populate table and s table. (zip) s datafile. genomes characterized by minion nanopore sequencing. table reports the annotated genomes (wild-type or defective) and the number of mapped reads in each passage. this the raw data used to populate the stacked-area plots in fig and s fig. (xlsx) chimeric s rrna sequence formation and detection in sanger and -pyrosequenced pcr amplicons why do rna viruses recombine? the mechanism of rna recombination in poliovirus rna recombination in vivo in the absence of viral replication sequencing and analyses of all known human rhinovirus genomes reveal structure and evolution widespread intra-serotype recombination in natural populations of dengue virus long-term circulation of vaccine-derived poliovirus that causes paralytic disease nucleotide-resolution profiling of rna recombination in the encapsidated genome of a eukaryotic rna virus by next-generation sequencing defective interfering rnas: foes of viruses and friends of virologists defective viral particles and viral disease processes a defective interfering influenza rna inhibits infectious influenza virus replication in human respiratory tract cells: a potential new human antiviral immunostimulatory defective viral genomes from respiratory syncytial virus promote a strong innate antiviral response during infection in mice and humans effects of defective interfering viruses on virus replication and pathogenesis in vitro and in vivo defective viral genomes arising in vivo provide critical danger signals for the triggering of lung antiviral immunity engineering of homologous recombination hotspots with au-rich sequences in brome mosaic virus identification and manipulation of the molecular determinants influencing poliovirus recombination defective interfering influenza virus rnas: time to reevaluate their clinical potential as broad-spectrum antivirals? analysis of efficiently packaged defective interfering rnas of murine coronavirus: localization of a possible rna-packaging signal defective virus drives human immunodeficiency virus infection, persistence, and pathogenesis biased hypermutation and other genetic changes in defective measles viruses in human brain infections defective interfering viral particles in acute dengue infections genetic analysis of hepatitis c virus with defective genome and its infectivity in vitro internally deleted wnv genomes isolated from exotic birds in new mexico: function in cells, mosquitoes, and mice sequence analysis of in vivo defective interfering-like rna of influenza a h n pandemic virus flock house virus: a nodavirus isolated from costelytra zealandica (white) (coleoptera: scarabaeidae) replication of flock house virus in three genera of medically important insects flock house virus: a model system for understanding non-enveloped virus entry and membrane penetration induction and suppression of rna silencing by an animal virus evidence that the packaging signal for nodaviral rna is a bulged stem-loop cis-acting requirements for the replication of flock house virus rna molecular characterization of drosophila cells persistently infected with flock house virus discovery of functional genomic motifs in viruses with virema-a virus recombination mapper-for analysis of next-generation sequencing data rna-mediated interference and reverse transcription control the persistence of rna viruses in the insect model drosophila host rnas, including transposons, are encapsidated by a eukaryotic single-stranded rna virus clickseq: fragmentation-free next-generation sequencing via click ligation of adaptors to stochastically terminated '-azido cdnas solid phase click ligation for the synthesis of very long oligonucleotides a first look at the oxford nanopore minion sequencer nanopore sequencing and assembly of a human genome with ultra-long reads rapid metagenomic identification of viral pathogens in clinical samples by real-time nanopore sequencing analysis real-time, portable genome sequencing for ebola surveillance minion nanopore sequencing of an influenza genome highly parallel direct rna sequencing on an array of nanopores. biorxiv improved data analysis for the minion nanopore sequencer covama: co-variation mapper for disequilibrium analysis of mutant loci in viral populations using next-generation sequence data poly(a)-clickseq: click-chemistry for next-generation -end sequencing without rna enrichment or fragmentation cutadapt removes adapter sequences from high-throughput sequencing reads ultrafast and memory-efficient alignment of short dna sequences to the human genome poretools: a toolkit for analyzing nanopore sequence data tablet-next generation sequence assembly visualization the sequence alignment/map format and samtools measuring hcv infectivity produced in cell culture and in vivo plaque assay for black beetle virus high-fidelity mass analysis unveils heterogeneity in intact ribosomal particles replication of flock house virus rnas from primary transcripts made in cells by rna polymerase ii packaging host rnas in small rna viruses: an inevitable consequence of an error-prone polymerase? double indexing overcomes inaccuracies in multiplex sequencing on the illumina platform common contaminants in next-generation sequencing that hinder discovery of low-abundance microbes generation and selection of coronavirus defective interfering rna with large open reading frame by rna recombination and possible editing flock house virus replicates and expresses green fluorescent protein in mosquitoes long-distance base pairing in flock house virus rna regulates subgenomic rna synthesis and rna replication requirements for the self-directed replication of flock house virus rna replication of the rna segments of a bipartite viral genome is coordinated by a transactivating subgenomic rna the cis-acting replication signal at the ' end of flock house virus rna is rna -dependent a ' terminal stem-loop structure in nodamura virus rna forms an essential cis-acting signal for rna replication characterization and template properties of rna dimers generated during flock house virus rna replication the oxford nanopore minion: delivery of nanopore sequencing to the genomics community continuous influenza virus production in cell culture shows a periodic accumulation of defective interfering particles nonhomologous rna recombination in tombusviruses: generation and evolution of defective interfering rnas by stepwise deletions massive genomic rearrangement acquired in a single catastrophic event during cancer development role of mitochondrial membrane spherules in flock house virus replication we thank thomas wood, steve widen and jill thomson from the utmb next-generation sequencing core and gillian lynch from the scsb computing facility for support. we are indebted to mariano garcia-blanco and shelton bradrick and the members of their lab for sharing lab space, helpful discussions and other research support. we thank eric wagner, jack johnson and bruce torbett for support and advice. visualization: ar ej. writing -review & editing: ar ej. key: cord- -hxxizipk authors: roberts, katherine e.; hadfield, jarrod d.; sharma, manmohan d.; longdon, ben title: changes in temperature alter the potential outcomes of virus host shifts date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: hxxizipk host shifts–where a pathogen jumps between different host species–are an important source of emerging infectious disease. with on-going climate change there is an increasing need to understand the effect changes in temperature may have on emerging infectious disease. we investigated whether species’ susceptibilities change with temperature and ask if susceptibility is greatest at different temperatures in different species. we infected species of drosophilidae with an rna virus and measured how viral load changes with temperature. we found the host phylogeny explained a large proportion of the variation in viral load at each temperature, with strong phylogenetic correlations between viral loads across temperature. the variance in viral load increased with temperature, while the mean viral load did not. this suggests that as temperature increases the most susceptible species become more susceptible, and the least susceptible less so. we found no significant relationship between a species’ susceptibility across temperatures, and proxies for thermal optima (critical thermal maximum and minimum or basal metabolic rate). these results suggest that whilst the rank order of species susceptibilities may remain the same with changes in temperature, some species may become more susceptible to a novel pathogen, and others less so. emerging infectious diseases are often the result of a host shift, where a pathogen jumps from one host species into another. understanding the factors underlying host shifts is a major goal for infectious disease research. this effort has been further complicated by the fact that host-parasite interactions are now taking place in a period of unprecedented global climatic warming. here, we ask how host shifts are affected by temperature by carrying out experimental infections using an rna virus across a wide range of related species, at three different temperatures. we find that as temperature increases the most susceptible species become more susceptible, and the least susceptible less so. this has important consequences for our understanding of host shift events in a changing climate as it suggests that temperature changes may affect the likelihood of a host shift into certain species. temperature is arguably the most important abiotic factor that affects all organisms, having both indirect and direct effects on physiology and life history traits [ ] [ ] [ ] . there is much to be learned about the impact of climate change on infectious diseases [ , , ] . changes in temperature can impact both host and parasite biology, leading to complex and difficult to predict outcomes [ , ] . host shifts, where a parasite from one host species invades and establishes in a novel host species, are an important source of emerging infectious disease [ ] . a successful host shift relies on a number of stages occurring [ ] . firstly, exposure of the host to the new pathogen species must occur in such a way that transmission is successful. secondly, the pathogen must be able to replicate sufficiently to infect the novel host. finally, there must be sufficient onwards transmission for the pathogen to become established in the new host species [ , , ] . some of the most deadly outbreaks of infectious diseases in humans including ebola virus, hiv and sars coronavirus have been linked to a host switch event [ ] [ ] [ ] [ ] and many others have direct animal vectors or reservoirs (e.g. dengue and chikungunya viruses) [ , ] . the potential for novel host shifts may increase with changing temperatures due to, fluctuations in host and/or parasite fitness, or changes in species distributions and abundances [ , ] . distribution changes may lead to new species assemblages, causing novel contacts between parasites and potential hosts [ ] [ ] [ ] . susceptibility to infection is known to vary with temperature, due to within individual physiological changes in factors such as the host immune response, metabolic rate or behavioural adaptations [ ] [ ] [ ] [ ] . thermally stressed hosts may face a trade-off between the resource investment needed to launch an immune response versus that needed for thermoregulation, or behavioural adaptations to withstand sub-optimal temperatures [ ] [ ] [ ] [ ] . temperature shifts could also cause asymmetrical or divergent effects on host and parasite traits [ ] . for example, changes in temperature may allow differential production and survival of parasite transmission stages, and changes in replication rates, generation times, infectivity and virulence [ ] [ ] [ ] . temperature is also known to impact vector-borne disease transmission through multiple effects on both vector life cycles and transmission behaviours [ , [ ] [ ] [ ] [ ] . host shifts have been shown to be more likely to occur between closely related species [ ] [ ] [ ] , but independently of this distance effect, clades of closely related hosts show similar levels of susceptibility [ , ] . thermal tolerances − like virus susceptibility − are known to vary across species, with groups of closely related species having similar thermal limits, with a large proportion of the variation in these traits being explained by the phylogeny [ ] [ ] [ ] [ ] . previous studies on host shifts have assayed the susceptibility of species at a single temperature [ , , , ] . however, if the host phylogeny also explains much of the variation in thermal tolerance, then phylogenetic patterns in virus susceptibility could be due to differences between species' natural thermal optima and the chosen assay temperatures. therefore, for experiments carried out at a single temperature, phylogenetic signal in thermal tolerance may translate into phylogenetic signal in thermal stress. any apparent phylogenetic signal in susceptibility could potentially be due to the effects of thermal stress, and may not hold true if each species was to be assayed at its optimal temperature. if this was indeed the case this would have implications for species distribution models that aim to use estimates of environmental conditions to predict host and pathogen ranges [ , , ] . here, we have asked how species' susceptibilities change at different temperatures and whether susceptibility is greatest at different temperatures in different species. we infected species of drosophilidae with drosophila c virus (dcv; dicistroviridae) at three different temperatures and measured how viral load changes with temperature. viral load is used here as a measure of dcv's ability to persist and replicate in a host, which has previously been shown to be tightly correlated to host mortality [ ] . we are therefore examining one of the steps ("ability to infect a novel host") needed for a host shift to successfully occur [ , , ] . we also examine how proxies for thermal optima and cellular function (thermal tolerances and basal metabolic rate) relate to virus susceptibility across temperatures, as increasing temperatures may have broad effects on both host and parasite [ ] [ ] [ ] . dcv is a positive sense rna virus in the family discistroviridae that was originally isolated from drosophila melanogaster and in the wild has been found in d. melanogaster and d. simulans [ ] [ ] [ ] . dcv infected flies show reduced metabolic rate and activity levels, develop an intestinal obstruction, reduced hemolymph ph and decreased survival [ ] [ ] [ ] [ ] . this work examines how temperature can influence the probability of host shifts, and looks at some of the potential underlying causes. we used drosophila c virus (dcv) clone b a, which is derived from an isolate collected from d. melanogaster in charolles, france [ ] . the virus was prepared as described previously [ ] ; briefly dcv was grown in schneider's drosophila line cells and the tissue culture infective dose (tcid ) per ml was calculated using the reed-muench end-point method [ ] . flies were obtained from laboratory stocks of different species. all stocks were maintained in multi generation populations, in drosophila stock bottles (dutscher scientific) on ml of their respective food medium at ˚c and % relative humidity with a hour lightdark cycle (table a in s text). each day, two vials of - day old male flies were randomly assigned to one of three potential temperature regimes; low, medium or high ( ˚c, ˚c and ˚c respectively) at % relative humidity. flies were tipped onto fresh vials of food after days, and after days of acclimatisation at the experimental temperature were infected with dcv. flies were anesthetized on co and inoculated using a . mm diameter stainless steel needle that was bent to a right angle~ . mm from the end (fine science tools, ca, usa) [ , , ] . the bent tip of the needle was dipped into the dcv solution (tcid = . × ) and pricked into the pleural suture on the thorax of the flies. we selected this route of infection as oral inoculation has been shown to lead to stochastic infection outcomes in d. melanogaster [ ] . however, once the virus passes through the gut barrier, both oral and pinpricked infections follow a similar course, with both resulting in the same tissues becoming infected with dcv [ ] . one vial of inoculated flies was immediately snap frozen in liquid nitrogen to provide a time point zero sample as a reference to control for relative viral dose. the second vial of flies were placed onto a new vial of fresh cornmeal food and returned to their experimental temperature. after days (+/- hour) flies were snap frozen in liquid nitrogen. this time point was chosen based on pilot data as infected flies showed little mortality at days post infection, and viral load plateaus from day at ˚c. temperatures were rotated across incubators in each block to control for incubator effects. all frozen flies were homogenised in a bead homogeniser for seconds (bead ruptor ; omni international, georgia, usa) in trizol reagent (invitrogen) and stored at - ˚c for later rna extractions. these collections and inoculations were carried out over three replicate blocks, with each block being completed over consecutive days. the order that the fly species were infected was randomized each day. we aimed for each block to contain a day and day replicate for each species, at each temperature treatment ( species × temperatures × experimental blocks). in total we quantified viral load in , flies over biological replicates (a biological replicate = change in viral load from day to day post-infection), with a mean of . flies per replicate (range across species = - ) . of the species, had biological replicates and three species had biological replicates. the change in rna viral load was measured using quantitative reverse transcription pcr (qrt-pcr). total rna was extracted from the trizol homogenised flies, reverse-transcribed with promega goscript reverse transcriptase (promega) and random hexamer primers. viral rna load was expressed relative to the endogenous control housekeeping gene rpl (rp ). rpl primers were designed to match the homologous sequence in each species and crossed an intron-exon boundary so will only amplify mrna [ ] . the primers in d. melanogaster were rpl qrt-pcr f ( '-tgctaagctgtcgcacaaatgg - ') and rpl qrt-pcr r ( '-tgcgcttgttcgatccgtaac - '). dcv primers were f ( '-gacactgccttt gattag- ') and r ( 'ccctctgggaactaaatg- ') as previously described [ ] . two qrt-pcr reactions (technical replicates) were carried out per sample with both the viral and endogenous control primers, with replicates distributed across plates in a randomised block design. qrt-pcr was performed on an applied biosystems steponeplus system using sensifast hi-rox sybr kit (bioline) with the following pcr cycle: ˚c for min followed by cycles of: ˚c for sec followed by ˚c for sec. each qrt-pcr plate contained four standard samples. a linear model was used to correct the cycle threshold (ct) values for differences between qrt-pcr plates. any samples where the two technical replicates had cycle threshold (ct) values more than cycles apart after the plate correction were repeated. to estimate the change in viral load, we first calculated Δct as the difference between the cycle thresholds of the dcv qrt-pcr and the rpl endogenous control. for each species the viral load of day flies relative to day flies was calculated as -ΔΔct ; where ΔΔct = Δct day -Δct day . the Δct day and Δct day are a pair of Δct values from a day biological replicate and a day biological replicate. calculating the change in viral load without the use of the endogenous control gene (rpl ) gave equivalent results (spearman's correlation between viral load calculated with and without endogenous control: ρ = . , p< . ) we carried out two assays to measure the thermal tolerances of species; a cold resistance measure to determine critical thermal minimum (ct min ) under gradual cooling, and a heat resistance measure through gradual heating to determine critical thermal maximum (ct max ). - day old males were collected and placed onto fresh un-yeasted cornmeal food vials. flies were kept for days at ˚c and % relative humidity and tipped onto fresh food every days. in both assays individual flies were placed in ml glass vials (st , ampulla, uk) and exposed to temperature change through submersion in a liquid filled glass tank (see fig a in s text). for ct max the tank was filled with water and for ct min a mixture of water and ethylene glycol ( : by volume) was used to prevent freezing and maintain a constant cooling gradient. five biological replicates were carried out for each species for both ct max and ct min . temperature was controlled using a heated/cooled circulator (txf , grant instruments, cambridgeshire, uk) submerged in the tank and set to change temperatures at a rate of . ˚c/min, always starting from ˚c (the rearing temperature for stock populations). flies were monitored continually throughout the assay and the temperature of knock down was ascertained by a disturbance method, whereby a fly was scored as completely paralysed if on gentle tapping of the vial wall the fly did not move any of its body parts. to examine how cellular function changes with temperature, we estimated the resting metabolic rate of each species at ˚c, ˚c and ˚c to examine if changes in general cellular processes were related to changes in viral load. following the same methods as the viral inoculation assay, groups of , - day old male flies from species were acclimatised at the three experimental temperatures for days (d. pseudoobscura was excluded as not enough individuals could be obtained from stocks for sufficient replication). every days flies were tipped onto fresh vials of cornmeal food. this was repeated in three blocks in order to get three repeat measures of metabolic rate for each of the species, at each of the three experimental temperatures. flies were collected in a randomly assigned order across the three blocks. closed system respirometry was used to measure the rate of co production (vco ) as a proxy for metabolic rate [ ] . flies were held in ml - airtight plastic chambers constructed from bev-a-line v tubing (cole-parmer instrument company, uk). all measures were carried out during the day inside a temperature controlled incubator, with constant light, that was set to each of the experimental temperatures that the flies had been acclimatised to. the set up followed that of okada et al. ( ) [ ] . compressed air of a known concentration of oxygen and nitrogen ( % o : % n ) was scrubbed of any co and water (with ascarite ii & magnesium perchlorate respectively) and pumped through a sable systems rm eight-channel multiplexer (las vegas, nv, usa) at ml/min - (± %) into the metabolic chambers housing the groups of flies. the first chamber was left empty as a reference cell, to acquire a baseline reading for all subsequent chambers at the start and end of each set of runs, therefore seven groups of flies were assayed in each run. air was flushed into each chamber for minutes, before reading the previous chamber. readings were taken every second for minutes by feeding the exiting air through a licor li- infrared gas analyser (lincoln, ne, usa). carbon dioxide production was measured using a sable systems ui analog-digital interface for acquisition, connected to a computer running sable systems expedata software (v . . ) [ ] . the metabolic rate was calculated from the entire -minute recording period by taking the co reading of the ex-current gas from the chamber containing the flies and subtracting the co measure of the incurrent gas entering the chamber. these values were also corrected for drift away from the baseline reading of the empty chamber. volume of co was calculated as vco = fr (fe co -fi co ) / ( -fi co ). where fr is the flow rate into the system ( ml/ min - ), fe co is the concentration of co exiting and fi co is the concentration co entering the respirometer. species were randomly assigned across the respiration chambers and the order in which flies were assayed (chamber order) was corrected for statistically (see below). to check for any potential effect of body size differences between species on viral load, wing length was measured as a proxy for body size [ ] . a mean of (range - ) males of each species were collected and immediately stored in ethanol during the collections for the viral load assay. subsequently, wings were removed and photographed under a dissecting microscope. using imagej software (version . ) the length of the iv longitudinal vein from the tip of the proximal segment to where the distal segment joins vein v was recorded, and the mean taken for each species. the host phylogeny was inferred as described in longdon et al ( ) [ ] , using the s, adh, amyrel, coi, coii, rpl and sod genes. briefly, any publicly available sequences were downloaded from genbank, and any not available we attempted to sanger sequence [ ] . in total we had rpl sequences for all species, s from species, adh from species, amyrel from species, coi from species, coii from species and sod from species (see www.doi.org/ . /m .figshare. full details). the sequences of each gene were aligned in geneious (version . . , [ ] ) using the global alignment setting, with free end gaps and a cost matrix of % similarity. the phylogeny was constructed using the beast program (version . . , [ ] ). genes were partitioned into three groups each with their own molecular clock models. the three partitions were: mitochondrial (coi, coii); ribosomal ( s); and nuclear (adh, sod, amyrel, rpl ). a random starting tree was used, with a relaxed uncorrelated lognormal molecular clock. each of the partitions used a hky substitution model with a gamma distribution of rate variation with categories and estimated base frequencies. additionally, the mitochondrial and nuclear data sets were partitioned into codon positions + and , with unlinked substitution rates and base frequencies across codon positions. the treeshape prior was set to a birth-death process. the beast analysis was run twice to ensure convergence for million mcmc generations sampled every steps. the mcmc process was examined using the program tracer (version . , [ ] ) to ensure convergence and adequate sampling, and the constructed tree was then visualised using figtree (version . . , [ ] ). all data were analysed using phylogenetic mixed models to look at the effects of host relatedness on viral load across temperature. we fitted all models using a bayesian approach in the r package mcmcglmm [ , ] . we ran trivariate models with viral load at each of the three temperatures as the response variable similar to that outlined in longdon et al. ( ) [ ] . the models took the form: where y is the change in viral load of the i th biological replicate of host species h, for temperature t (high, medium or low). β are the fixed effects, with β being the intercepts for each temperature, β being the effect of basal metabolic rate, β the effect of wing size, and β and β the effects of the critical thermal maximum (ct max ) and minimum (ct min ) respectively. u p are the random phylogenetic species effects and e the model residuals. we also ran models that included a non-phylogenetic random species effect (u np:ht ) to allow us to estimate the proportion of variation explained by the host phylogeny [ , , ] . we do not use this term in the main model as we struggled to separate the phylogenetic and non-phylogenetic terms. our main model therefore assumes a brownian motion model of evolution [ ] . the random effects and the residuals are assumed to be multivariate normal with a zero mean and a covariance structure v p � a for the phylogenetic affects and v e � i for the residuals (� here is the kronecker product). a is the phylogenetic relatedness matrix, i is an identity matrix and the v are × (co)variance matrices describing the (co)variances between viral titre at different temperatures. the phylogenetic covariance matrix, v p, describes the inter-specific variances in each trait and the inter-specific covariances between them. the residual covariance matrix, v e, describes the within-species variance that can be both due to real within-species effects and measurement or experimental errors. the off-diagonal elements of v e (the covariances) can not be estimated because no vial has been subject to multiple temperatures and so were set to zero. we excluded d. pseudoobscura from the full model as data for bmr was not collected, but included it in models that did not include any fixed effects, which gave equivalent results. diffuse independent normal priors were placed on the fixed effects (means of zero and variances of ). parameter expanded priors were placed on the covariance matrices resulting in scaled multivariate f distributions, which have the property that the marginal distributions for the variances are scaled (by ) f , . the exceptions were the residual variances for which an inverse-gamma prior was used with shape and scale equal to . . the mcmc chain was run for million iterations with a burn-in of million iterations and a thinning interval of , . we confirmed the results were not sensitive to the choice of prior by also fitting models with inverse-wishart and flat priors for the variance covariance matrices (described in [ ] ), which gave qualitatively similar results ( . /m .figshare. ). all confidence intervals (ci's) reported are % highest posterior density intervals. using similar model structures we also ran a univariate model with bmr and a bivariate model with ct min and ct max as the response variables to calculate how much of the variation in these traits was explained by the host phylogeny. both of these models were also run with wing length as a proxy for body size as this is known to influence thermal measures [ ] . we observed significant levels of measurement error in the metabolic rate data; this was partially caused by respiratory chamber order during the assay. we corrected for this in two different ways. first, we fitted a linear model to the data to control for the effect of respiratory chamber number and then used this corrected data in all further models. we also used a measurement error model that controls for both respiratory chamber number effects and random error. both of these models gave similar results although the measurement error model showed broad cis suggesting the bmr data should be interpreted with caution. all datasets and r scripts with the model parameterisation are provided as supporting information (s text). to investigate the effect of temperature on virus host shifts we quantified viral load in , flies over biological replicates, from species of drosophilidae at three temperatures ( fig ) . dcv replicated in all host species, but viral load differed between species and temperatures (fig ) . species with similar viral loads cluster together on the phylogeny (fig ) . measurements were highly repeatable (table ) , with a large proportion of the variance being explained by the inter-specific phylogenetic component (v p ), with little within species or measurement . we also calculated the proportion of between species variance that can be explained by the phylogeny as v p /(v p + v s ) [ ] , which is equivalent to pagel's lambda or phylogenetic heritability [ , ] . we found the host phylogeny explains a large proportion of the inter-specific variation in viral load across all three temperatures, although these estimates have broad confidence intervals due to the model struggling to separate the phylogenetic and non-phylogenetic components (low = . , % ci: . to examine if species responded in the same or different way to changes in temperature we examined the relationships between susceptibilities across the different temperatures. we found strong positive phylogenetic correlations between viral loads across the three temperatures (table ). our models showed that the variance in viral load increased with temperature, however the mean viral load showed no such upward trend (table ). this suggests that the changes in variance are not simply occurring due to an increase in the means, that is then driving an increase in variance. the high correlations suggest the rank order of susceptibility of the species is not changing with increasing temperature. however, the change in variance suggests that although the intercepts are the temperature-specific intercepts when the other covariates (e.g. wing size) are set to their temperature specific means. they can be interpreted as the expected viral loads at the root of the phylogeny at each temperature. v p is the variance in between-species effects, which are structured by the phylogeny, and v r is the variance in within species effects attributable to between individual differences and measurement error. reaction norms are not crossing they are diverging from each other as temperature increases i.e. the most susceptible species are becoming more susceptible with increasing temperature, and the least susceptible less so [ ] . for example, d. obscura and d. affinis are the most susceptible species at all three temperatures. the responses of individual species show that some species have increasing viral load as temperature increases (fig , e.g. z. taronus, d. lummei) , while others decease (e.g. d. littoralis, d. novamexicana). the changes we observe could be explained by the increase in temperature effectively increasing the rate at which successful infection is progressing (i.e. altering where in the course of infection we have sampled). however, this seems unlikely as at days post infection at the medium temperature ( ˚c), viral load peaks and then plateaus [ ] . therefore, in those species where viral load increases at higher temperatures the peak viral load itself must be increasing, rather than us effectively sampling the same growth curve but at a later time point. likewise, in those species where viral load decreased at higher temperatures, viral load would need to first increase and then decrease, which we do not observe in a time course at ˚c [ ] . to check whether this also holds at higher temperatures we carried out a time course of infection in a subset of six of the original experimental species at ˚c, where we would expect the fastest transition between the rapid viral growth and the plateau phase of infection to occur (fig b in s text) . this allowed us to confirm that the decreasing viral loads observed in some species at higher temperatures are not due to general trend for viral loads to decline over longer periods of (metabolic) time. we quantified the lower and upper thermal tolerances (ct min and ct max ) across all species with replicates per species. neither ct max nor ct min were found to be significant predictors of viral load (ct min - . , % ci: - . , . , pmcmc = . and ct max . , % ci: - . , . , pmcmc = . ). when treated as a response in models we found the host phylogeny explained a large proportion of the variation in thermal maximum (ct max : . , % ci: . , ) and thermal minima (ct min : . , % ci: . , . , see s text fig c) . we also measured the basal metabolic rate of flies from species, across the three experimental temperatures, to examine how cellular function changes with temperature. bmr was not found to be a significant predictor of viral load when included as a fixed effect in our model (slope = . , % ci = - . , . , pmcmc = . ). bmr increased with temperature across all species (mean bmr and se: low . ± . , medium . ± . , high . ± . co ml/min - , see s text fig d) . when bmr was analysed as the response in models, the phylogeny explained a small amount of the between species variation (low . , % ci: × − , . , medium . , % ci: × − , . , high . , % ci: × − - . , s text fig e) indicating high within species variation or large measurement error. consequently the mean bmrs for each species, at each temperature, were used in the analysis of viral load will be poorly estimated and so the effects of bmr will be underestimated with too narrow credible intervals. to rectify this we ran a series of measurement error models, the most conservative of which gave a slope of - . but with very wide credible intervals (- . , . ) . full details of these models are given in the supporting information (s text). we found that susceptibilities of different species responded in different ways to changes in temperature. the susceptibilities of different species showed differing responses as temperatures increased (fig ) . there was a strong phylogenetic correlation in viral load across the three experimental temperatures (table ) . however, the variance in viral load increased with temperature, whereas the mean viral load did not show the same trend. this suggests that the rank order of susceptibility of the species remains relatively constant across temperatures, but as temperature increases the most susceptible species become more susceptible, and the least susceptible less so. changes in global temperatures are widely predicted to alter host-parasite interactions and therefore the likelihood of host shifts occurring [ , , , , ] . the outcome of these interactions may be difficult to predict if temperature causes a different effect in the host and pathogen species [ , , [ ] [ ] [ ] . our results show that changes in temperature may change the likelihood of pathogens successfully infecting certain species, although they suggest that it may not alter which species are the most susceptible to a novel pathogen. the increase in phylogenetic variance with temperature is effectively a form of genotypeby-environment interaction [ , [ ] [ ] [ ] . however, it varies from the classically considered ecological crossing of reaction norms, as we do not see a change in the rank order of species susceptibly across the range of experimental temperatures. instead, we find the species means diverge with increasing temperatures and so the between species differences increase [ , ] . it is also important to note that temperature may not simply be causing a change in effect size when considering the biological processes occurring during host-parasite interactions [ , ] . for example, virus replication may plateau at higher temperatures due to resource limitation. the observed level of susceptibility may be the combined outcome of both host and parasite traits, which may interact nonlinearly with temperature. we also note that by using a limited range of temperatures for practical reasons we may have not captured all unimodal relationships between viral load and temperature. as temperature is an important abiotic factor in many cellular and physiological processes, we went on to examine the underlying basis of why viral load might change with temperature. previous studies that found phylogenetic signal in host susceptibility were carried out at a single experimental temperature [ , ] . therefore, the patterns observed could potentially be explained by some host clades being assayed at sub-optimal thermal conditions. we used ct max and ct min as proxies for thermal optima which, due to its multifaceted nature, is problematic to measure directly [ ] [ ] [ ] . we also measured basal metabolic rate across three temperatures to see if the changes in viral load could be explained by general increases in enzymatic processes. we found that these measures were not significant predictors of the change in viral load with temperature. this may be driven by the fact that all temperature related traits are likely to be more complex than what any single measure can explore. traits such as host susceptibility are a function of both the host and parasite thermal optima, as well as the shape of any temperature-trait relationship [ , ] . the host immune response and cellular components utilised by the virus are likely to function most efficiently at the thermal optima of a species, and several studies have demonstrated the outcomes of host-pathogen interactions can depend on temperature [ , , , ] . however, the mechanisms underlying the changes in susceptibility with temperature seen in this study are uncertain and a matter for speculation. our results show that in the most susceptible species, viral load increases with temperature; this may be due to the virus being able to successfully infect and then freely proliferate, utilizing the host cells whist avoiding host immune defences. in less susceptible species viral load does not increase with temperature, and in some cases it actually appears to decreases. here, temperature may be driving an increase in biological processes such as enhanced host immunity, or simply increasing the rate of degradation or clearance of virus particles that have failed to establish an infection of host cells. we have investigated how an environmental variable can alter infection success following a novel viral challenge. however, temperature is just one of the potential environmental factors that will influence the different stages of a host shift event [ ] . using a controlled method of viral inoculation allows us to standardize inoculation dose so we can ask, given equal exposure, how does temperature affect the ability of a pathogen to persist and replicate in a given host? however, in nature hosts will be faced with variable levels of pathogen exposure, infected through various modes of transmission and often by multiple strains or genotypes [ ] . such variables may have consequences for the establishment and subsequent infection success of any potential host shift event. it is known that oral infection by dcv is stochastic and immune barriers such as the gut are important [ , , ] , therefore establishing the relevance of infection in the wild in this system would require further study using different potential routes of infection. the geographical distribution of a host will also influence factors such as diet and resource availability [ , [ ] [ ] [ ] [ ] , and so further work on the role of nutrient and resource availability would therefore be needed to further explore the impact of these on potential host shifts. in conclusion, we have found changes in temperature can both increase or decrease the likelihood of a host shift. our results show the rank order of species' susceptibilities remain the same across temperatures, suggesting that studies of host shifts at a single temperature can be informative in predicting which species are the most vulnerable to a novel pathogen. changing global temperatures may influence pathogen host shifts; for example changes in distributions of both host and pathogen species may generate novel transmission opportunities. our findings suggest that increases in global temperature could increase the likelihood of host shifts into the most susceptible species, and reduce it in others. climate change may therefore lead to changing distributions of both host and pathogens, with pathogens potentially expanding or contracting their host range. understanding how environmental factors might affect broader taxonomic groups of hosts and pathogens requires further study if we are to better understand host shifts in relation to climate change in nature. climate warming and disease risks for terrestrial and marine biota global warming and temperature-mediated increases in cercarial emergence in trematode parasites climate change and evolutionary adaptation climate change and infectious diseases: from evidence to a predictive framework environmental-mechanistic modelling of the impact of global change on human zoonotic disease emergence: a case study of lassa fever. freckleton r, editor climate oscillations and the structure of natural communities emerging pathogens: the epidemiology and evolution of species jumps the evolution and genetics of virus host shifts host phylogeny determines viral persistence and replication in novel hosts population biology of emerging and re-emerging pathogens virus rna structure specialization facilitates host adaptation rapid spread of emerging zika virus in the pacific area ebola in west africa: the outbreak able to change many things impact of climate change on global malaria distribution chikungunya virus emergence is constrained in asia by lineage-specific adaptive landscapes the many projected futures of dengue rapid range shifts of species associated with high levels of climate warming host and parasite thermal ecology jointly determine the effect of climate warming on epidemic dynamics how will global climate change affect parasite-host assemblages? global temperature constraints on aedes aegypti and ae. albopictus persistence and competence for dengue virus transmission. parasit vectors evolution in action: climate change, biodiversity dynamics and emerging infectious disease thermal biology in insect-parasite interactions host thermal biology: the key to understanding host-pathogen interactions and microbial pest control? variation in the immune state of gammarus pulex (crustacea, amphipoda) according to temperature: are extreme temperatures a stress? environmental temperature variation influences fitness trade-offs and tolerance in a fish-tapeworm association the influence of ambient temperature on the course of myxomatosis in rabbits some like it hot: the effects of climate change on reproduction, immune function and disease resistance in the cricket gryllus texensis host-parasite and genotype-by-environment interactions: temperature modifies potential for selection by a sterilizing pathogen environmental stressors alter relationships between physiology and behaviour empirical evidence that metabolic theory describes the temperature dependency of within-host parasite dynamics parasites and global warming: net effects of temperature on an intertidal host-parasite system some (worms) like it hot: fish parasites grow faster in warmer water, and alter host thermal preferences global change, parasite transmission and disease control: lessons from ecology understanding uncertainty in temperature effects on vector-borne disease: a bayesian approach detecting the impact of temperature on transmission of zika, dengue and chikungunya using mechanistic models short title: temperature predicts zika, dengue, and chikungunya transmission impact of human mobility on the emergence of dengue epidemics in pakistan rethinking vector immunology: the role of environmental temperature in shaping resistance phylogenetic signal in plant pathogen-host range phylogenetic determinants of potential host shifts in fungal pathogens host phylogeny constrains cross-species emergence and establishment of rabies virus in bats the causes and consequences of changes in virulence following pathogen host shifts phylogenetic studies of coadaptation:preferred temperatures versus optimal performance temperature of lizards phylogenetic constraints in key functional traits behind species' climate niches: patterns of desiccation and cold resistance across drosophila species upper thermal limits of drosophila are linked to species distributions and strongly constrained phylogenetically upper thermal limits in terrestrial ectotherms: how constrained are they? fox c, editor infection success in novel hosts: an experimental and phylogenetic study of drosophila -parasitic nematodes spatial, seasonal and climatic predictive models of rift valley fever disease across africa global trends in emerging infectious diseases studies on drosophila c and a viruses in australian populations of drosophila melanogaster the discovery, distribution, and evolution of viruses associated with drosophila melanogaster twenty-five new viruses associated with the drosophilidae (diptera) physiological and metabolic consequences of viral infection in drosophila melanogaster drosophila c virus systemic infection leads to intestinal obstruction the novel genome organization of the insect picorna-like virus drosophila c virus suggests this virus belongs to a previously undescribed virus family the toll-dorsal pathway is required for resistance to viral oral infection in drosophila existence in drosophila of groups of picornavirus with different biological and serological properties host shifts result in parallel genetic changes when viruses evolve in closely related species a simple method of estimating fifty per cent endpoints measuring metabolic rates: a manual for scientists longevity, calling effort, and metabolic rate in two populations of cricket genetic architecture of metabolic rate: environment specific epistasis between mitochondrial and nuclear genes in an insect sexual size dimorphism in a drosophila clade, the d. obscura group geneious basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data bayesian phylogenetics with beauti and the beast . mcmc methods for multi-respoinse generalized linear mixed models: the mcmcglmm r package r: a language and environment for statistical computing. vienna, austria: r foundation for statistical computing the phylogenetic mixed model maximum-likelihood estimation of evolutionary trees from continuous characters phylogenetic analysis and comparative data: a test and review of evidence inferring the historical patterns of biological evolution the role of genotype-by-environment interactions in sexual selection how will global climate change affect parasite-host assemblages? identifying climate drivers of infectious disease dynamics: recent advances and challenges ahead complex effects of temperature on mosquito immune function infection risk decreases with increasing mismatch in host and pathogen environmental tolerances the thermal mismatch hypothesis explains host susceptibility to an emerging infectious disease genotype-environment interaction and the evolution of phenotypic plasticity quantitative genetics and the evolution of reaction norms genotype-by-environment interactions and adaptation to local temperature affect immunity and fecundity in drosophila melanogaster coexistence of similar genotypes of daphnia magna in intermittent populations: response to thermal stress temperature checks the red queen? resistance and virulence in a fluctuating environment phenotypic variance, plasticity and heritability estimates of critical thermal limits depend on methodological context making sense of heat tolerance estimates in ectotherms: lessons from drosophila validity of thermal ramping assays used to assess thermal tolerance in arthropods the evolution of transmission mode costs and benefits of sublethal drosophila c virus infection entry is a rate-limiting step for viral infection in a drosophila melanogaster model of pathogenesis impact of environmental variation on host performance differs with pathogen identity: implications for host-pathogen interactions in a changing climate host nutrition alters the variance in parasite transmission potential measuring parasite fitness under genetic and thermal variation immunity in a variable world many thanks to darren obbard and frank jiggins for useful discussion and vanessa kellerman and johannes overgaard for discussion about thermal assay methods. thanks to dave hosken for use of bmr chambers and to the drosophila species stock centre for supplying flies. thanks to two anonymous reviewers for constructive comments. key: cord- - u tdrj authors: geoghegan, jemma l.; duchêne, sebastián; holmes, edward c. title: comparative analysis estimates the relative frequencies of co-divergence and cross-species transmission within viral families date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: u tdrj the cross-species transmission of viruses from one host species to another is responsible for the majority of emerging infections. however, it is unclear whether some virus families have a greater propensity to jump host species than others. if related viruses have an evolutionary history of co-divergence with their hosts there should be evidence of topological similarities between the virus and host phylogenetic trees, whereas host jumping generates incongruent tree topologies. by analyzing co-phylogenetic processes in virus families and their eukaryotic hosts we provide a quantitative and comparative estimate of the relative frequency of virus-host co-divergence versus cross-species transmission among virus families. notably, our analysis reveals that cross-species transmission is a near universal feature of the viruses analyzed here, with virus-host co-divergence occurring less frequently and always on a subset of viruses. despite the overall high topological incongruence among virus and host phylogenies, the hepadnaviridae, polyomaviridae, poxviridae, papillomaviridae and adenoviridae, all of which possess double-stranded dna genomes, exhibited more frequent co-divergence than the other virus families studied here. at the other extreme, the virus and host trees for all the rna viruses studied here, particularly the rhabdoviridae and the picornaviridae, displayed high levels of topological incongruence, indicative of frequent host switching. overall, we show that cross-species transmission plays a major role in virus evolution, with all the virus families studied here having the potential to jump host species, and that increased sampling will likely reveal more instances of host jumping. emerging infectious diseases are often characterized by host switching events, in which a pathogen jumps from its original host to infect a novel species. however, given the ecological and genetic barriers a virus must overcome to jump species and adapt to new hosts, it might be reasonable to assume that successful cross-species transmission is a relatively rare occurrence and that viruses are instead more likely to co-diverge with their hosts. using a comparative co-phylogenetic analysis performed at the scale of virus family a a a a a emerging pathogens that cross the species barrier to infect new hosts can profoundly affect human and animal health, as well as wildlife and the agricultural industries. although most emerging diseases seemingly result from such a process of cross-species transmission, it is also the case that some viruses seem to rarely jump the species barrier and instead co-diverge with their hosts over long stretches of evolutionary time. for example, long-term virus-host codivergence has been suggested to play a key role in the evolution of vertebrate herpesviruses over periods of~ million years [ ] and insect baculoviruses over a time-scale of~ million years [ ] . indeed, it has been proposed that a number of families of dna viruses have codiverged with their hosts over long evolutionary time-scales [ ] [ ] [ ] , and do so more frequently than rna viruses, which in contrast display a combination of co-divergence and host switching [ ] . in particular, while phylogenetic trees for some rna viruses, such as particular retroviruses, are generally congruent with those from their hosts suggesting long-term codivergence [ ] , for others, such as flaviviruses, host jumping appears to be relatively frequent [ ] . in the case of flaviviruses this likely in part reflects the fact that many are transmitted by arthropod vectors and characterized by short durations of infection. the situation appears to be even more complex in cases such as the hantaviruses where there is evidence of both codivergence and host jumping [ ] . given the evolutionary and ecological barriers a virus must overcome to cross the species barrier and successfully establish itself in a new host, it might seem reasonable to assume that successful cross-species transmission is a relatively rare occurrence [ ] . indeed, many emerging diseases are in reality 'spill-over' infections, in which onward transmission between members of a new host species is limited such that extinction of the novel virus occurs rapidly [ ] . nevertheless, it is possible that an increased sampling of hosts and their viruses will reveal more instances of host jumping, in turn implying that cross-species transmission is a fundamental aspect of virus evolution [ ] . as a case in point, although there is strong evidence that hepadnaviruses have co-diverged with their vertebrate hosts over hundreds of millions of years [ ] , the recent identification of hepadnaviruses in fish and amphibians has revealed more instances of cross-species transmission, potentially including that from aquatic to terrestrial vertebrates [ ] . clearly, identifying the relative frequencies of co-divergence versus cross-species transmission is of central importance to understanding the basic mechanisms of virus evolution and disease emergence. in particular, it is important to determine whether some virus families have a greater propensity to jump hosts than others and, if so, what factors govern this pattern. currently, however, there is no quantitative or comparative measure of the frequency of crossspecies transmission versus co-divergence, so that determining whether one virus family is more likely to jump species boundaries than another is difficult to assess. one simple and powerful way to estimate these key evolutionary parameters is via 'co-phylogenetic' analysis that assesses the degree of phylogenetic congruence (i.e. similarity) between hosts and their parasites [ ] . in particular, a clear congruence between the host and virus phylogenies provides strong evidence for a history of co-divergence, whereas phylogenetic incongruence (i.e. discordance) is compatible with cross-species transmission. to date, co-phylogenetic studies of viruses have largely focused on the evolution of a subset of viruses within a particular virus family, and have not been performed in a comparative manner. for example, although there has been much work dedicated toward describing co-divergence in herpesviruses, these studies generally only encompass one particular host type (e.g. primates [ ] ) and so may fail to capture the broader picture of potential host jumps among more distantly related species. hence, there has been no attempt to use analyses of this kind to provide a broad-scale comparative and quantitative measure of the frequency of co-divergence and cross-species transmission in virus evolution. herein, we provide such an analysis. specifically, using a normalized tree topology distance metric based on the penny and hendy distance metric that enables comparisons between pairs of virus and host trees with different numbers of tips [ ] , which we now term the 'nph ' distance (where n = normalized), we compare phylogenies of virus families and their hosts. while this method does not explicitly model host-switching events, it does provide a simple means to compare multiple topologies of virus-host pairs, and accounts for differences in sample size and the fact that several viruses from a specific family can infect a single host species. to provide a quantitative measure of host switching we compared virus families, incorporating viruses infecting a diverse sample of eukaryotic hosts including mammals, birds, reptiles, amphibians, fish, plants and insects. under the measure we utilize here, when nph = between the virus and host trees it implies that their topologies are identical such that there is very strong evidence for co-divergence ( fig a) . conversely, if nph = , there are no clades in common such that co-divergence is implausible ( fig b) . crucially, this metric does not depend on where the mismatched clades are located in the tree. for example, for a pair of virus and host trees that differ in one clade, the nph is the same whether species jumping events were recent (i.e. shallow nodes fig c) or ancient (i.e. deep nodes fig d) . importantly, the nph distance increases as the number of incongruent nodes (i.e. nodes that differ) between the virus and host trees increases ( fig e) . a phylogenetic measure of the relative frequency of virus-host codivergence our analysis considered a total of seven dna and rna virus data sets that provided sufficient data to perform a quantitative co-phylogenetic analysis. hence, the study relied heavily on specific selection criteria (see materials and methods) that necessarily limited data availability. despite these rigorous criteria, the majority of data sets encompassed a diverse collection of viruses and host species, and hence can be regarded as illustrative of the broad-scale frequency of co-divergence versus cross-species transmission. these data contained no evidence for recombination. to determine the prevalence of host switching between different viruses, we inferred family-level viral phylogenies and compared these to phylogenies of their hosts. importantly, our analytical approach-which utilizes the nph distance-provides a relative measure of phylogenetic congruence that is directly comparable between data sets that differ in size (i.e. different number of viruses and host species). our method assumes that viruses that have codiverged with their hosts will share the same tree topology. in contrast, an increasing number of host jumping events should lead to greater phylogenetic incongruence. the reasoning behind this assumption is that there exists a very large number of possible phylogenetic tree topologies even for data sets with a few samples, such that similarities between a pair of virushost trees (i.e. congruence) are highly unlikely to arise by chance. of course, phylogenetic events other than cross-species transmission might also lead to phylogenetic incongruence and we test the validity of this assumption later in the manuscript. across the data set as a whole we found that all virus families displayed relatively large tree topological distances with nph values of ! . , suggesting that cross-species transmission is widespread, at least at the family-level (fig ; s table) . while all families showed distances at the upper end of the scale, the hepadnaviridae (double-stranded dna) had the shortest distance (nph = . ), indicating that this family experiences more frequent co-divergence than any other studied here. at the other end of the spectrum both the rhabdoviridae and picornaviridae (single-stranded rna) displayed nph > . , indicative of frequent host switching and hence little evidence for virus-host co-divergence. we also investigated when the species jumping events occurred in the evolutionary history of the virus families. to do this, we determined whether phylogenetic incongruences tended to cross-species transmission among viral families occur in deeper sections of the phylogeny or to more shallow nodes in the tree. accordingly, we considered the number of nodes subtending clades in the host tree that are not present in the virus tree, a metric known as 'node depth'. nodes that are deep correspond to clades that are more diverse, and often older, than those clades subtended by shallower nodes. for each pair of virus-host trees we calculated the depth of every node that differed within each virushost pair and divide each depth by the maximum node depth (fig ) . this normalized metric, which we term 'relative node depth', ranges between near for phylogenetic incongruences at shallow nodes, and for incongruences at deeper nodes. most incongruences corresponded to shallow nodes, which is expected because there are naturally more shallow nodes than deep nodes in phylogenetic trees. however, that incongruences were found in both shallow and deep nodes suggests that co-divergence is relatively rare in these virus families, even over long evolutionary time-scales. tanglegrams depicting pairs of rooted phylogenetic trees display the evolutionary relationship between each virus family and their host species (fig ; phylogenies with the individual tip labels visible are shown in s fig) . despite the obvious widespread occurrence of host jumping, a number of co-phylogenies reveal the occurrence of at least some co-divergence, as expected from the nph distances. for example, the tanglegrams for the hepadnaviridae and poxviridae exhibit some clear matches with the evolutionary histories of their respective hosts. most notably, their co-phylogenies show a clear segregation between distinct clades that are associated with a specific host type (mammals, birds, etc.). conversely, the phylogenies of most rna viruses appear to largely mismatch those of their hosts. our fundamental assumption is that incongruences between virus and host topologies imply the occurrence of cross-species transmission. to test the validity of this assumption, we reconciled the viruses with the phylogenetic history of their hosts. by associating 'event costs' with host-jumping, as well as with lineage duplication and extinction events, we found the range of optimal co-phylogenetic solutions for each virus family ( fig a) . as with the analysis of topological distances, this revealed that cross-species transmission was the most common evolutionary event in all virus families studied here, with co-divergence consistently less frequent (with the possible exception of the hepadnaviridae-see below), and lineage duplication and extinction playing a much more minor role. we next reconstructed the history of these evolutionary events in detail in the hepadnaviridae (i.e. the most co-divergent virus family). this revealed that under the most likely co-phylogenetic scenario the proportion of crossspecies transmission represents . of all events (i.e. co-divergence = events; duplications = ; extinction = ; host-jumping = ; fig b) . since the nph distance for the hepadnavirus data set was . , we suggest that our method generates results consistent with the reconciliation analysis. in addition, one important disadvantage of performing full reconciliation analysis is that co-phylogenetic methods such as that implemented in jane [ ] and tarzan [ ] are not straightforward since they offer many combinations of possible events and are difficult to compare between families, especially in cases with more than~ viruses where there are many possible co-phylogenetic scenarios. despite these limitations, our reconciliation analysis did reveal the possible causes of the topological incongruence between the virus and host phylogenies. we next determined whether there was any association between the relative frequency of codivergence and larger scale biological properties, such as the number of viruses per family and whether the viruses in question possess rna or dna genomes. to better display this analysis branches on the co-phylogenetic trees were colored according to host type, which comprised mammals, fish, birds, reptiles, amphibians, invertebrates, and plants (fig ) , such that each cophylogeny incorporated between one (i.e. potyviridae) and five (i.e. togoviridae) host types. notably, we found a significant association between the number of viruses per virus family and the nph (p< . ) (fig a) . importantly, because we expect no association between [ ] . boxplots illustrate the range of the proportion of possible events. the 'event costs' associated with incongruences between trees were conservative towards co-divergence and defined here as: for co-divergence, for duplication, for host-jumping and for extinction. virus families are ranked in order of highest mean co-divergence to lowest mean co-divergence. abbreviations on the x-axis are as follows: 'co-div' = co-divergence, 'dup' = duplication, 'hj' = host-jumping, 'ext' = extinction. (b) reconciliation of the hepadnaviridae phylogeny with that of their vertebrate hosts, again utilizing the co-phylogenetic method implemented in jane [ ] . the figure illustrates all possible codivergence, extinction and host-jumping events (no lineage duplication events were reconstructed in this case). the number of viruses and hosts per family and the nph under our tree distance metric, this result implies that sampling more viruses increases the likelihood of detecting host jumping events. in addition, we found that dna viral families had, on average, a shorter nph distance than families of rna viruses (p< . ) (fig b) . note that there is no significant difference (p = . ) between the number of viruses in families of dna viruses compared to those in rna virus families. in this context it is striking that the five families with the shortest topological distances all possessed dna genomes. this analysis also revealed that segmented viruses had a significantly larger nph distance than non-segmented viruses (p< . ), and that negative-sense rna viruses had a larger nph distance than positive-sense rna viruses (p< . ); however, the sample sizes within all these categories were small so that these results should be treated with caution. finally, we note that although the duration of infection (for example, the division between acute versus chronic infections) is clearly a parameter that would likely affect the frequency of host jumping [ , ] , we were unfortunately unable to perform any analyses of this variable on the data available here as it tends to be host-specific rather than a general characteristic of individual virus families. understanding how viruses and their hosts co-evolve is central to revealing the nature of virus evolution and the determinants of disease emergence. in particular, we lack a quantitative understanding of whether some types of virus, such as those classified into different families or that possess genomes of different nucleic acid types, are better able to jump species boundaries compared to others. to investigate the comparative prevalence of cross-species transmission among viruses we measured the congruence between virus and host phylogenetic trees using a normalized tree topological distance-based approach (nph , [ ] ). if taxonomically related viruses have an evolutionary history of co-divergence with their hosts the virus and host phylogenetic trees should be similar in topology, whereas phylogenetic incongruence is the signature of species jumping. overall, our analysis revealed absolute departure from co-divergence among all the virus families studied here (nph ! . and supported by the reconciliation analysis) suggesting that cross-species transmission occurs frequently, at least at the level of virus family. particularly striking was that even the most slowly evolving dna viruses, which have previously been suggested to represent exemplars of virus-host co-divergence [ ] , exhibit relatively common cross-species transmission. hence, at their most basic, these results indicate that viruses are often exposed to a variety of susceptible host species that provide opportunities for cross-species transmission. despite the overall large nph distances observed among all virus families, our data also revealed that the hepadnaviridae, polyomaviridae, poxviridae, papillomaviridae and adenoviridae had the shortest nph distances and were thus relatively more host-specific than the other virus families analyzed here. this is supportive of earlier suggestions that some dna viruses have a long history of co-divergence with their hosts [ ] , which in some cases may be a reflection of relatively long durations of infection. indeed, long-term virus-host associations have been observed in the herpesviridae [ ] , the poxviridae [ ] and the polyomaviridae [ ] . however, it is also important to note that we found these viruses contain more instances of host jumping than previously thought. for example, although the tanglegram shown in fig suggests co-divergence in the case of some primate hepadnaviruses, cross-species transmission seemingly occurs more frequently among those hepadnaviruses that infect birds. in addition, it was recently observed that a fish (bluegill) hepadnavirus clusters more closely with mammalian hepadnaviruses than to other fish viruses [ ] (see figs and b). similarly, early studies of rna viruses suggested that virus-host co-divergence was important in the evolution of two members of the flaviviridae that infect primates-the pegiviruses and hepaciviruses, [ ] [ ] [ ] . however, more recent phylogenetic analyses of expanded data sets have revealed multiple cross-species transmissions events, including the recent emergence of hepaciviruses in domestic dogs, horses and donkeys [ ] , and a newly described pegiviruses in rodents, bats and horses [ ] . despite the obvious caveat of sample size, it seems that rna viruses generally experience more frequent cross-species transmission than their dna counterparts. indeed, the rna viral families analyzed here had an overall mean nph distance of . , compared to dna viruses with a mean of . . this may, in part, be due to the fact that rna viruses are generally characterized by very high rates of mutation and replication [ ] . intuitively, high rates of evolutionary change should confer more rapid adaptation to new environments, which, coupled with the frequency of exposure to new hosts, will facilitate host-switching. in addition, many rna viruses are characterized by short durations of infection that will limit the opportunities for virus-host co-divergence [ ] . an informative exception among rna viruses are the simian foamy viruses (sfv), in which hosts may develop long-term latent infections and the virus has been associated with long-term co-divergence [ ] . indeed, it is notable that among the retroviridae analyzed here those assigned to sfv seem to display relatively similar evolutionary histories to those of their primate hosts (see s fig) . it is also possible that successful cross-species transmission occurs more frequently among phylogenetically related hosts, likely because it is easier to infect and replicate in genetically similar hosts that share less divergent cell receptors [ ] . in addition, related hosts may sometimes inhabit the same geographic region, increasing the probability of cross-species transmission through more frequent exposure [ ] . indeed, a useful generality in studies of disease emergence is that the closer the phylogenetic relationship between hosts, then, given appropriate exposure, the more likely that a pathogen will be able to jump between them, in turn leading to preferential host switching [ ] . if true, so that cross-species transmission results in a viral phylogeny that mirrors that of their hosts, then any phylogeny-based approach such as that utilized here will underestimate the true frequency of host jumping. as a case in point, although there is a general concordance between the phylogenies of simian immunodeficiency virus (siv) and their primate hosts, in which four species of african green monkey harbor distinct forms of siv that is clearly suggestive of co-divergence [ ] , it has been argued that the evolutionary history of siv may also have been shaped by preferential host switching [ ] , although these mechanisms are not mutually exclusive. in contrast, incomplete lineage sorting among closely related viruses may produce a false signal for cross-species transmission when co-divergence has in fact occurred [ ] . in addition, because there is growing evidence that viruses can have complex evolutionary histories with genes derived from multiple sources [ ] , it is important to note that our virus phylogenies are necessarily gene trees rather than species trees. it is therefore possible that other virus gene trees will exhibit a stronger topological match with host phylogenies than those presented here, and hence provide more evidence for co-divergence. finally, while our analysis was only based on robust phylogenetic patterns, because nodes that were topologically uncertain were excluded from the analysis, it is possible that our virus trees contain topological errors reflecting the use of sometimes small numbers of highly divergent sequences. another important aspect of assessing virus-host co-divergence is that the evolutionary time-scales of viruses and their hosts are consistent [ ] . although such a comparison is valuable, it is problematic for the present study because high rates of evolution lead to substitutional saturation in virus genomes at a much faster rate than in cellular organisms. indeed, it is likely that many of the cross-species transmission events implied here have occurred on timescales of many millions of years. as a result, temporal signal is rapidly lost, precluding accurate estimates of their long-term evolutionary time-scales, even though the topology is often accurately recovered [ ] . we therefore suggest that simpler topological comparisons such as those performed here may be a more informative way to proceed in family-level studies of cross-species transmission versus co-divergence. overall, we have observed frequent cross-species transmission across the virus families studied here, with relatively little evidence for virus-host co-divergence. hence, our study suggests that, at the virus family scale in the data analyzed here, host switching plays a major role in the evolution and diversification of viruses and, importantly, that it can occur in viruses of all types. interestingly, we found that increased sampling of viruses from different host species reveals more frequent species jumping events among viral families. as such, the discovery of new viruses is likely to reveal more instances of cross-species transmission. undoubtedly, the analysis presented here should be extended to a wider range of data sets as they become available, particularly because increased taxon sampling results in a larger tree space and increases the statistical power of these analyses. gene sequence data of viruses were obtained from genbank (table ; see s table for all gen-bank accession numbers). following a broad and comprehensive survey of all virus genomic data available on genbank, a total of family-level virus data sets passed our selection criteria and were included in the analysis. these selection criteria, which are independent of whether the viruses have evolved by co-divergence or cross-species transmission, were: (i) the availability of virus sequence data that included a wide range of distinct and diverse virus species that is representative of the virus genera currently available; (ii) the availability of data with informative genomic regions that can be used to reveal evolutionary relationships (e.g. the rnadependent rna polymerase-see table ) and that were not so divergent as to prevent reliable sequence alignment; and (iii) the virus sequence data met a minimum length requirement of amino acids following alignment and the removal of any ambiguously aligned regions. the virus families that passed these selection criteria were the adenoviridae, bunyaviridae, caliciviridae, coronaviridae, flaviviridae, hepadnaviridae, herpesviridae, orthomyxoviridae, papillomaviridae, paramyxoviridae, parvoviridae, picornaviridae, polyomaviridae, potyviridae, poxviridae, reoviridae, retroviridae, rhabdoviridae and togaviridae. each data set contained between - viruses from a diverse range of eukaryotic hosts, including mammals, birds, reptiles, amphibians, fish, invertebrates, and plants. for the purposes of this study we regarded a virus isolated from a particular host species as a distinct virus sample worthy of analysis: for example, rabies virus isolated from a human host was deemed distinct from rabies virus isolated from a canine host. the resulting virus and host data sets included in this study comprised a diverse sample of the available data (see s table for a summary of the virus and host diversity). most data sets contained more viruses than those from their corresponding hosts because they included multiple viruses from a family that can infect the same host. for each virus family nucleotide sequences were first translated to amino acid data using seqotron v. . . [ ] , aligned with muscle v. . [ ] , and poorly aligned regions then eliminated using trimal [ ] , ensuring that all remaining sequences were at least amino acids in length (table ) . amino acid sequences were aligned because there is widespread substitutional saturation at the nucleotide level. although our data sets utilize single genes, we ensured that they were free of inter-specific virus recombination using rat [ ] . to estimate phylogenetic trees for the virus data sets we selected the optimal amino acid substitution model identified using the bayesian information criterion as implemented in modelgenerator v . [ ] and analyzed the data using phyml v . [ ] , employing the spr branch-swapping tree search algorithm (see table for the substitution models used). we assessed the support for individual nodes using the approximate likelihood ratio test (alrt) implemented in phyml v . [ ] , with alrt values ranging between (no support) and (strong support). studies involving simulations and empirical data have demonstrated that this statistic has similar false-positive rates to other metrics, such as the non-parametric bootstrap [ ] . cladograms were constructed for all host species from which the viruses of interest were isolated. in each case the host tree topologies used were the most up-to-date available in the literature [ ] [ ] [ ] [ ] [ ] . for the vector-borne viruses studied here, in which viruses pass between arthropods and vertebrates, the appropriate vertebrate species were assigned as the hosts. in contrast, for insect-specific viruses, where there is no evidence for vertebrate involvement, the relevant invertebrate species were assigned as the hosts. since there were often multiple viruses that infected the same host species, multiple lineages within a single host (i.e. polytomies) were added to the host phylogenetic tree to ensure the number of hosts equaled that of the virus tree. the addition of these polytomies does not influence the nph distance metric (described in detail below) because the distance between a polytomous clade and one that is fully resolved is zero [ ] . all virus and host phylogenetic trees and virus sequence alignments are available at github. com/jemmageoghegan. we measured the extent of virus-host co-divergence (and by exclusion host-jumping) by comparing, in a quantitative manner, the tree topologies for viruses and their corresponding hosts. to this end we calculated a normalized ph tree topological distance [ ] , referred to here as the 'nph ' distance (this function has been included in nelsi v . [ ] ). specifically, the nph distance, which utilizes two phylogenetic trees as its input, describes the number of bipartitions (clades) that are not shared between two tree topologies. importantly, it does not depend on the nodes where the topological differences occur in the tree (fig ) . in addition, this metric considers the tree topology of unrooted trees, but not the branch lengths of the tree. first, the ph metric is calculated as the topological distance between a pair of unrooted trees. it can be understood in terms of the following: where t and t are the clades contained within the host and virus trees, respectively. let the expression t \ t denote the clades that are shared between both trees so that (t \ t ) corresponds to the clades that are not shared between the pair (i.e. those that are unique to each tree). the actual ph distance is twice the number of unique clades. to normalize this metric we divide ph by the maximum distance by considering the two tree topologies, randomizing the tips for one of the trees times, and calculating ph for each replicate (where randomizations was shown to be robust even for very large trees; see s fig) . the largest value of the randomizations is approximately the maximum ph distance in tree topologies. therefore, nph ranges between , for identical trees, and , for trees that have no clades in common (fig ) . the advantages of this method over other tree distance metrics is that it is comparable for pairs of trees with different numbers of tips, it maintains the backbone of the tree (i.e. the tree structure remains constant, unlike in [ ] ), and it is comparable for trees with polytomous nodes. to address phylogenetic uncertainty, we collapsed all nodes with alrt of less than . , which corresponds to a false-positive rate of < . [ ] . in such cases, we randomly resolved the polytomies times and calculated the nph . accordingly, we report the overall normalized topology distance, as well as the mean and % percentile range of values (s table) . to determine whether host jumping occurred more often toward the root or tips of the trees, we calculated the relative node depth for incongruent nodes between virus-host pairs of trees (see fig c and d ). this metric counts the number of nodes contained within each clade in the host tree that are not present in the virus tree. because this number can depend on the size of the tree, we divide each of the node depths by the largest value in the tree. accordingly, this metric is decreased if incongruent clades correspond to shallow nodes ( fig c) compared to deep nodes (fig d) . for example, the maximum node depth is if a pair of trees differs in the deepest node and approaches if they differ only in very shallow nodes. an important assumption of the current study is that incongruence between virus and host topologies is a result of cross-species transmission. in some instances, however, it might be possible to explain the lack of virus-host co-evolutionary history through multiple instances of lineage duplication and extinction, without such host-switching events. to address this issue, we reconciled the co-phylogenetic relationship between viruses and their hosts. in particular, we determined the optimal solutions for co-phylogenetic reconstruction for all families, including the possibility of lineage duplication and extinction, using the jane co-phylogenetic software package [ ] . this uses a polynomial time dynamic programming algorithm in conjunction with a genetic algorithm to find optimal solutions to reconcile cophylogenies. although this is a simple heuristic method, it is able to generate results on relatively large data sets (although it is most effective for trees with less that~ - tips). importantly, we used 'event costs' associated with incongruences between trees that were conservative towards co-divergence and defined here as: for co-divergence, for duplication, for host-jumping and for extinction. utilizing this reconciliation, we also examined the evolution of the hepadnaviridae in more detail as this family contains the best evidence for co-divergence (see results). finally, to assist in visualization of these data, tanglegrams for each virus family were constructed using treemap v . [ ] . lines between the trees connect the host (left) with its virus (right). we utilized the 'untangle' function, which rotates the branches of one tree, to minimize the number of crosses lines. if viruses and hosts have congruent topologies then the number of crossed lines, and hence cross-species transmission events, will obviously be reduced. required to obtain the maximum topological distance (black lines) for the hepadnaviridae and the parvoviridae phylogenies, which represent the minimum and maximum number of viruses in our data sets, respectively. the red, dashed line illustrates the ph distance of the non-randomized data, while the black, solid line is the ph distance after randomizing the data after n randomizations. (tif) s table. genbank accession numbers for the virus and host genetic sequence data utilized here. (docx) virus genera were excluded either due to lack of available data or because we were unable to obtain a reliable alignment of sufficient length for phylogenetic analysis (i.e. at least amino acids after trimal pruning). (docx) s table. overall nph distances, means and % percentiles between two unrooted phylogenetic trees for each virus family determined using the normalized penny and hendy [ ] topological distance method, implemented in in nelsi v . [ ] . the overall nph distances are illustrated in fig in integrating reptilian herpesviruses into the family herpesviridae paleozoic origin of insect large dsdna viruses acute and persistent viral life strategies and their relationship to emerging diseases evolution and emergence of rna viruses virological factors that increase the transmissibility of emerging human viruses the evolution and emergence of hantaviruses. current opinion in virology a cophylogenetic perspective of rna-virus evolution family level phylogenies reveal modes of macroevolution in rna viruses pathogen population bottlenecks and adaptive landscapes: overcoming the barriers to disease emergence early mesozoic coexistence of amniotes and hepadnaviridae distinct viral lineages from fish and amphibians reveal the complex evolutionary history of hepadnaviruses tangled trees: phylogeny, cospeciation and coevolution absence of frequent herpesvirus transmission in a nonhuman primate predator-prey system in the wild the use of tree comparison metrics jane: a new tool for the cophylogeny reconstruction problem reconstruction of the cophylogenetic history of related phylogenetic trees with divergence timing information time scale evolution of avipoxviruses comparing phylogenetic codivergence between polyomaviruses and their hosts evaluating the evidence for virus/host co-evolution. current opinion in virology convergent evolution of escape from hepaciviral antagonism in primates phylogenetic analysis of gb viruses a and c: evidence for cospeciation between virus isolates and their primate hosts. the journal of general virology differential infection patterns and recent evolutionary origins of equine hepaciviruses in donkeys bats are a major natural reservoir for hepaciviruses and pegiviruses viral mutation rates island biogeography reveals the deep history of siv cross-species virus transmission and the emergence of new epidemic diseases viral evolution and the emergence of sars coronavirus preferential host switching by primate lentiviruses can account for phylogenetic similarity with the primate phylogeny redefining the invertebrate rna virosphere substitution model adequacy and assessing the reliability of estimates of virus evolutionary rates and time scales model selection in phylogenetics seqotron: a user-friendly sequence editor for mac os x muscle: multiple sequence alignment with high accuracy and high throughput trimal: a tool for automated alignment trimming in large-scale phylogenetic analyses recombination analysis tool (rat): a program for the highthroughput detection of recombination assessment of methods for amino acid matrix selection and their use on empirical data shows that ad hoc assumptions for choice of matrix are not justified new algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of phyml . . systematic biology approximate likelihood-ratio test for branches: a fast, accurate, and powerful alternative survey of branch support methods demonstrates accuracy, power, and robustness of fast likelihood-based approximation schemes. systematic biology a comprehensive phylogeny of birds (aves) using targeted next-generation dna sequencing the tree of life and a new classification of bony fishes angiosperm phylogeny poster-flowering plant systematics resolving conflict in eutherian mammal phylogeny using phylogenomics and the multispecies coalescent model reevaluating the arthropod tree of life simulating and detecting autocorrelation of molecular evolutionary rates among lineages distributions of tree comparison metrics-some new results. systematic biology writing -review & editing: jlg sd ech. key: cord- -pw xqhwa authors: feeley, eric m.; sims, jennifer s.; john, sinu p.; chin, christopher r.; pertel, thomas; chen, li-mei; gaiha, gaurav d.; ryan, bethany j.; donis, ruben o.; elledge, stephen j.; brass, abraham l. title: ifitm inhibits influenza a virus infection by preventing cytosolic entry date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: pw xqhwa to replicate, viruses must gain access to the host cell's resources. interferon (ifn) regulates the actions of a large complement of interferon effector genes (iegs) that prevent viral replication. the interferon inducible transmembrane protein family members, ifitm , and , are iegs required for inhibition of influenza a virus, dengue virus, and west nile virus replication in vitro. here we report that ifn prevents emergence of viral genomes from the endosomal pathway, and that ifitm is both necessary and sufficient for this function. notably, viral pseudoparticles were inhibited from transferring their contents into the host cell cytosol by ifn, and ifitm was required and sufficient for this action. we further demonstrate that ifn expands rab and lamp -containing structures, and that ifitm overexpression is sufficient for this phenotype. moreover, ifitm partially resides in late endosomal and lysosomal structures, placing it in the path of invading viruses. collectively our data are consistent with the prediction that viruses that fuse in the late endosomes or lysosomes are vulnerable to ifitm 's actions, while viruses that enter at the cell surface or in the early endosomes may avoid inhibition. multiple viruses enter host cells through the late endocytic pathway, and many of these invaders are attenuated by ifn. therefore these findings are likely to have significance for the intrinsic immune system's neutralization of a diverse array of threats. the h n pandemic provided a strong reminder of the threat that influenza a virus poses to world health (http://www. cdc.gov/h n flu/cdcresponse.htm). the most effective means of protection against influenza is the seasonal vaccine. however, if the vaccine does not match the viral strains, its effectiveness can be reduced to % or less [ , ] . among small molecules, only two approved influenza drugs remain effective, zanamivir (relenza) and oseltamivir (tamiflu). although resistance to zanamivir is rare, there has been an increase in oseltamivir-resistant flu strains [ ] . of concern, both drugs target viral neuraminidase (na), precluding combinatorial therapy to minimize resistance [ , ] . thus, research to identify new anti-influenza strategies would be useful. the influenza a virus is - nm in size, encodes for up to proteins, and contains eight segments of negative single-stranded genomic rna ( ) . influenza a virus infection initiates with the cleavage and activation of the viral hemaglutinnin (ha) envelope receptor by host proteases [ , , , ] . ha then binds to sialylated proteins on the cell surface, eliciting endocytosis of the viral particle. endocytosed viruses are transported through the early and late endosomes, with late endosomal acidification triggering a conformational change in ha which results in viral-host membrane fusion [ , ] . fusion transitions from a hemifusion intermediate into a fusion pore through which the virus' eight viral ribonucleoproteins (vrnps) enter the cytosol. the vrnps are subsequently guided by the host cell's karyopherins into the nucleus [ , , ] , wherein the viral rna-dependent rna polymerase synthesizes viral genomes (vrna) and mrnas, both of which are exported to the cytosol, culminating in the production of viral progeny. genetic screens have identified multiple host factors and pathways which modulate influenza a virus infection in vitro [ , , , ] . using such a genetic screen, we identified the ifitm protein family members ifitm , and as antiviral factors capable of blocking influenza a viruses [ ] . we further tested the antiviral activity of ifitm protein using the seasonal influenza a strains, a/uruguay/ / (h n ) and a/brisbane/ / (h n ), and found similar levels of ifitm mediated viral inhibition [ ] . ifitm accounts for a significant portion ( - %) of ifn's (type i or ii) ability to decrease influenza a virus infection in vitro, and ifitm resides in vesicular compartments that are ifn-inducible [ ] . in addition, the ifitm family inhibits infection by the flaviviruses, dengue virus and west nile virus [ , ] , as well as the filoviruses, ebola and marburg, and the sars coronavirus [ ] . the ifitm proteins also block vesicular stomatitis virus-g protein (vsv-g)-mediated entry, but do not substantially alter the replication of moloney leukemia virus (mlv), several arena viruses, or hepatitis c virus (hcv, [ , ] ). the human ifitm proteins were identified years ago based on their expression after ifn stimulation [ , , ] . the ifitm , , and genes are clustered on chromosome , and all encode for proteins containing two transmembrane domains (tm and ), separated by a conserved intracellular loop (cil, [ ] ), with both termini extra-cellular or intra-vesicular [ , ] . tm and the cil are well conserved between the ifitm proteins and a large group of proteins representing the cd protein family. cd family members exist from bacteria ( members) to man ( members, with members in chordata), with no in depth functional data available for any member other than the ifitm proteins. ifitm , and are present across a wide range of species including amphibians, fish, fowl and mammals. the ifitm proteins have been described to have roles in immune cell signaling and adhesion, cancer, germ cell physiology, and bone mineralization [ , , , , , ] . ifitm expression can inhibit the growth of some ifn-responsive cancer cells [ ] . genetic evidence also points to ifitm /bril being required for early bone mineralization [ , ] . ifitmdel mice, which are null for all five of the murine ifitm genes, display a % perinatal mortality among null pups, but thereafter grow and develop normally in a controlled setting [ ] . however, cells derived from these ifitmdel mice are more susceptible to influenza a virus infection in vitro [ ] . ifitm inhibited infection by all influenza a virus strains tested including a pandemic isolate and two contemporary seasonal vaccine viruses [ ] . we have found ifitm to be the most potent of the ifitm protein family members in decreasing influenza a virus replication [ ] . viral pseudoparticles are differentially inhibited by the ifitm proteins based on the specific viral receptors expressed on their surfaces [ , ] . therefore, we have hypothesized that ifitm proteins inhibit susceptible virus families (orthomyxoviridae, flaviviridae, rhabdoviridae, filoviridae, and coronaviridae) during the envelope-dependent early phase of the infection cycle, which extends from viral binding to cell surface receptors through the creation of the fusion pore between viral and host membranes [ , , ] . in support of this notion, recent work demonstrated that ifitm protein overexpression did not prevent influenza a virions from accessing acidified compartments [ ] . consistent with its acting on endocytosed viruses, a portion of ifitm resides in structures that contain host cell endosomal and lysosomal proteins [ ] . furthermore, inhibition of influenza a virus infection depends on the palmitoylation of ifitm , a posttranslational modification that targets proteins to membranous compartments [ ] . here we directly test the idea that ifitm restricts influenza a viral infection during the envelope-dependent early phase of the viral lifecycle. consistent with previous studies, we find that ifitm inhibits influenza a viral infection after viral-host binding and endocytosis, but prior to primary viral transcription [ , ] . moreover, using a combination of assays, we find that either ifn or high levels of ifitm impede influenza a viruses from transferring their contents into the host cell cytosol, and that ifitm is necessary for this ifn-mediated action. therefore, we conclude that ifn is acting predominantly through ifitm to block viral fusion. we also find that ifn expands the late endosomal and lysosomal compartments, and that ifitm overexpression is sufficient for this phenotype. this study also presents data showing that ifitm overexpression leads to the expansion of enlarged acidified compartments consisting of lysosomes and autolysosomes. interestingly, we observe that viruses trapped in the endocytic pathway of ifitm -overexpressing cells are trafficked to these expanded acidified compartments. based on these results and those of others [ , ] , we present a model whereby ifn acts via ifitm to prevent viral fusion, thereby directing endocytosed viruses to lysosomes and autolysosomes, for subsequent destruction. collectively this study expands our understanding of how ifitm restricts a growing number of viruses by exploiting a shared viral vulnerability arising from their use of the host's endocytic pathway. the inhibition of ha-expressing pseudoparticles by the ifitm proteins pointed towards restriction occurring during the envelope-dependent phase of the viral lifecycle [ ] . therefore we tested ifitm 's impact on the most proximal phase of infection, viral binding, by incubating influenza a virus a/wsn/ h n (wsn/ , multiplicity of infection (moi) ) with a lung carcinoma cells either stably overexpressing ifitm (a -ifitm ) or an empty vector control cell line (a -vector, fig. a ). samples were incubated on ice to permit viral binding but prevent endocytosis. after incubation, cells were washed with cold media, fixed and stained for ha. when analyzed by flow cytometry, we observed no appreciable difference in surface bound ha between the vector and ifitm cells. there was also no difference in surface-bound virus over a series of ten-fold dilutions of viral supernatant (data not shown). we also determined that the stable expression of ifitm did not alter the surface levels of (a , ) or (a , ) sialylated cell-surface proteins (fig. s ) . to investigate ifitm 's impact on initial viral mrna production, we infected canine kidney cells, either expressing ifitm (mdck-ifitm ) or the empty vector (mdck-vector), with inf luenza a virus (a/puerto rico/ / h n (pr ), moi ). we used pr because of the purified high titer stocks available. next, the viral supernatant was removed and warm media was added ( min). at the indicated times, cells were processed and stained for the positive stranded np mrna of pr using a specific rna probe set (red, fig. b) , then imaged on a influenza epidemics exact a great toll on world health. thus research to identify new anti-influenza virus strategies would be useful. each of our cells contains antiviral factors that work to inhibit infection. a large component of this antiviral program is regulated by the interferon family of signaling molecules. here, we seek to better understand how one of these antiviral factors, ifitm , contributes to both baseline, as well as interferon-induced, antagonism of influenza a viral infection. we found that interferon prevents influenza a virus from entering our cells by blocking the virus' fusion with the cellular membrane. furthermore, we learned that ifitm is required for this antiviral action of interferon, and that high levels of ifitm alone can produce a similar viral inhibition. together, these results improve our understanding of how ifitm serves to defend us against viral invasion at a very early stage of infection. confocal microscope. based on np mrna staining, primary viral transcription begins by min. p.i. in the vector control, with the np mrna signal increasing through to min., when the export of viral mrnas to the cytosol can be observed. a decrease in primary viral transcription can be seen when comparing the ifitm cells to the vector control line. therefore, ifitm inhibits influenza a viral infection after viral-host binding but before primary viral mrna transcription. ifn interferes with vrnp nuclear entry and ifitm is necessary and sufficient for this antiviral defense we next used confocal imaging to track the nuclear translocation of vrnps ( fig. [ , ] ). at the start of infection, the np within infected cells is complexed with viral genomic rna forming vrnps. therefore, immunostaining for np permitted us to follow vrnp distribution intracellularly [ , , ] . normal diploid human lung fibroblasts (wi- cells) were stably transduced with empty vector (vector), ifitm cdna (ifitm ), or short hairpin rnas (shrna) either against ifitm (shifitm ) or a scrambled non-targeting control (shscramble, fig. , s ). wi- s were chosen because of their normal karyotype and relatively larger and flatter morphology. cells were first incubated on ice with pr (moi ). next, the viral supernatant was removed and warm media was added ( min). at the indicated times after warming, cells were fixed, permeabilized, stained for np and dna, and imaged on a confocal microscope. image analysis software was used to create an outline of each cell's periphery (white lines) and nucleus (blue lines). based on np staining, vrnps arrive in the nuclei by min in the vector control, shifitm , and in the shscramble cells, with the np signal increasing through to min ( fig. a, s a-d) . in contrast, we observed decreased nuclear and increased cytosolic np staining in the ifitm cells (fig. , s c) . moreover, in the ifitm cells greater than % of the cytosolic np colocalized with lysotracker red (ltred), a dye which marks acidic cellular compartments (late endosomes, lysosomes, ph# . ), and which was added to the warm media at time zero (fig. s a, d) . the increased np in the cytosol of the ifitm cells likely arises in part from an increase in the local concentration of viruses because a-np western blots (after trypsinizing the cells to remove adherent np) did not show substantial differences in internalized np levels between cell lines for up to min post infection (p.i., data not shown). because ifitm is required for the anti-viral actions of ifn in vitro [ ] , we performed a companion experiment with the wi- cells treated with ifn-a ( fig. b ). ifn-a treatment also decreased np nuclear staining in the wi- -vector cells, however this block was not as complete nor was it associated with similar levels of cytosolic np staining as those seen with high levels of ifitm . consistent with the gain-offunction data, the depletion of ifitm decreased ifn's ability to block vrnp trafficking to the nucleus ( fig. a and b , compare top and bottom rows). similar results were obtained either using a cells (fig. s ) or using mdck cells, with the latter experiments employing additional influenza a viral strains (x: , a/aichi/ (aichi h n ), fig. s a -c, wsn/ and a/victoria/ / h n , data not shown). it is important to note that the levels of ifitm protein in the a -ifitm cells are higher than those seen after treatment with ifn-a or -c (fig. s c) . however, we have not observed that other overexpressed proteins have either protected against viral infection or expanded the lysosome/autolysosome compartment (data not shown), arguing that this is a specific effect. to better assess the expanded ltred compartments observed with ifitm overexpression, we created mdck cells stably expressing the lysosomal protein, lamp , fused to a red fluorescence protein (lamp -rfp) and ifitm . as compared . ifn prevents vrnp nuclear entry, and ifitm is necessary and sufficient for this action. a) normal diploid human fibroblasts (wi- cells) were stably transduced with retroviruses containing either ifitm (ifitm ), a shrna against ifitm (shifitm ), an empty viral vector alone (vector), or a non-targeting control shrna (shscramble, fig. s ). cells were incubated with pr on ice, and then warm media was added at time zero. cells were fixed at the indicated times p.i. and stained for np (green) and dna and analyzed by confocal microscopy. image analysis software was used to define each cell's cytosolic (white lines) and nuclear peripheries (blue lines, based on dic images and dna staining, respectively). red arrows: cytosolic compartments containing np. images are representative of four independent experiments. (scale bar: mm). b) as in (a) except that cells were treated with ifn-a prior to infection. doi: . /journal.ppat. .g to control cells, the ifitm cells demonstrated extensive colocalization (. %) between the np and lamp -rfp signals, revealing that the entering viruses are trafficked to lysosomal compartments (fig. s ) . we extended this analysis by directly tracking the location of the vrna contained in the incoming vrnps. mdck cells stably expressing an empty vector or ifitm , were used in time-course experiments as above (fig. a-d) . at the indicated times, cells were processed and stained for the negative stranded np vrna of pr using a specific rna probe set (green). as seen with the wi- cells, we observed the nuclear translocation of vrna by min p.i. in the mdck-vector cells (fig. a) . the nuclear vrna signal was strongly decreased with ifitm overexpression based on the average number of vrna particles present per nucleus (fig. c) . consistent with the wi- results, the vrnas accumulated in the cytosol of the ifitm cells, with . % co-localizing with ltred-staining acidic structures (fig. d ). similar levels of retained cytosolic vrnps were observed in experiments without ltred (data not shown). interestingly, we observed the loss of the vrna signal in the acidic inclusions of the mdck-ifitm cells between and min. p.i. (fig. b ). by comparison, the vrnas in the control cells increased in number in both the nucleus and cytosol, as would be expected with the nuclear export of newly synthesized viral genomes [ ] . we next evaluated vrnp translocation in murine embryonic fibroblasts (mefs) derived from animals that have had all five ifitm genes deleted (ifitmdel / , [ , ] ). compared to wild-type (wt) matched litter mate controls, the ifitmdel / mefs displayed - fold more nuclear np staining, with or without ifn-c treatment (fig. , s c) . ifn-mediated viral restriction was restored when we transduced the null mefs with a retrovirus expressing ifitm (ifitmdel / ifitm , fig. s ). similar to what was observed with the ifitm overexpressing cell lines, the majority of the vrnp signal in the ifn-c-treated wt and ifitm rescued cells localized to acidic compartments (red, fig. s b ). an increase in acidic compartments occurred after ifn-c treatment with either the wt or the ifitmdel / ifitm mefs, but not in the ifitmdel / cells, suggesting that ifitm is required for this event (fig. , s ) . similar results were obtained with ifn-a (data not shown). we conclude from these experiments using orthologous reagents (cell lines and influenza a viruses) and methods, that ifn impedes vrnp nuclear entry, and ifitm is necessary and sufficient for this activity. viral pseudoparticle fusion mediated by either ha or vsv-g envelope proteins is decreased by ifn, and ifitm is necessary and sufficient for this activity to further characterize the mechanism of ifitm -mediated restriction, we used an established viral fusion assay [ , ] . lentiviral pseudoparticles containing the b-lactamase protein fused to the hiv- accessory protein vpr (blam-vpr) and expressing either ha and na (h n , wsn/ ), or vsv-g envelope proteins, were incubated for h with cells, which were then loaded with the b-lactamase flourogenic substrate, ccf . upon viral pseudoparticle fusion, blam-vpr enters the cytosol and cleaves ccf , producing a wave length shift in emitted light (from green to blue) when analyzed by flow cytometry (fig. a , [ ] ). in mdck-ifitm cells we observed a decrease in both ha-and vsv-g-directed fusion, which was comparable to the block produced by poisoning of the host vacuolar atpase (vatpases) with a low dose of bafilomycin a (baf, fig. b ). the inhibition of vatpases prevents the low-ph activation required by these two viral envelope proteins to produce membrane fusion. a block to fusion of pseudoparticles expressing h (pr ), h (a/udorn/ ), h (a/thai/ ) or h (a/fpv/ rostock/ ) subtypes of ha was also detected with mdck cells mefs, either a) wild type (wt) or b) ifitmdel / , which are missing all five of the mouse ifitm proteins, were either left untreated (left panels, buffer), or treated (right panels) with ifn-c. the following day cells were incubated with pr on ice. cells were next incubated in warm media containing ltred. cells were then fixed at the indicated times and immunostained with anti-np antibodies (green), stained for dna (blue), and imaged by confocal microscopy. image analysis software was used to define the nuclear boundaries (blue lines). images are representative of three independent experiments. (scale bar: mm). doi: . /journal.ppat. .g [ , ] comprised of lentiviral pseudoparticles (pps) containing the b-lactamase protein fused to the hiv- accessory protein vpr (blam-vpr, shown in orange/red) and expressing ha and na (wsn/ ) on their surfaces. the h n pps were incubated for h with cells, which were subsequently loaded with the b-lactamase flourogenic substrate, ccf . upon viral fusion, blam-vpr enters the cytosol and can cleave ccf , producing a wavelength shift from green to blue in emitted light when analyzed by flow cytometry ( [ ] ). b) mdck cells stably overexpressing ifitm (mdck-ifitm ) or empty vector control cells (mdck-vector) were exposed for h to viral pseudoparticles containing a blam-vpr and expressing either the ha and na envelope proteins of influenza a virus (wsn/ , h n pp) or the vsv-g envelope protein (vsv-gpp), then loaded with ccf . after incubation with the indicated pseudoparticles, the cells were fixed and assayed for cleavage of ccf by determining the conversion of the fluorescence emission from nm (uncleaved ccf ) to nm (cleaved ccf ) using flow cytometry. fusion of the pseudoparticles was inhibited by bafilomycin a (baf). these results are representative of six independent experiments. c) ifitm inhibits fusion of h n pps in normal diploid fibroblasts. wi- fibroblasts stably transduced with ifitm (wi- m ) or the empty vector (wi- v) were exposed for h to serial dilutions of h n pps containing blam-vpr, with or without baf. these results are representative of four independent experiments. d) fusion of h n pps increases after ifitm knockdown. wi- fibroblasts stably transduced with a shrna against ifitm (wi- shm ), a shrna control with a scrambled sequence (wi- shscr), or the ifitm cdna (wi- m ) were exposed to either no virus, h n pps or vsv-gpps containing blam-vpr. these results are representative of two independent experiments. e) fusion of h n pps is inhibited by ifn-c. wi- fibroblasts were treated with ifn-c for h or buffer alone prior to incubation with h n pps containing blam-vpr. these results are representative of three independent experiments. doi: . /journal.ppat. .g or with chicken embryonic fibroblasts (chefs), in which ifitm strongly inhibited viral replication (fig. s a , b, c). in the case of the mdck cells, the block to fusion closely paralleled the level of inhibition seen when the pseudoparticles were tested for productive infection using hiv- p expression as a readout (fig. s e) . consistent with earlier findings, pseudoparticles expressing an amphotropic mlv envelope protein were insensitive to ifitm , showing the specificity of these results (fig. s d) . similarly to its effect on h -expressing pseudoparticles, ifitm inhibited replication of infectious avian h n influenza a virus, a/vietnam/ / (vn/ ), isolated from a fatal human infection (fig. s f-h) . to enhance our analysis, we tested two additional cell lines, wi- and hela cells. a strong block to fusion in wi- -ifitm cells, similar to that of the baf and uninfected control samples, was seen at a range of serial dilutions of pseudoparticles, as well as an increase in fusion with ifitm depletion (shifitm , fig. c, d) . ifn treatment inhibited fusion of the h n pseudoparticles, albeit to a lesser extent than ifitm overexpression (fig. e) , and this effect was largely absent when ifitm was stably depleted in hela cells (fig. s ). similar results were obtained with ifn-a (data not shown). based on these experiments using multiple cell lines and ha, vsv-g, and mlv envelope-expressing pseudoparticles, we conclude that ifitm is required and sufficient for an ifn-mediated block of viral pseudoparticle fusion. importantly, the increase in pseudoparticle fusion seen when endogenous ifitm was depleted in either the hela or wi- shifitm cell lines argues that fusion inhibition underlies the first line defense provided by endogenous, as well as overexpressed, ifitm . mxa is an ifn-inducible large gtpase which interferes with secondary transcription during influenza a viral replication [ ] . a cells express mxa and have been used extensively in influenza a viral replication studies [ ] . therefore to clarify the antiviral roles of ifitm and mxa, we tested the levels of viral replication in a cells stably expressing one of three shrnas targeting ifitm (shifitm - , - , or - ). all three shifitm cell lines showed increased infection (wsn/ strain) and strong ifitm knockdown, when compared to the negative control cell line expressing a shrna against firefly luciferase (shluc), with or without ifn treatment (fig. s a, b) . the majority of the protective effect of either ifn-a or c was lost in the shifitm cell lines. we next confirmed both the baseline levels, as well as the ifn-inducibility of mxa in the a cells (fig. s c) . we also determined that mxa was both present and ifn-inducible in wi- normal fibroblasts, another cell line used in loss-of-function experiments in this work (fig. s d) . furthermore, if studies of wi- cells showed that mxa is expressed in an ifn-inducible vesicular pattern and that these structures did not appreciably colocalize with vesicles containing ifitm (fig. s e , [ ] ). we conclude that mxa is expressed in the a and wi- cell lines, but cannot fully compensate for loss of the antiviral actions of ifitm . ifitm is present in endosomes and lysosomes and these compartments are expanded with ifitm overexpression or ifn treatment our data demonstrate that ifn or ifitm inhibit viral fusion. influenza a virus fuses with the host membrane in late endosomes when the ph decreases to [ , , ] . rab is a late endosomal/lysosomal small gtpase that is required for the fusion of many ph-dependent viruses, including influenza a virus [ , ] . previous reports have shown that ifitm colocalizes with lamp and cd , components of lysosomes and multivesicular bodies, respectively [ ] . however, the relationship of ifitm and rab within the host cell infrastructure remains unknown. therefore we investigated the location of ifitm , by undertaking immunoflourescence (if) studies using antibodies that recognize ifitm , rab , or lamp [ ] . although the baseline level of ifitm in the a -vector cells was low, there was partial colocalization observed with either rab or lamp (fig. a-d, a,) . ifitm also partially colocalized with lamp and ltred-containing structures seen with ifitm overexpression (fig. a, b, a) . interestingly, either ifitm overexpression or ifn increased the staining intensity of rab and lamp (fig. a, b, s a ). partial colocalization of ifitm was also seen with either endogenous lamp , or an exogenously expressed rab -yellow fluorescence fusion protein (rab -yfp) in mdck cells (fig. e-i) . however, in all cases, co-localization was not complete because cells contained areas that uniquely labeled for each of the proteins. western blots indicated that ifitm overexpression led to modest increases in both lamp and rab proteins in the a -ifitm cells (fig. c) . however, these blots also showed that while ifn treatment of the a -vector cells increased ifitm protein levels as expected, the amount of rab and lamp remained unchanged. we conclude that ifitm partially resides in the late endosomal and lysosomal compartments along with rab and lamp , and that ifitm overexpression or ifn treatment expands these compartments through a mechanism that cannot be fully explained by increased protein expression alone. our assays showed that incoming influenza a viruses were retained in the expanded acidic compartments of both the ifitm overexpressing cell lines as well as the ifn-c-treated mefs, and that ifitm partially localized to these structures ( fig. - , s - , s ) . therefore, we extended our investigation of these compartments. an increase in acidic structures was seen in mdck and a cells overexpressing ifitm as compared to control cell lines, using either the vital acidophilic stain, acridine orange (ao), ltred, or a cathepsin-l substrate that fluoresces only after it is proteolyzed, when compared to the corresponding vector control cells (fig. a, b, a, b) . cathepsins are a family of lysosomal zymogens active in acidic environments (ph# . ) which are required for both the degradation of endocytic substrates and for the entry of several ifitm -susceptible viruses [ ] . flow cytometry revealed an increase in the total ltred fluorescent signal in both the mdck and a ifitm cell lines when compared to controls (fig. c ). this expanded compartment represents a heterogeneous population of lysosomes and autolysosomes, based on confocal imaging showing the colocalization of the autophagosome marker, microtubule-associated protein light chain (lc ), with either ltred or with cd , with the latter being a resident of multivesicular bodies, amphisomes and autolysosomes (fig. d, e) . furthermore, mdck-ifitm cells stably transduced with an lc protein fused to both a red fluorescent protein (mcherry) and an enhanced green fluorescence protein (egfp) showed a predominantly red signal, which occurs when the mcherry-egfp-lc protein resides inside the acidified interior of an autolysosome (fig. f, [ ] ). in keeping with previous reports that ifn-c induces autophagy [ , ] , we detected enhanced ltred staining in either ifn-c treated mefs or a cells (fig. a, s a) . we conclude that increases in ifitm levels expand the lysosomal/autolysosomal compartment. here we report several novel findings regarding the antiviral actions of ifn and the transmembrane ieg, ifitm . first, this study demonstrates that ifn inhibits the nuclear translocation of vrnps, and that ifitm is required for this ifn-mediated block, with both endogenous and overexpressed ifitm inhibiting vrnp nuclear entry. second, either endogenous or overexpressed ifitm , as well as ifn treatment, block the fusion of viral pseudoparticles expressing various influenza a virus envelope proteins (h , h , h and h subtypes of ha), or the vsv-g envelope protein; this block is specific because the fusion of pseudoparticles expressing mlv envelope is not inhibited by ifitm . third, our work reveals that ifitm partially resides with rab in late endosomes, thus placing it in position to block influenza a virus' cytosolic access. fourth, ifitm overexpression or ifn induce the expansion of late endosomal and lysosomal compartments containing rab and lamp . fifth, we show that similar to ifn-c treatment, ifitm overexpression expands the number and size of autolysosomes, and it is into these compartments that trapped viruses are trafficked and subsequently degraded. consistent with previous reports, our data show that high levels of ifitm do not prevent viral access to acidified compartments and that ifitm colocalizes with cd and lamp [ ] . this is in contrast to a report noting the exclusion of overexpressed ifitm from lamp -containing structures [ ] . therefore, this work adds substantially to our interpretation of previous reports by demonstrating that key downstream events in the viral lifecycle, fusion and vrnp nuclear translocation, are prevented by either ifn or ifitm . ifitm thus represents a previously unappreciated class of anti-viral effector that permits viral entry into the endosomal compartment, but prevents egress into the cytosol. these studies also raise new questions including i) how do ifn and ifitm prevent viral fusion? ii) how do ifn and ifitm alter the endosomal and autolysosomal compartments? and iii) is the latter action required for viral restriction, or alternatively does it arise as an outcome of ifitm 's potential cellular role? based on the substantial loss in ifn's potency observed when ifitm is depleted ( - % loss of viral inhibition, fig. s a , b, [ ] ) we conclude that inhibition of viral emergence from the endosomal pathway is a prominent component of ifn's antagonism of influenza a virus replication in vitro. our data also show that mxa cannot fully compensate for the loss of ifitm in ifn-treated cells challenged with influenza a virus. recent work by dittmann et al. [ ] and zimmermann et al. [ ] reveal that human influenza a viral strains have evolved a means to evade mxa, suggesting a possible explanation for the cellular reliance on ifitm for protection in vitro. similarly the ieg, ifit , prevents viral replication by targeting viral triphosphate-rnas (ppp-rna) for destruction [ , ] . given that ifitm is necessary for the majority of ifn-mediated restriction of influenza a virus in vitro, it may be that the virus has also evolved a means to at least partially nullify ifit , perhaps via the massive production of short ''decoy'' ppp-rnas, as previously postulated [ , ] . ifitm primarily resides in the endosomal compartment and partly colocalizes with rab and lamp . ifitm overexpression or ifn stimulation caused the endocytosed viruses to accumulate in acidic compartments that contained both ifitm and lamp . together with the blam-vpr fusion assay data, these results reveal that ifitm prevents viral-host membrane fusion within late endosomes, and likely within lysosomes as well, in light of studies showing ifitm-mediated restriction of filoviruses and coronaviruses, which depend on cathepsin-mediated activation prior to fusion [ ] . in doing so, ifitm traps the virus on a path which terminates in a degradative environment [ ] . in support of this, our experiments show the eventual loss of a detectable vrna signal in the ltred-positive compartments of the ifitm transduced cells, thus revealing the fate of viral fitness under those conditions. these studies also reveal that elevated levels of ifitm correlate with the expansion of host cell structures containing rab and lamp , and that ifitm was also present in these structures. in the mef and a experiments, ifn produced increased rab and lamp immunostaining, in addition to an increase in acidic structures. at present, we cannot explain the increased rab and lamp signals seen after ifn stimulation or ifitm overexpression solely on the slight elevations in the abundance of these proteins detected by immunoblotting. two possible explanations for the increased immunostaining observed, are that ifn stimulation induced these proteins to cluster together or alternatively unmasked sequestered epitopes; we find the latter possibility less likely since lamp and rab flourescent fusion proteins also showed larger and more intense signals under similar conditions. we envision that ifitm -mediated clustering of organelles and their protein cargoes might contribute to the host cell's antiviral state. earlier work reported no correlation between the size of the ifitm -induced acidified compartments and the level of viral restriction [ ] , however, we observe that increasing levels of ifitm result in both an expansion of lysosomes/ autolysosomes and increased viral inhibition. these observations might be explained by a common mechanism underlying the increase in these structures and viral inhibition, in addition to raising the possibility that they play a role in ifitm-mediated viral restriction. is there a common characteristic shared by ifitm -susceptible viruses? the late endosomal-and lysosomal-associated small gtpase, rab , is required for influenza a virus infection [ , ] . the ifitm -resistant viruses previously tested (mlv, the arena viruses and the hepacivirus, hcv) are all rab -independent, while the entry of the ifitm -susceptible viruses (influenza a, dengue, ebola, marburg, and sars) relies on rab [ , , , , , ] . standing against this hypothesis, is the lack of effect on vsv-g-mediated entry with expression of a dominant negative rab [ , , ] ). however, additional studies have shown that vsv-g-directed entry is dependent on transport to the late endosomes [ , ] ; these latter results, together with those of huang et al. and weidner et al. [ , ] , are consistent with the prediction that viruses that fuse in late endosomes or lysosomes are vulnerable to ifitm 's actions, while viruses whose genomes enter at the cell surface or in the early endosomes may avoid ifitm 's full effect. of note, we have been unable to demonstrate that ifitm blocks hiv- replication using tzm-bl hela cells and are working to address these differences with a published study ( [ ] , data not shown). this study, together with previous work, demonstrates that ifitm permits endocytosis of viruses, but prevents viral fusion and the subsequent entry of viral contents into the cytosol [ , ] . while the blam-vpr fusion assay demonstrates inhibition of fusion by ifn or by ifitm , we note that this assay uses an indirect readout to assess entry of viral contents. therefore several possibilities could explain the containment and neutralization of viruses within the endosomal pathway, including alterations in endosomal trafficking, acidification, or the host membrane's fusion characteristics (bending modulus, elasticity). while additional work is required to further define the mechanism, the lack of toxicity seen with cells stably overexpressing high levels of ifitm suggests that gross alterations in endogenous trafficking or ph control are unlikely (data not shown). therefore overexpressing or activating ifitm to produce an enhanced antiviral state may be an effective prevention strategy during high risk periods in vulnerable populations. we propose that ifn causes the degradation of endocytosed viruses by preventing their contents from entering the host cytosol, and that ifitm is necessary and sufficient for this defense ( figure g ). ifitm 's mode of defense could be envisioned as an effective means to neutralize pathogens during an organism-wide threat. such actions might confer an advantage to the host because if ifitm simply decreased viral attachment and/or entry, the repulsed viruses would be free to attack neighboring cells. of course while there are considerable differences between this simple scenario and the directed phagocytosis of pathogens by specialized immune cells, i.e. macrophages, the similarities none-the-less suggest an early prototype for a more evolved defense mechanism. cell lines and culture conditions u os, a , mdck, hela cells (all from atcc), and chicken embryonic fibroblasts (chefs, from charles river labs) were grown in complete media (dmem, invitrogen cat# ) with % fbs (invitrogen). wi- cells (atcc) were cultured in dmem (invitrogen cat# ), containing non-essential amino acids (invitrogen cat# ) and % fbs. wild type and matched ifitmdel / mefs were from adult ifitmdel+/ mice [ ] that were intercrossed and mefs derived from embryos at day . of gestation, as described previously [ ] . the mefs were genotyped by pcr and western blot, and the generation of the ifitmdel / ifitm cells have been previously described [ ] . the ifitm retroviral vector, pqcxip-ifitm and empty vector control (clontech) have been previously described [ ] . . dna = blue. e) mdck cells stably transduced with ifitm or with the vector alone were immunostained for confocal imaging of lc (endogenous, red) and cd (endogenous, green). dna = blue. f) confocal images of mdck cells overexpressing ifitm or the empty vector alone showing the distribution and fluorescence intensities of a stably expressed mcherry-egfp-lc b fusion protein using fluorescence channels that detect light emitted from the mcherry protein, egfp or both (merge). dna = blue. g) model of ifitm -mediated restriction of virus replication. endocytosed viruses enter late endosomes where ifitm is present. ifitm prevents viral fusion within the endosomes and likely lysosomes via an unknown mechanism, perhaps by altering ph, membrane characteristics, lipid composition, transport speed or destination. trapped viruses are trafficked to lysosomes and/or autolysosomes where they undergo degradation. doi: . /journal.ppat. .g # ) and was kindly deposited by jayanta debnath. plzs-rab -yfp and plvx-rfp-lamp were generously provided by walther mothes, section of microbial pathogenesis, yale university school of medicine. the following shrna sequences (sense strand sequence provided) were cloned into the papm shrna-expression lentiviral vector [ ] , to create the viruses used to generate the a ifitm knockdown cell lines in fig. s : ifitm - : -tcctcatgaccattctgctcat- ifitm - : -cccacgtactccaacttccatt- ifitm - : -tttctacaatggcattcaataa- influenza a virus a/puerto rico/ / (h n ) (pr , charles river labs) and a/wsn/ (h n ) (kind gift of dr. peter palese, microbiology dept., mt. sinai school of medicine, ny, ny) were propagated and assessed for viral infectivity as previously described [ ] . influenza a virus a/vietnam/ / (h n ) was propagated and characterized as previously described [ ] . human interferon (ifn)-c (invitrogen) was used at - ng/ml, human ifn-aa (pbl interferon source) was used at - u/ml. cells were incubated with cytokines for - h prior to if or viral infection experiments unless otherwise noted. murine ifn-c (pbl interferon source) was used at - ng/ml. whole-cell extracts were prepared by cell lysis, equivalent protein content boiled in sds sample buffer, resolved by sds/ page, transferred to immobilon-p membrane (millipore), and probed with the indicated antibodies. cells were seeded on glass coverslips for influenza a virus infection experiments. cells were incubated on ice with pr for min. at time zero, the viral supernatant was removed and uc media was added with or without lysotracker red dnd- (invitrogen). at the indicated time points post-warming, cells were washed twice with d-pbs (sigma) and incubated for seconds with room temperature . % trypsin (invitrogen). the cells were then washed with complete media twice and fixed with % formalin (pfa, sigma) in d-pbs. image analysis for quantitation of vrnp nuclear translocation was done using imaris . (bitplane scientific software). we generated a mask of the nucleus and applied this mask to the channel containing the viral signal (puncta) to determine vrna puncta contained in each nucleus. cells were incubated at uc and % co for min. with either lysotracker red dnd- or acridine orange (immuno-chemistry technologies). hoechst (dna stain, invitrogen) was incubated ( : , ) with the cells for the final min. the cathepsin l flourogenic substrate assay was performed as per the manufacturer's instructions (cathepsin l -magic red, immuno-chemistry technologies). cells were visualized live by confocal microscopy. cells were fixed in % pfa in d-pbs, and then incubated sequentially in . % tween (sigma), then % bsa with . m glycine (sigma), both in d-pbs. primary and secondary antibodies are listed below. slides were mounted in vectashield with dapi counterstain (vector labs). slides were imaged using a zeiss lsm , laser scanning inverted confocal microscope equipped with the following objectives: zeiss c-apochro-mat uv-vis-ir water, . na, zeiss plan-apochro-mat dic oil, . na, and zeiss plan-apochromat dic oil, . na. image analysis was performed using zen software (zeiss). laser intensity and detector sensitivity settings remained constant for all image acquisitions within a respective experiment. nuclear outlines were generated using metamorph software suite (molecular devices) using the kirsch/prewitt filter to define boundaries and then subtracting out the original binary images. the following antibodies were used in this study for either these experiments employ the quantigene viewrna slidebased assay kit from affymetrix (cat #qv ) with all components from that source unless noted. rna was visualized following a modified manufacturer protocol; changes made include the omission of the ethanol dehydration step, and use of vectashield mounting media. post-fixation with % pfa, cells adherent on coverslips were incubated with detergent solution or incubated in . % pbs-tween . cells were then incubated with proteinase k. next cells were incubated at uc in hybridization solution a containing a viewrna probe set designed against either the negative stranded rna np genome (vrna) of pr (affymetrix vx - - qg viewrna type probe set against np influenza a virus (a/puertorico/ / (h n )) at : ) or a probe set against the positive stranded np mrna. cells were then incubated in hybridization preamplifiers ( : in hybridization buffer b) at uc. finally cells were incubated with labeled probes ( : in hybridization buffer c), washed and imaged as above. all steps were followed by two d-pbs washes. sialic acid linkage expression studies a cells stably transduced to overexpress ifitm or with empty expression vector (pqcxip, clontech) were grown to , % confluency, dissociated with trypsin-free edta-based dissociation buffer (invitrogen) for min. at uc. cells were incubated at uc with fitc-conjugated sambucus nigra lectin (sna, vector labs #fl- ) to detect (a- , ) sialic acid linkages, and biotinylated maackia amurensis lectin ii (mal, vector labs #b- ) to detect (a- , ) sialic acid linkages, followed by streptavidin-pe-cy (invitrogen). cells were incubated with lectins individually and in combination, and the results of staining were indistinguishable. all cells were stained with violet cell-impermeable dye (invitrogen #l ), and cells were included in the analysis if viable by fsc/ssc and viability dye. binding assay a cells transduced with ifitm or the empty vector pqxcip were detached using enzyme free pbs-based dissociation buffer, and then washed in cold pbs extensively. cells and virus (wsn/ ) were pre-chilled on ice for min. and mixed at a moi of and incubated at uc for h with rotation. cells were washed extensively with ice cold pbs and then fixed using % pfa. the cells were then probed with anti-ha mouse monoclonal antibody (wistar collection, coriell institute, clone h -s , wc , if) for h at room temperature, followed by antimouse alexaflour- conjugated antibody (invitrogen) for h with pbs washes in between, then analyzed by flow cytometry. figure s ifitm overexpression does not alter the surface levels of (a- , ) or (a- , ) sialylated proteins. a cells stably transduced with ifitm or the empty vector were incubated with biotinylated maackia amurensis lectin ii (mal) to detect (a- , ) sialic acid linkages, followed by streptavidin-pe-cy , as well as fitc-conjugated sambucus nigra lectin (sna) to detect (a- , ) sialic acid linkages. a) the percentage of ifitm or vector cells staining positive for both sialic acid linkages (upper right hand quadrant), compared to unstained controls. b) ifitm overexpressing and vector cells are compared with regard to each sialic acid linkage in the double-stained populations. (pdf) figure s ifitm arrests influenza a virus in acidic cytosolic inclusions preventing vrnp nuclear translocation. a) normal diploid human fibroblasts (wi- cells) were stably transduced with retroviruses containing ifitm (wi- ifitm ) or (b) a non-targeting control shrna (wi- shscramble). cells were incubated with pr on ice, and then warm media containing ltred (red) was added at time zero. cells were fixed at min. p.i. and stained for np (green) and dna, then analyzed by confocal microscopy. image analysis software was used to define each cell's cytosolic (white lines) and nuclear peripheries (blue lines, based on dic images and dna staining, respectively). images are representative of four independent experiments. figure s ifitm expression rescues ifn-c-mediated inhibition of vrnp nuclear translocation in ifitmdel / mefs. a) ifitmdel / mefs stably overexpressing ifitm (ifitmdel / ifitm ), were left untreated (left panels, buffer), or treated (right panels) with ifn-c. the following day cells were incubated on ice with pr (moi ). cells were next incubated in warm media containing ltred ( min.). cells were then fixed at the indicated times p.i., immunostained with anti-np antibodies (green) and imaged by confocal microscopy. image analysis software was used to define the nuclear boundaries (blue lines). images are representative of three independent experiments. (scale bar cm) . b) percent colocalization of vrnp and ltred compartments in the indicated mef cell lines, with or without ifn-c treatment, are shown for the indicated times p.i. c) quantitation of nuclear vrnp particles. the number of vrnp particles per nucleus of the mef cell lines, with or without ifn-c treatment, at the indicated time points are shown. values represent the mean +/ the sd of three independent experiments. d) western blot of whole cell lysates from the indicated mefs probed with anti-mouse ifitm and using gapdh as a loading control. (pdf) figure s fusion of viral pseudoparticles expressing ha envelope subtypes, but not a mlv envelope, is decreased by ifitm . ifitm inhibits the replication of infectious h n virus. a) mdck cells stably transduced with ifitm or empty vector were incubated with pseudoparticles expressing n and ha subtypes (h n pp, h n pp, or h n pp). cells were then fixed and assayed for cleavage of ccf using flow cytometry. these results are representative of three independent experiments. b) chicken embryonic fibroblasts (chef) cells stably expressing the empty vector control or ifitm were incubated with pseudoparticles expressing n and either of the two avian influenza a viral ha subtypes, h or h , as in (a). these data are representative of three independent experiments. c) chef cells stably transduced with the empty vector control or overexpressing ifitm , were infected with wsn/ for h then stained for ha protein (red) and dna (blue). average percent infection is given for three independent experiments +/ sd. magnification. d) mdck-vector or mdck-ifitm cells were incubated with pseudoparticles expressing the amphotropic mlv envelope protein (mlvpp) and then assayed for cleavage of ccf using flow cytometry. these results are representative of two independent experiments. e) infectivity of ha-expressing pseudoparticles is decreased by ifitm . mdck-vector or mdck-ifitm cell lines were infected with the indicated pseudoparticles for h. cells were then immunostained for expression of hiv- p protein expressed from the integrated lentiviral genomes. percent infection is provided. these results are representative of three independent experiments. magnification. f) a cells were stably transduced with retroviruses containing ifitm or empty viral vector alone, then infected with a/vietnam/ / (h n ) influenza a virus (vn/ ). after h, the cells were fixed and stained for viral np expression (green) and for dna (blue). values given are percentage infected cells and are representative of two independent experiments. magnification. g) western blot of lysates from a -ifitm or a -vector cell lines probed with the indicated antibodies. h) a cell lines were infected with increasing amounts of h n vn/ . twelve hours after infection the cells were immunostained for np expression and scored for infection status. values are representative of two independent experiments. (pdf) figure s ifitm is required for ifn's inhibition of ha-mediated fusion. a) hela cells were stably transduced with retroviruses containing either ifitm , a shrna against ifitm (shifitm ), or a non-targeting control shrna (shscr). cells were left untreated (left panels), or treated with ifn-c (right panels), then exposed for h to h n pps (wsn/ ) containing blam-vpr. after incubation with the pseudoparticles, the cells were fixed and assayed for cleavage of ccf by flow cytometry. figure s ifn treatment both expands rab -and ifitm -containing structures, and increases the size and number of acidified organelles. a) confocal images of wi- cells treated with buffer, ifn-a, or ifn-c, and then immunostained for either ifitm (endogenous, red) or rab (endogenous, green), and dna (blue). arrows denote larger structures staining for rab and ifitm that were seen predominantly with ifn-c treatment. (scale bar: mm). b) a cells treated with either buffer or ifn-c, then incubated with ltred before fixation and dna staining (blue) followed by confocal imaging. images in this figure are representative of three independent experiments. (pdf) effectiveness of the - influenza vaccine among children months to years of age effectiveness and cost-benefit of influenza vaccination of healthy working adults: a randomized controlled trial the evolution of influenza resistance and treatment new approaches to influenza chemotherapy the mist (management of influenza in the southern hemisphere trialists) study group ( ) randomised trial of efficacy and safety of inhaled zanamivir in treatment of influenza a and b virus infections virus entry by endocytosis endocytosis of influenza viruses enhancement of the infectivity of influenza a and b viruses by proteolytic cleavage of the hemagglutinin polypeptide the cleavage site of the hemagglutinin of fowl plague virus driving a wedge between viral lipids blocks infection trafficking of viral genomic rna into and out of the nucleus: influenza, thogoto and borna disease viruses nuclear transport of influenza virus ribonucleoproteins: the viral matrix protein (m ) promotes export and inhibits import role of the influenza virus m protein in nuclear export of viral ribonucleoproteins the ifitm proteins mediate cellular resistance to influenza a h n virus, west nile virus, and dengue virus genomewide rnai screen identifies human host factors crucial for influenza virus replication human host factors required for influenza virus replication a physical and regulatory map of host-influenza interactions reveals pathways in h n infection identification of five interferon-induced cellular proteins that inhibit west nile virus and dengue virus infections distinct patterns of ifitm-mediated restriction of filoviruses, sars coronavirus, and influenza a virus interferoninduced cell membrane proteins, ifitm and tetherin, inhibit vesicular stomatitis virus infection via distinct mechanisms transcriptional and posttranscriptional regulation of interferon-induced gene expression in human cells molecular analysis of a human interferon-inducible gene family the small interferon-induced transmembrane genes and proteins the cd / cd signal transducing complex of human b lymphocytes includes the target of antiproliferative antibody- and leu- molecules expression of the mouse fragilis gene products in immune cells and association with receptor signaling complexes normal germ line establishment in mice carrying a deletion of the ifitm/fragilis gene family cluster the fragilis interferon-inducible gene family of transmembrane proteins is associated with germ cell specification in mice cloning of ip , a pancreatitis-induced gene whose expression inhibits cell growth ifn-alpha induces homotypic adhesion and leu- expression in human b lymphoid cells bril: a novel bone-specific modulator of mineralization inhibition of proliferation by - u in interferon-alpha-responsive and non-responsive cell lines characterization of the osteoblast-specific transmembrane protein ifitm and analysis of ifitm -deficient mice palmitoylome profiling reveals s-palmitoylation-dependent antiviral activity of ifitm the ubiquitin-vacuolar protein sorting system is selectively required during entry of influenza virus into host cells human host factors required for influenza virus replication orthomyxoviridae: the viruses and their replication nef does not affect the efficiency of human immunodeficiency virus type fusion with target cells an enzymatic virus-like particle assay for sensitive detection of virus entry human mxa protein: an interferon-induced dynamin-like gtpase with broad antiviral activity regulation of ifn-alpha/beta, mxa, , -oligoadenylate synthetase, and hla gene expression in influenza a-infected human lung epithelial cells differential requirements of rab and rab for endocytosis of influenza and other enveloped viruses sequential roles for phosphatidylinositol -phosphate and rab in tethering and fusion of early endosomes via their interaction with eea ) p / sqstm binds directly to atg /lc to facilitate degradation of ubiquitinated protein aggregates by autophagy traf and a regulate lysine -linked ubiquitination of beclin- to control tlr -induced autophagy autophagy is a defense mechanism inhibiting bcg and mycobacterium tuberculosis survival in infected macrophages influenza a virus strains differ in sensitivity to the antiviral action of mx-gtpase the viral nucleoprotein determines mx sensitivity of influenza a viruses where, in antiviral defense, does ifit fit ifit is an antiviral protein that recognizes -triphosphate rna influenza a virus expresses high levels of an unusual class of small viral leader rnas in infected cells acid ribonuclease from hela cell lysosomes different mechanisms of cell entry by human-pathogenic old world and new world arenaviruses dissecting the cell entry pathway of dengue virus by single-particle tracking in living cells endocytosis of chikungunya virus into mammalian cells: role of clathrin and early endosomal compartments a spatio-temporal analysis of matrix protein and nucleocapsid trafficking during vesicular stomatitis virus uncoating hepatitis c virus entry requires a critical postinternalization step and delivery to early endosomes via clathrincoated vesicles viral entry: a detour through multivesicular bodies endosome-tocytosol transport of viral nucleocapsids the ifitm proteins inhibit hiv- infection trim is an innate immune sensor for the retrovirus capsid lattice evolution of highly pathogenic h n avian influenza viruses in vietnam between we thank thomas murooka and thorsten mempel (center for immunology and inflammatory diseases, massachusetts general hospital) for the kind gift of the modified pbr ieg-nef+ plasmid. we thank marina boyarina, karen rogers, melissa hinely and cynthia hanes for their managerial support. we thank the university of iowa hybridoma bank and the nih aids reagent repository for the generous supplying of reagents. we thank richard sutton and walther mothes (yale university medical school) and gregory melikian (emory university medical school) for helpful discussions. we thank alex forrest-hay, kim crawford, quan nguyen and ankit patel of panomics/affymetrix for their expertise and guidance with the viewrna studies. the findings and conclusions in this report are those of the authors and do not necessarily represent the views of the centers for disease control and prevention or the agency for toxic substances and disease registry. key: cord- -jld ygxt authors: neidermyer, william j.; whelan, sean p. j. title: global analysis of polysome-associated mrna in vesicular stomatitis virus infected cells date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: jld ygxt infection of mammalian cells with vesicular stomatitis virus (vsv) results in the inhibition of cellular translation while viral translation proceeds efficiently. vsv rna synthesis occurs entirely within the cytoplasm, where during transcription the viral polymerase produces mrnas that are structurally indistinct to cellular mrnas with respect to their ′ cap-structure and ′-polyadenylate tail. using the global approach of massively parallel sequencing of total cytoplasmic, monosome- and polysome-associated mrna, we interrogate the impact of vsv infection of hela cells on translation. analysis of sequence reads in the different fractions shows > % of total cytoplasmic and polysome-associated reads map to the viral genes by hours post-infection, a time point at which robust host cell translational shut-off is observed. consistent with an overwhelming abundance of viral mrna in the polysome fraction, the reads mapping to cellular genes were reduced. the cellular mrnas that remain most polysome-associated following infection had longer half-lives, were typically larger, and were more au rich, features that are shared with the viral mrnas. several of those mrnas encode proteins known to positively affect viral replication, and using chemical inhibition and sirna depletion we confirm that the host chaperone heat shock protein (hsp ) and eukaryotic translation initiation factor a (eif a)—encoded by such mrnas—support viral replication. correspondingly, regulated in development and dna damage (redd ) encoded by a host mrna with reduced polysome association inhibits viral infection. these data underscore the importance of viral mrna abundance in the shut-off of host translation in vsv infected cells and link the differential translatability of some cellular mrnas with pro- or antiviral function. introduction infection of mammalian cells by vesicular stomatitis virus (vsv) results in a profound shut-off of host cell gene expression. this host cell shut-off occurs at the level of mrna transcription through inhibition of rna polymerase ii by the viral-encoded matrix protein (m) [ ] [ ] [ ] . the m protein also forms a complex with ribonucleic acid export (rae ) and nucleoporin (nup ) [ ] thus suppressing host cell mrnp export from the nucleus, including that of mature cellular mrnas [ ] [ ] [ ] [ ] . vsv infection also inhibits protein synthesis by manipulation of the host-cell translation machinery, particularly at the level of translation initiation [ , ] . eukaryotic initiation factor e (eif e)-the rate limiting factor for translation initiation-recognizes the m gpppn mrna cap structure as part of the eif f complex, and in concert with other translation initiation factors facilitates the recruitment of the small s ribosomal subunit to the mrna prior to scanning to the initiating methionine where the s subunit joins [ , ] . vsv infection results in the rapid dephosphorylation of eif e itself, for which the functional consequences are unclear, and of its binding protein (eif e-bp ) leading to eif e sequestration and the suppression of translation initiation [ , ] . viral gene expression evades the shut-off mechanisms employed to suppress host gene expression. as vsv rna synthesis occurs entirely within the cytoplasm, viral rna synthesis is not subject to the inhibitory effects of m on rna polymerase ii and mrna export from the nucleus. the vsv rna synthesis machinery comprises a ribonucleoprotein complex of the negative-sense genomic rna completely encased by a nucleocapsid protein (n) sheath and associated with the viral polymerase complex [ ] . the viral transcriptase copies the n-rna template into monocistronic mrnas that are structurally indistinct to those of the host-cell with respect to their cap and polyadenylate tail [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the enzymes necessary for mrna synthesis, namely an rna dependent rna polymerase (rdrp) and a set of capping enzymes, reside within the viral large protein (l) [ , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] . vsv l protein cannot engage the n-rna template directly, but instead depends on the viral phoshoprotein (p) to facilitate the interaction [ ] [ ] [ ] [ ] [ ] [ ] . messenger rna polyadenylation is also catalyzed by l through reiterative transcription by the rdrp of a u tract that resides at the end of each gene [ ] [ ] [ ] [ ] [ ] . this program of viral transcription results in the cytoplasmic synthesis of mrnas that depend upon the host machinery for their translation, and must therefore avoid the shut-down mechanisms that effectively suppress host mrna translation. metabolic labeling studies demonstrate that hours post vsv infection of baby hamster kidney cells in culture, total translation is suppressed to about % the level of uninfected controls [ ] . extraction of mrna from infected cells coupled with its in vitro translation confirmed that the cellular mrnas remain intact and are competent for translation [ ] . the vsv mrnas are present in an approximately - fold excess of the total cellular mrna, leading to the model that competition between viral and cellular mrnas for ribosomes results in the dominance of viral translation [ , ] . polysome analysis also demonstrates that the cellular mrnas are associated with significantly fewer ribosomes in infected cells [ ] . for example, infection results in the movement of actin mrna from polysomes containing or more ribosomes to those containing [ ] . this movement reflects the competition between viral and cellular mrna for ribosomes and the limited pool of eif e. the competition model predicts that the kinetics of viral mrna synthesis and the levels of viral mrna should correlate closely with host shut-off. tests of this prediction yielded conflicting results. the kinetics of host shut-off and viral mrna accumulation correlate well for many strains of vsv, consistent with the competition model [ , ] . inhibition of host protein synthesis is, however, largely unaffected following coinfection of cells with increasing quantities of defective interfering (di) particles that suppress viral mrna levels up to -fold [ ] . a similar result was obtained for a vsv mutant that is restricted for genome replication at ˚c, and yields only % of the wild type levels of viral mrna [ ] . collectively, these studies suggest additional mechanisms may contribute to the shut-off of host cell protein synthesis. specific features of the viral mrnas that contribute to their efficient translation have not been defined. the untranslated regions of vsv mrnas are short, being - nucleotides for the viral n, p and l mrnas that encode the proteins required for rna replication [ , ] . how such short utrs serve as effective initiators of translation is unclear. evidence for differential translation of viral mrna comes from small interfering rna suppression of eif e, which inhibits host gene expression but has no impact on viral gene expression [ ] . viral translation is also hypersensitive to the loss of ribosomal protein l , suggesting different mrna features facilitate translation of viral versus host mrna [ ] . flanking cellular or reporter genes by the conserved viral -nt gene-start and -nt gene-end sequences, and inserting them into the viral genome is sufficient to mediate their efficient translation [ ] . by contrast, expression of the same genes following transfection of plasmid dna into cells and subsequent vsv infection does not offer this translational advantage [ ] . thus, transcription of the mrnas from the viral genome appears to contribute to their efficient translation. in the present study, we interrogate global mrna translation in vsv infected cells using rnaseq analysis of the cytoplasmic mrna transcriptome, and parallel sequencing of polysome-associated mrnas. we obtain support for the model that an overabundance of viral mrna contributes to host shut-off by leading to a re-distribution of cellular ribosomes onto viral mrna. by combining this rnaseq analysis with examining the distribution of specific viral and cellular mrnas within polysomes, we also demonstrate that mrnas shift to smaller polysomes. analysis of cellular mrnas less-sensitive to this global shut-down of translation identifies several host proteins that promote viral replication. similar analyses revealed the abundance of viral mrna contributes to the host-cell shut-off for other viruses including coronaviruses, influenza and vaccinia [ ] [ ] [ ] . to interrogate the impact of vsv infection on global translation we isolated total cytoplasmic, monosome-and polysome-associated mrna from hela cells at and hpi and compared the relative sequence reads obtained by deep-sequencing ( fig a) . statistical analysis of sequencing reads between biological replicates from each fraction yields a pearson correlation of > . for cytoplasmic, monosome-and polysome-associated mrna pools validating reproducibility between the replicates. as visible in the polysome profiles (fig b) , vsv infection results in a small but reproducible increase in the pool of monosomes and large polysomes at hpi, and a collapse of large polysomes and an increase in monosomes by hpi. mapping of the sequence reads to the viral and host genome highlights that by hpi > % of the total reads in the cytoplasmic and polysome fractions are viral (fig c) . this increase from the < % observed at hpi (fig c) emphasizes the impact of the exponential phase of viral rna replication and secondary transcription of the viral genome on mrna production. the viral sequence reads map to all genes, with clear dips in coverage at gene-junctions (s fig) . consistent with the order of transcription of the viral genome and the localized transcriptional attenuation at gene-junctions [ ] [ ] [ ] [ ] , the relative reads that map to each viral gene generally diminish with distance from the single promoter ( fig d and s fig) . analysis of the sequencing reads that map to cellular genes supports that like the viral mrnas, the level of reads in the polysome fraction mirrors that in the total cytoplasmic fraction at and hpi ( fig c) . this result demonstrates that the majority of mrnas are polysome-associated in proportion to their abundance. the abundance of the viral mrnas at hpi supports the model that viral mrnas outcompete cellular mrnas for ribosomes [ ] . we note that viral mrnas are, however, underrepresented ( %) and cellular mrnas overrepresented ( %) in the monosome fraction at hpi, compared to their cytoplasmic abundance ( fig c) . this finding is consistent with a differential effect on viral versus host mrna translation. to determine how vsv infection affects the distribution of the population of mrnas between total cytoplasmic, monosome or polysome fractions, we plotted the transcript per million (tpm) for each individual mrna mapped to the human and viral genome in all fractions (fig ) . at hpi, reads that map to the viral genes in each fraction are similar in abundance to those reads that map to highly expressed cellular genes (fig a) . the reads that map to any given cellular gene alter within a relatively narrow range, with few genes showing a greater than -fold change in the relative number of sequence reads (fig b) . for the population of mrnas, the relative reads obtained from the polysome fraction mirrored the relative reads in the total cytoplasmic fraction, consistent with the abundance of an mrna being a determinant of its translatability. by hpi, reads that map to each of the viral genes-with the exception of l-exceed the reads that map to any individual cellular gene (fig c, red triangles) . this is concurrent with a decrease in reads that map to the majority of cellular genes in cytoplasmic, monosome and polysome-associated fractions (fig c) . there were, however, some distinctions between the monosome and polysome fractions. for the majority of the population of cellular mrnas, hela cells were infected with vsv at a moi of and cytoplasmic extracts were prepared at and hpi for mrna isolation and polysome profiling. messenger rna was isolated from fractions corresponding to s monosomes, or polysomes containing or more ribosomes, and used for deep sequencing. (b) polysome analysis of uninfected (black) or vsv (red) infected hela cells. cytoplasmic extracts were sedimented through a - % sucrose gradient and . ml fractions were collected while continuously monitoring absorbance at λ = nm. (c) distribution of fragments mapping to the concatenated hg (human) and vsv genomes for cytoplasmic, monosome, and polysome samples at and hpi. trimming and mapping was performed in clc genomics workbench. (d) distribution of reads among the viral genes at and hpi. expression level is presented as transcripts per kilobase million (tpm) to normalize for gene length and library size, error bars denote standard deviation from two biological replicates. reads were most reduced in the polysome fraction compared to the total cytoplasmic fraction ( fig d) . a smaller reduction in reads was observed in the monosome fraction, and some cellular mrnas even showed an increase in reads compared to the total cytoplasmic fraction ( fig c) . this may reflect differences in the movement of cellular mrnas from large polysomes to monosomes or out of the pool of translating ribosomes. we next mined our sequence data for evidence of differential translation of cellular mrnas following vsv infection. for this analysis we divided the polysome tpm by the total cytoplasmic tpm as an indicator of the efficiency with which any given mrna is translated. we also performed a similar analysis for the monosome pool. we are cognizant of the fact that such ratios ignore the movement of any given mrna from larger to smaller polysomes, and will likely represent an underestimate of the extent of any translational regulation. to identify the subset of the population of cellular mrnas with the highest probability for translational regulation in infected cells, we plotted the fold change in tpm at and hpi (fig a- d ). at hpi the monosome or polysome-associated reads changed within a narrow range for the majority of cellular genes ( fig a) . the marked shut-off of host protein synthesis observed by metabolic labeling suggests that at hpi the association of cellular mrna with polyribosomes would alter significantly at the population level. although we observe a global reduction in polysome-associated reads for the bulk of the population of cellular mrnas the reduction is less than - fold. accompanying this global reduction in polysome-associated reads, we also observe an increase in monosome-associated reads with more than half the mrnas within the population exhibiting a > -fold increase (fig b and d ). from the above ratios we selected the subset of cellular mrnas that exhibit the largest changes in relative polysome-associated reads at hpi to determine whether those mrnas shared any common features. for this purpose, we selected those mrnas that change > standard deviations of the mean and thus exceed the % confidence interval. this analysis identified cellular mrnas as candidates for translational upregulation and cellular mrnas as candidates for translational downregulation following vsv infection (fig b and d ). consistent with monosome and polysomeassociated reads at hpi changing within a narrow range, only genes with increased and with decreased polysome association, overlap between and hpi (fig c and d ). within the monosome fraction genes with increased and with decreased monosome association overlap from to hpi. we next determined whether shared functional or sequence elements are present within the specific subsets of mrnas with increased polysome association, or the mrnas with decreased polysome association (fig b and d , blue and green dots). for the genes with significantly increased polysome-associated reads, gene ontology analysis identifies functions in rna binding, helicase and ntpase activities, among others ( fig e and s dataset) . the genes with decreased polysome-associated reads are associated with cellular responses to stimuli and signaling activities ( fig f and s dataset) . this gene ontology analysis reveals that the up and down regulated transcripts comprise distinct functional groups. at hpi the cytoplasmic abundance of cellular mrnas correlates with their polysome association at hpi ( fig a and b ), consistent with mrna abundance being a determinant of translatability. as described above, we use as an indicator of translation efficiency (te) of an mrna the ratio of polysome to total cytoplasmic associated reads. to determine whether there are shared features between the mrnas with evidence of enhanced polysome association or the with reduced polysome association, we extracted mrna sequences and annotations from the ucsc genome browser. assisted by published datasets we examined whether the half-life, size, gc content or poly(a) tail length correlate with increased or decreased polysome association (fig c- f and s fig) [ , ] . cellular mrnas with increased polysome-associated reads tended to have a longer half-life, were typically larger, and were more au-rich ( fig c- e and s fig) . correspondingly, those with decreased polysome-associated reads tended to have shorter half-lives, higher gc content, and were typically smaller. the correlation between higher au content and increased polysome-associated reads was most evident for the coding region and utr (s fig). the effect of length was predominantly a determinant of the orf and not the or utr (s fig). there was no correlation between poly(a) tail length and polysome-associated reads at hpi ( fig f) . this analysis highlights that the cellular mrnas that exceed the % confidence interval for increased polysome-associated reads in response to vsv infection are most similar to the viral mrnas in that they are typically longer and more au rich. we next examined whether cellular mrnas that exhibit increased polysome association encode proteins that are pro-or antiviral. among the cellular mrnas with increased polysome association several encode known proviral factors including the heat shock proteins (hsp) , , and . previous work demonstrated that inhibition of hsp inhibits viral replication, and linked inhibition of those chaperones to defects in l protein folding [ ] [ ] [ ] . we independently verified the proviral function of hsp using the inhibitor -[ -(dimethylamino)ethyl]amino- -desmethoxygeldanamycin ( -dmag) [ , ] . infection of hela cells with vsv that expresses egfp as a marker of infection demonstrates that -dmag has no effect on the fraction of cells infected, but slows the rate of egfp expression ( fig a and s fig) . this was not simply due to defects in egfp folding, as metabolic labeling of viral rna substantiates the defect in gene expression (s fig) . we also found that polysome association of the mrna encoding eukaryotic initiation factor subunit a (eif a), increases after infection. to test whether this reflects a specific proviral function of eif a, we used sirna depletion to reduce eif a and measured viral gene expression using reporter viruses expressing egfp or luciferase. both reporter viruses displayed a sensitivity to the loss of eif a (figs b and s ). as expected, depletion of eif a also reduced cellular translation in uninfected cells, but that reduction was modest as evidenced by expression of a cmv promoter driven renilla luciferase reporter ( fig b) . translation of the cmv driven reporter, however, reflects the accumulated steady-state levels of luciferase mrna. we therefore measured the effect of eif a depletion on viral vs host translation by metabolic incorporation of [ s]-methionine in infected and uninfected cells (fig b) . following eif a depletion we observed a % reduction in viral m protein synthesis over a minute time period, which is similar to the % reduction in host protein synthesis measured in mock infected cells. this analysis supports a proviral role for cellular mrnas that encode proteins with important house-keeping functions that remain polysome-associated following vsv infection. among the cellular mrnas that exhibit reduced polysome association following infection was dna-damage inducible transcript (ddit ) which encodes regulated in development and dna-damage response (redd ) [ ] [ ] [ ] . existing studies demonstrate that ddit /redd restricts the replication of negative-strand rna viruses, including vsv. depletion of ddit /redd by sirna increased viral gene expression as evidenced from infection of cells with vsv-egfp, and by metabolic labeling of viral protein synthesis (fig c, s fig) . consistent with the enhancement of viral gene expression following ddit depletion, we obtained an approximately -fold increase in viral titers ( fig c) . depletion of ddit /redd also increases cellular protein synthesis likely reflecting its role as a negative regulator of mtor ( fig c and s fig) . the above analysis supports that the polysome association of some host mrnas following vsv infection correlates with their pro-or antiviral functions, but does not directly demonstrate that the level of polysome association is associated with a change in synthesis of the corresponding protein. to independently examine whether changes in polysome association of host mrnas affect synthesis of the corresponding protein we selected the heat shock protein (hsp ) and y-box binding protein (ybx ) as representative mrnas with increased and decreased polysome association, respectively. we selected those mrnas based on their highlevels of expression, stability, and availability of antibodies suitable for the selective immunoprecipitation of the corresponding protein. we compared the effect of vsv infection on protein synthesis by selective immunoprecipitation of proteins following metabolic incorporation of [ s]-methionine from - hours post infection (fig d) . synthesis of hsp - hpi is indistinguishable to that synthesized during a h period from mock infected cells (fig d) . by contrast, ybx synthesis decreases more than two-fold ( fig d) . this result confirms for cellular mrnas that the extent of polysome association observed by our rnaseq analysis is reflected in synthesis of the corresponding host proteins. analysis of cellular mrnas with high cytoplasmic abundance (purple) or low cytoplasmic abundance (orange) as compared to mrnas with cytoplasmic abundance within standard deviations of the mean abundance (gray) in uninfected cells. cytoplasmic abundance by tpm is from the data set published with this paper. ��� p< . x − ; �� p< . x − ; � p< . ; all others p> . as determined by the wilcoxon rank sum test compared to mrnas with relative abundance levels within the % confidence interval of the mean. hinges correspond to the th- th percentiles, and whiskers denote . times the inter-quartile range. (b) analysis as in a for cytoplasmic abundance in infected cells. (c-f) mrna characteristics for mrnas with increased polysome association (blue) or decreased polysome association (green) at hpi, as defined in fig . data for rna half-life and poly(a) tail length were from [ , ] . analysis was performed as in a. for our experiments we pooled fractions that contained or more ribosomes prior to sequencing of the polysome-associated mrna. as a result, we do not assess the impact of the redistribution of mrnas toward smaller polysomes. we therefore selected a subset of cellular mrnas (fig a) , and interrogated their distributions across polysomes using reverse transcription and quantitative pcr. as controls, we analyzed the distribution of n and g mrnas as representative viral transcripts translated by soluble and endoplasmic reticulum-associated ribosomes, respectively [ ] . consistent with the robust production of viral proteins at hpi, the vsv n and g mrnas were localized in fractions corresponding to or more ribosomes (fig b) . for two cellular mrnas with increased polysome tpm-collagen type iv alpha (col a ) and alpha-actinin- mrna (actn )-the mrnas remained associated with larger polysomes in infected cells (fig c) . two cellular transcripts that were largely unaltered in their polysome associated tpm-β-actin (actb) and glyceraldehyde -phosphate dehydrogenase (gapdh)-remained polysome-associated, although there was a shift toward smaller polysomes and some gapdh transcripts exited polysomes (fig d) . for two representative cellular mrnas with decreased polysome tpm-transforming growth factor b induced factors (tgif ) and ubiquitin conjugating enzyme e b (ube b)-the mrnas largely exited the polysome fractions, and those that remained were predominantly present on smaller polysomes ( fig e) . in all cases examined, dissociation of polysomes with edta shifted the mrna distribution toward the fractions corresponding to free ribosomal subunits (s fig). these qpcr data highlight the shift towards smaller polysome fractions for some cellular mrnas, which also likely contributes to suppression of host protein synthesis. this shift might also explain our finding that hsp protein synthesis is relatively unaffected by viral infection (fig d) , although the mrna exhibits increased polysome association. the abundance of viral mrna and the suppression of translation initiation through reducing the pool of eif e will both contribute to the movement of mrnas toward smaller polysomes. recognition of the mrna cap-structure by eif e requires that the guanine-n- position of the mgpppnmn cap is methylated [ ] . we previously reported a panel of recombinant vsvs containing amino acid substitutions within the l-encoded mrna cap methylase domain that are defective in viral mrna cap methylation [ ] . mutants vsv-l g a and vsv-l g a contain substitutions in the binding site for the methyl donor s-adenosyl methionine (sam) and ablate all viral mrna cap methylation (vsv-l g a ) or guanine-n but not ribose- -o methylation (vsv-l g a ) [ ] . as vsv mrna is relatively insensitive to the loss of eif e [ ] , we would anticipate that the methylation status of the mrna cap-structure would have little impact on polysome association. analysis of the distribution of vsv n and g mrna within polysomes at hpi revealed a similar distribution in cells infected with wild-cells infected with wild-type vsv. the position of viral proteins is noted to the right. presented is a representative gel from two independent replicates. (c) vsv gene expression and replication in ddit depleted cells. a representative histogram of egfp expression is shown along with the mfi of cells normalized to a non-targeting sirna control. error bars denote the standard deviation from the mean of three independent replicates. for metabolic labeling a representative gel of two independent replicates is presented. kinetics of viral replication were measured by titration of yields at various times post infection of sirna treated hela cells. error bars denote the standard deviation from the mean of independent replicates. (d) immunoprecipitation of cellular proteins synthesized post-infection with vsv wt . shown is a representative gel from two independent replicates. a quantitative analysis of the hsp and ybx bands is shown in the bottom two panels, error bars denote the standard deviation from two independent replicates. https://doi.org/ . /journal.ppat. .g control of vsv protein synthesis type virus as well as those infected with vsv-l g a and vsv-l g a (fig a- c) . correspondingly, the rate of viral protein synthesis in cells infected with vsv-l wt and vsv-l g a measured by a -minute pulse of [ s]-methionine is similar (s fig). these results demonstrate that defects in viral mrna cap methylation do not significantly alter the rate of viral protein synthesis, consistent with a reduced dependence on eif e [ ] . to directly test whether manipulating eif e levels affects viral translation we depleted eif e levels approximately -fold using a peptide-conjugated morpholino (ppmo) and measured the rates of vsv-l wt and vsv-l g a viral protein synthesis by a -minute pulse of [ s]-methionine at various times post-infection (fig d- f) . depletion of eif e decreased the rate of viral protein synthesis in vsv-l a infected cells, but not l wt infected cells ( fig e and f) . this was not due to sequestration of eif e by differential activation of eif e-bp between vsv-l wt and vsv-l g a infected cells, as the kinetics of eif e-bp dephosphorylation are the same during both infections (s fig). we previously reported that although vsv-l a is defective in mrna cap methylation, up to % of the mrna cap-structures are guanine-n methylated. we interpret this finding as indicative of an eif e dependent mechanism of translation early in infection. we obtained two snapshots into the complex battle for control of protein synthesis in cells infected with vesicular stomatitis virus by sequencing of polysome-associated mrnas at and hpi. those snapshots provide further evidence that the abundance of vesicular stomatitis virus mrnas is a determinant of the dominance of viral protein synthesis in infected cells, but highlight several additional attributes of this complex relationship. those include the demonstration that some host mrnas that remain polysome associated encode proteins that support viral replication, and some of those that exhibit reduced polysome association encode proteins that are antiviral. we also obtained further insight into the seemingly paradoxical observations that viral infection results in a reduction of the available pool of eif e -the rate limiting factor for translation initiation-yet viral mrnas contain a cap structure that is indistinct to that of host mrnas. through the use of a viral mutant defective in mrna cap methylation, and suppression of eif e levels we provide evidence consistent with a transition from an eif e dependent phase of viral translation to one less-dependent on eif e. the sequence data reported here provides some support for the model that the vsv mrnas overwhelm the pool of cellular mrna leading to a redistribution of ribosomes onto viral messages [ ] . evidence in support of this model is based on massively parallel sequencing of mrnas associated with polysomes, compared with those present in the cytoplasm. as a fraction of the total cytoplasmic mrna, the vsv mrnas represent~ % by hpi, but more than % by hpi, illustrating the power of exponential amplification of the viral genome. as a result, the viral n, p, m and g mrnas far exceed the abundance of any given cellular mrna, and even the least abundant viral mrna-that encoding the l polymerase-is present at similar levels to the most abundant cellular mrna. thus, one contributor to host cell shut-off in vsv infected cells appears to relate to the synthetic capabilities of the viral polymerase in transcription of viral mrna. similar conclusions have recently been reached for other viruses. infection of cells with mouse hepatitis virus (mhv) a positive-strand rna coronavirus that replicates within the cytoplasm results in - % of the cytoplasmic mrna being viral by hpi [ ] . for influenza a virus, a segmented negative-strand rna virus that replicates in the nucleus, > % of the total mrna in the cytoplasm is viral [ ] . in this case however, the viral endonuclease pa-x degrades cellular mrna which further contributes to the dominance of viral mrna [ , ] . for vaccinia virus, a dna virus that replicates entirely within the cytoplasm, degradation of host mrna through the viral encoded decapping enzymes d and d also helps the viral mrnas overwhelm those of the host cell [ , , ] . collectively these studies indicate that one shared mechanism for host cell shut-off in virus-infected cells is competition for host cell ribosomes through tipping the balance between viral and host mrna. earlier work concludes that viral mrna abundance is not the determinant of host cell shut-off [ ] . when vsv mrna levels were suppressed up to -fold by using defective interfering particles of vsv or a viral mutant defective in transcription, host shut-off was still observed. we did not directly test how suppressing viral mrna levels impacts host shut-off in this study, but rather conclude that abundance is only part of the mechanism by which the virus induces host cell shut-off-as discussed below. we also obtained evidence in support of additional mechanisms that contribute to host cell shut-off in vsv infected cells. we confirmed earlier work that demonstrated a suppression of the pool of the rate limiting factor for initiation, eif e, by altering the phosphorylation status of its negative regulator, eif e-bp , which results in eif e sequestration [ ] . differences in sensitivity to reductions in eif e may contribute to the overrepresentation of cellular mrna we observe in monosome fractions during infection. we also provide new evidence in support of a phase of vsv gene expression that is dependent on eif e through the use of a viral mutant partially defective in guanine-n methylation [ ] and by the suppression of cellular pools of eif e. when eif e levels are suppressed -fold, we unmask a defect in viral protein synthesis in cells infected with vsv-vsv-l g a a mutant with a -fold defect in methylation at the guanine-n -position of the cap-structure. we suspect that this significantly underestimates the eif e dependent phase of viral replication since transformed cell lines, like the hela cells used here, have higher constitutive levels of eif e [ ] . our findings are consistent with a model where viral mrnas initially compete with cellular mrnas and translate in an eif e dependent manner. as infection progresses and the shut-down of host transcription, mrna export and eif e sequestration continue, the process of initiation is increasingly less dependent on eif e. the mechanism by which the viral mrnas become less dependent on eif e remains uncertain, but earlier studies demonstrate that neither the or utr of viral mrnas facilitate this efficient translation. ongoing transcription of viral mrna from the viral genome has also been linked to efficient protein synthesis [ , ] . whether this reflects the fact that the virus is an efficient producer of mrna that supports the competition model, or whether there is a temporal requirement for continued viral mrna synthesis is uncertain. as obligate intracellular parasites, viruses depend upon host cell functions for their replication. our sequence analysis provides evidence that vsv infection differentially impacts the polysome association of cellular mrnas. several host mrnas increased in polysome association include genes with known "proviral" functions for entry and replication including heparan sulfate, clathrin, and hsp [ , [ ] [ ] [ ] . similarly, host mrnas with decreased polysome association included genes with published roles in restricting vsv replication such as interferon regulatory factor (irf ), ddit , and txnip [ , , ] . it is difficult to definitively determine whether this reflects evolution of the virus to contend with the environment in which it finds itself or a bona-fide pro and antiviral effect of a given host protein. our efforts to address this are confounded by the essential house-keeping nature of many of the proteins encoded by host mrnas that remain polysome associated. an example of this is provided by enhanced polysome association of eif a on vsv infection-a protein that is required for assembly of the multisubunit eif complex. that complex also includes eif d which has demonstrated cap-binding ability, and directs eif e-independent translation of select mrnas [ ] [ ] [ ] [ ] . depletion of eif a, however, resulted in an equivalent reduction in the rates of viral and host translation-inconsistent with a specialized need for eif components in vsv protein synthesis. in this study, we validated that the effect of vsv infection on the polysome association of host mrnas-hsp and ybx -had a concordant impact on protein synthesis. although synthesis of hsp did not increase per se, this is likely explained by the shifting of many cellular mrnas towards smaller polysomes. this finding highlights the fact that the designation of rnas as having "increased" or "decreased" polysome association is imprecise, and reflects the complexities of how any given host gene is regulated. nevertheless, the general finding that mrnas with "increased" polysome association on vsv infection are typically larger, have longer half-lives and higher au content-features that are shared with the viral mrna-highlights commonalities among mrnas that remain polysome-associated and thus are more efficient in competing for ribosomes during host shut-off. the cellular mrnas that exhibit reduced translation efficiency were shorter, have shorter half-lives and higher gc content. although we validated changes in translatability and differential impacts on viral gene expression for a few cellular genes, it would be of significant interest to perform stable isotope labelling by amino acids in cell culture (silac) to non-radioactively label newly synthesized cellular proteins, quantify them on a genome-wide scale and correlate those data with the rnaseq results presented here. in addition to suppression of host translation through mrna synthesis and eif e manipulation, the vsv matrix protein inhibits host rna polymerase ii transcription [ , ] , and blocks nuclear export of mature mrnas through complex formation with the nuclear pore components rae and nup [ ] [ ] [ ] [ ] [ ] . a well characterized viral mutant (vsv-m m r ) fails to interact with the nuclear pore complex and exhibits a delayed kinetics of host shut-off [ , , ] . a similar analysis to that described here of cells infected with such a mutant may help delineate the extent to which ongoing synthesis and export of cellular mrna impacts host cell shut-off. we anticipate that over the time course of vsv infection, the contribution of ongoing synthesis and export of host mrna from the nucleus will result in a relatively modest increase in the fraction of the cytoplasmic mrna that is cellular. this study highlights a strategy shared among distinct viruses to commandeer the host translational machinery by outcompeting cellular mrnas. precisely where the tipping point between viral and host mrna levels with respect to this shut-off occurs is uncertain. for vsv, a viral mutant that makes less mrna than the wild type yet still exhibits host cell shut-off suggests that shut-off can be achieved with less than the % of total cytoplasmic mrna observed here [ ] . additional work will be required to define whether a specific tipping point exists and how this is influenced by other viral strategies such as eif e suppression or blocking host gene transcription. hela cells (a gift from james hogle) were maintained in dulbecco's modified eagle medium (dmem; invitrogen, carlsbad, ca) supplemented with % fetal bovine serum (fbs; tissue culture biologicals, tulare, ca). viral stocks were grown on syrian golden hamster kidney bsrt cells (a gift from k. conzelmann), and purified on linear - % sucrose gradients prepared in nte ( mm tris ph . , mm nacl, mm edta). viral titers were determined by plaque assay on african green monkey kidney vero cells (atcc, ccl- ). for infections, cells were first washed with hanks' balanced salt solution (hbss) and incubated with virus for hour at ˚c in serum free medium, washed with hbss and subsequently incubated with medium supplemented with % fbs. for polysome profiling, hela cells were treated with dmem containing μg ml - cycloheximide at ˚c for minutes. cells were washed twice with x ice-cold phosphate buffered saline (pbs) containing μg ml - cycloheximide, and kept on ice or at ˚c. cells were scraped into a . ml microcentrifuge tube in x pbs with μg ml - cycloheximide, and pelleted at × g for minutes. cells were resuspended in μl of a hypotonic buffer of mm tris (ph . ), . mm mgcl , . mm kcl, and rnasin (promega, madison, wi), supplemented with cycloheximide to μg ml - and dl-dithiothreitol (dtt) to μm. the detergents triton x- . % (vol/vol) and sodium deoxycholate . % (wt/vol) were then added sequentially, cells were briefly vortexed, and incubated for minutes on ice and clarified by centrifugation at , × g for min. polysomes were separated on sucrose gradients prepared on a gradient master station (biocomp, fredericton, canada) using % and % (wt/ vol) sucrose dissolved in mm tris (ph . ), mm mgcl , and mm nacl supplemented with rnasin and μg ml - cycloheximide. following centrifugation for hours at , × g in a beckman coulter ultracentrifuge using an sw ti rotor, μl fractions were collected from the top of the gradient while monitoring absorbance at λ = nm on a gradient master station. rna was extracted from total cytoplasmic, polysome, or monosome fractions using ls trizol (invitrogen) according to the manufacturer's protocol. equal amounts of rna as determined by spectrophotometry using absorbance at nm on a nanodrop (thermo fisher, waltham, ma) were subject to library preparation using the illumina truseq vii rna library preparation kit (illumina, san diego, ca), and sequenced at the whitehead institute (cambridge, ma) on an illumina hiseq . reads were trimmed and mapped to the concatenated hg and vsv genomes using clc genomics workbench (qiagen, redwood city, ca). mapping parameters were as follows; mismatch cost , insertion cost , deletion cost , length fraction . , similarity fraction . , and a maximum of hits per read. raw sequence data is available from the ncbi sequence read archive (sra) under the primary accession code srp . transcripts per kilobase million (tpm) was calculated for genes with or more mapped reads in the cytoplasmic fraction of both uninfected and infected cells using the total number of mapped exon reads. to identify cellular mrnas that were potential targets for translational regulation in infected cells, we determined the tpm in the polysome fraction/tpm in the total cytoplasmic fraction for each individual mrna. this ratio was determined for uninfected and infected cells, and presented as the log fold change. gene ontology analysis was performed in r using goseq. utrs and cds sequences were downloaded from the ucsc table browser using "knowncanonical" mrna identifiers. non-protein coding rnas were excluded from the analysis. poly (a) tail length and mrna half-lives were from published data sets [ , ] . graphs and statistical analyses were performed in r using the "wilcox_test" statistical test, the "density" kernel density estimation, and "geom_boxplot" or "geom_density" functions in ggplot and cowplot. total rna was recovered from polysome fractions using ls trizol according to the manufacturer's protocol. rna ( ng) was annealed with oligo d(t) and reverse-transcribed using superscript iii reverse transcriptase (thermo fisher) at ˚c for hour. following digestion of the rna strand with rnasea and rnaseh for min at ˚c, reactions were diluted : for cellular gene-specific qpcr or : for viral gene-specific qpcr. quantitative pcr was performed using power sybr green (thermo fisher) and relative amounts determined by images of rvsv-egfp infected cells were acquired using a × objective on a nikon eclipse te microscope (nikon instruments, melville, ny) equipped with a spot rt se monochrome camera (diagnostic instruments, sterling heights, mi). for cytometry, cells were washed in hbss, trypsinized, fixed in % paraformaldehyde at ˚c for minutes and measured using a facscalibur (cytek development, freemont, ca). cytometry data was analyzed using flowjo (flowjo inc, ashland, or). for mean fluorescence intensity (mfi) we gated on live cells identified by forward and side-scatter. to measure the % infected cells we subtracted those cells that fell within the gate established from uninfected control cells. for luciferase assays, where indicated hela cells were transfected with sirna, and the medium replenished at h. cells were transfected h later with ng prl-cmv (promega), and activity measured h later. for viral driven luciferase reporters sirna transfected cells were infected at h and monitored h later. luciferase expression was measured in a spec-tramax l microplate reader using the appropriate reagents according to the manufacturer's instructions (promega, e and e ). for depletion of host factors by peptide-conjugated morpholinos (ppmos) approximately . x hela cells per well of a well plate were treated h later with μm of the indicated ppmo. at h post treatment, the media was replaced with fresh medium containing μm ppmo, and used for testing h later. cells were washed twice with ice-cold x pbs and lysed in rose lysis buffer consisting of mm tris-hcl (ph . ), mm edta, . % w/v sodium deoxycholate, and % v/v np- on ice for minutes. rose lysis buffer was supplemented with phosphatase inhibitor cocktail (sigma-aldrich, st. louis, mo) and halt protease and phosphatase inhibitor cocktail (thermo fisher) for detection of phospho-eif e-bp . lysates were clarified, protein input was normalized by bradford assay and proteins resolved on polyacrylamide gels- % for eif a and eif e or %, eif e-bp . proteins were transferred to nitrocellulose membranes for minutes, eif e and eif e-bp , or minutes, eif a, at v. membranes were blocked with odyssey blocking buffer in pbs (li-cor, lincoln, ne) for h at room temperature, and incubated overnight at ˚c with the following primary antibodies: rabbit anti-eif a (cell signaling, # ), rabbit anti-eif e (cell signaling, # ), rabbit anti- e-bp (cell signaling, # ), rabbit anti-phospho- e-bp ser (cell signaling, # ), rabbit anti-phospho- e-bp thr / (cell signaling, # ), mouse anti-actin (millipore, #mab ), mouse anti-actin (sigma, #a ). membranes were washed x with x pbs-t for minutes at room temperature, and incubated with the relevant secondary antibodies: goat anti-mouse irdye rd (li-cor, # - ) or goat anti-rabbit irdye cw (li-cor, # - ), for hour at room temperature. membranes were washed again and kept in x pbs, and scanned on an odyssey clx (li-cor). hela cells were starved in dmem lacking l-methionine (corning, # - -cl) for minutes, prior to addition of [ s] express protein labeling mix (perkin elmer, waltham, ma) at . mci ml - . cell lysates were prepared as described above and separated on a low-bis % polyacrylamide gel. the gel was dried for . h at ˚c in a vacuum gel drier and detected using a phosphoimager. quantitative analyses of band intensities was performed in image-quant tl v . (ge healthcare, marlborough, ma). for radioimmunoprecipitations, . x hela cells plated in cm dishes (corning) were starved of methionine for h at h post-plating, and labeled with [ s]-express for h. cells were washed twice with ice-cold x pbs, collected by scraping and subsequent centrifugation for minutes at ˚c and , × g and lysed in ml of mm tris (ph . ), mm nacl , mm edta, % v/v np , mm dtt, supplemented with pierce protease inhibitor (thermo fisher) on ice for minutes. protein input was normalized by bradford assay, sds was added to . %, and μl lysate was pre-cleared for h at ˚c on a nutator with μl prewashed pierce protein a agarose beads. protein a beads were pelleted by centrifugation for minutes at ˚c and , × g and the labeled supernatant incubated with primary antibody at ˚c overnight. the antibodies used for immunoprecipitation were μg anti-yb (abcam, #ab ) and μg anti-hsp (enzo, #adi-spa- ). immune complexes were isolated using μl pre-washed pierce protein a agarose beads, by incubating for hours at ˚c. bead complexes were collected by centrifugation for minutes at ˚c and , × g, washed x with μl ice-cold ip lysis buffer with . % sds, on an orbital shaker for minutes at room temperature. protein-antibody complexes were eluted by boiling in x sds loading buffer, the beads pelleted for minutes at ˚c in a microcentrifuge and the supernatant loaded on a % polyacrylamide gel. after electrophoresis the gel was dried and imaged using a phosphoimager. approximately . x hela cells were plated per well of a -well dish and hours later incubated with phosphate free media (gibco, - ) for h. thirty minutes before infection, actinomycin d-mannitol (sigma, #a ) was added to a final concentration of μg ml - . infections were carried out in phosphate free media supplemented with μg ml - actinomycin d and . μm -dmag. at hpi cells were washed, fresh medium added, and supplemented h later with [ p]-orthophosphoric acid (perkin elmer, #nex h) . μci μl - . cells were harvested at hpi in rose lysis buffer, and total rna was extracted using ls trizol. rna was separated on a m urea-agarose gel as previously described, and detected using a phosphoimager [ ] . vesicular stomatitis virus matrix protein inhibits host cell-directed transcription of target genes in vivo the role of vesicular stomatitis virus matrix protein in inhibition of host-directed gene expression is genetically separable from its function in virus assembly effect of vesicular stomatitis virus matrix protein on transcription directed by host rna polymerases i, ii, and iii vesiculoviral matrix (m) protein occupies nucleic acid binding site at nucleoporin pair (rae * nup ) inhibition of ran guanosine triphosphatase-dependent nuclear transport by the matrix protein of vesicular stomatitis virus the matrix protein of vesicular stomatitis virus inhibits nucleocytoplasmic transport when it is in the nucleus and associated with nuclear pore complexes vesicular stomatitis virus matrix protein inhibits host cell gene expression by targeting the nucleoporin nup vsv disrupts the rae / mrnp mrna nuclear export pathway vesicular stomatitis virus infection alters the eif f translation initiation complex and causes dephosphorylation of the eif e binding protein e-bp inhibition of host and viral translation during vesicular stomatitis virus infection. eif is responsible for the inhibition of viral but not host translation the mechanism of eukaryotic translation initiation: new insights and challenges quantitative studies of mrna recruitment to the eukaryotic ribosome dissociation and reconstitution of the transcriptase and template activities of vesicular stomatitis b and t virions ribonucleic acid synthesis of vesicular stomatitis virus. . multiple complementary messenger rna molecules polysomal ribonucleic acid of vesicular stomatitis virus-infected hela cells translation of vesicular stomatitis messenger rna by extracts from mammalian and plant cells in vitro synthesis of methylated messenger rna by the virionassociated rna polymerase of vesicular stomatitis virus translation and identification of the mrna species synthesized in vitro by the virion-associated rna polymerase of vesicular stomatitis virus methylated and blocked ' termini in vesicular stomatitis virus in vivo mrnas ribonucleic acid synthesis of vesicular stomatitis virus, ii. an rna polymerase in the virion l protein requirement for in vitro rna synthesis by vesicular stomatitis virus transcriptional activity and mutational analysis of recombinant vesicular stomatitis virus rna polymerase amino acid residues within conserved domain vi of the vesicular stomatitis virus large polymerase protein essential for mrna cap methyltransferase activity a single amino acid change in the l-polymerase protein of vesicular stomatitis virus completely abolishes viral mrna cap methylation a unique strategy for mrna cap methylation used by vesicular stomatitis virus unconventional mechanism of mrna capping by the rna-dependent rna polymerase of vesicular stomatitis virus a conserved motif in region v of the large polymerase proteins of nonsegmented negative-sense rna viruses that is essential for mrna capping rebinding of transcriptase components (l and ns proteins) to the nucleocapsid template of vesicular stomatitis virus location of the binding domains for the rna polymerase l and the ribonucleocapsid template within different halves of the ns phosphoprotein of vesicular stomatitis virus structure of the vesicular stomatitis virus nucleoprotein-rna complex structure of the vesicular stomatitis virus nucleocapsid in complex with the nucleocapsid-binding domain of the small polymerase cofactor molecular architecture of the vesicular stomatitis virus rna polymerase critical phosphoprotein elements that regulate polymerase architecture and function in vesicular stomatitis virus synthesis of poly(a) in vitro by purified virions of vesicular stomatitis virus in vitro synthesis of rna that contains polyadenylate by virion-associated rna polymerase of vesicular stomatitis virus site on the vesicular stomatitis virus genome specifying polyadenylation and the end of the l gene mrna aberrant polyadenylation by a vesicular stomatitis virus mutant is due to an altered l protein cis-acting signals involved in termination of vesicular stomatitis virus mrna synthesis include the conserved auac and the u signal for polyadenylation translational control of protein synthesis after infection by vesicular stomatitis virus vesicular stomatitis virus mrna and inhibition of translation of cellular mrna-is there a p function in vesicular stomatitis virus? effect of intracellular vesicular stomatitis virus mrna concentration on the inhibition of host cell protein synthesis complete intergenic and flanking gene sequences from the genome of vesicular stomatitis virus complete sequences of the ribosome recognition sites in vesicular stomatitis virus mrnas: recognition by the s and s complexes translation of mrnas from vesicular stomatitis virus and vaccinia virus is differentially blocked in cells with depletion of eif gi and/or eif gii a ribosome-specialized translation initiation pathway is required for cap-dependent translation of vesicular stomatitis virus mrnas preferential translation of vesicular stomatitis virus mrnas is conferred by transcription from the viral genome high-resolution analysis of coronavirus gene expression by rna sequencing and ribosome profiling a systematic view on influenza induced host shutoff ribosome profiling reveals translational upregulation of cellular oxidative phosphorylation mrnas during vaccinia virus-induced host shutoff localized attenuation and discontinuous synthesis during vesicular stomatitis virus transcription transcriptional mapping of vesicular stomatitis virus in vivo order of transcription of genes of vesicular stomatitis virus determination of molar ratios of vesicular stomatitis virus induced rna species in bhk cells genome-wide determination of rna stability reveals hundreds of short-lived noncoding transcripts in mammals tail-seq: genome-wide determination of poly(a) tail length and ' end modifications antiviral activity and rna polymerase degradation following hsp inhibition in a range of negative strand viruses hsp inhibitors exhibit resistance-free antiviral activity against respiratory syncytial virus hsp chaperoning in addition to phosphoprotein required for folding but not for supporting enzymatic activities of measles and nipah virus l polymerases inhibition of heat shock protein hsp -pp v-src heteroprotein complex formation by benzoquinone ansamycins: essential role for stress proteins in oncogenic transformation crystal structure and molecular modeling of -dmag in complex with human hsp txnip potentiates redd -induced mtor suppression through stabilization of redd chemical inhibition of rna viruses reveals redd as a host defense factor apobec g-regulated host factors interfere with measles virus replication: role of redd and mammalian torc inhibition separate pathways of maturation of the major structural proteins of vesicular stomatitis virus biophysical studies of eif e cap-binding protein: recognition of mrna ' cap structure and synthetic fragments of eif g and e-bp proteins an overlapping protein-coding region in influenza a virus segment modulates the host response selective degradation of host rna polymerase ii transcripts by influenza a virus pa-x host shutoff protein characterization of a second vaccinia virus mrna-decapping enzyme conserved in poxviruses vaccinia virus d protein has mrna decapping activity, providing a mechanism for control of host and viral gene expression malignant transformation by a eukaryotic initiation factor subunit that binds to mrna ' cap new mrnas are preferentially translated during vesicular stomatitis virus infection pathway of vesicular stomatitis virus entry leading to infection entry pathway of vesicular stomatitis virus into different host cells vesicular stomatitis virus enters cells through vesicles incompletely coated with clathrin that depend upon actin for internalization systematic identification of type i and type ii interferoninduced antiviral factors eif targets cell-proliferation messenger rnas for translational activation or repression ' utr m( )a promotes cap-independent translation eif d is an mrna cap-binding protein that is required for specialized translation initiation dynamic m( )a mrna methylation directs translational control of heat shock response ability of the matrix protein of vesicular stomatitis virus to suppress beta interferon gene expression is genetically correlated with the inhibition of host rna and protein synthesis rna molecular weight determinations by gel electrophoresis under denaturing conditions, a critical reexamination we thank members of the whelan lab for scientific discussions and suggestions; we further thank carl novina and cailin joyce for help with polysome profiling, keith ketterer for assistance with clc genomics workbench, and alos diallo for help with r and statistical analyses. we thank david stein and hong moulton, of oregon state university, for design and production of the ppmos used in this study. key: cord- - zh jmc authors: caignard, grégory; komarova, anastassia v.; bouraï, mehdi; mourez, thomas; jacob, yves; jones, louis m.; rozenberg, flore; vabret, astrid; freymuth, françois; tangy, frédéric; vidalain, pierre-olivier title: differential regulation of type i interferon and epidermal growth factor pathways by a human respirovirus virulence factor date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: zh jmc a number of paramyxoviruses are responsible for acute respiratory infections in children, elderly and immuno-compromised individuals, resulting in airway inflammation and exacerbation of chronic diseases like asthma. to understand the molecular pathogenesis of these infections, we searched for cellular targets of the virulence protein c of human parainfluenza virus type (hpiv -c). we found that hpiv -c interacts directly through its c-terminal domain with stat and grb , whereas c proteins from measles or nipah viruses failed to do so. binding to stat explains the previously reported capacity of hpiv -c to block type i interferon signaling, but the interaction with grb was unexpected. this adaptor protein bridges epidermal growth factor (egf) receptor to mapk/erk pathway, a signaling cascade recently found to be involved in airway inflammatory response. we report that either hpiv infection or transient expression of hpiv -c both increase cellular response to egf, as assessed by elk transactivation and phosphorylation levels of erk / , s ribosomal subunit protein s and translation initiation factor e (eif e). furthermore, inhibition of mapk/erk pathway with u prevented viral protein expression in infected cells. altogether, our data provide molecular basis to explain the role of hpiv -c as a virulence factor and determinant of pathogenesis and demonstrate that paramyxoviridae have evolved a single virulence factor to block type i interferon signaling and to boost simultaneous cellular response to growth factors. viruses need to interact with host macromolecules to hijack the cellular machinery and replicate. these interactions are essential for viruses to target endocytic pathways and penetrate host cells, to recruit cellular transcription and/or translation machinery, and to achieve intracellular migration and viral particles assembly. but viruses also encode virulence factors that induce a substantial alteration of host cell functions and genetic programs to increase virus replication and spreading. for example, specific viral factors stimulate survival pathways to prevent apoptosis of infected cells or inhibit cell signaling involved in immune response. among these pathways, ifn-a/b signaling represents a prime target for viruses because of its critical role in the induction of both innate and adaptive antiviral immune responses [ ] . ifn-a/b transduce signals through direct binding to a cell surface receptor composed of two transmembrane subunits, ifnar and ifnar c [ ] . this interaction activates ifnar /ifnar c associated kinases tyk and jak that subsequently phosphorylate stat and stat transcription factors. activated stat and stat , altogether with irf , form the interferon-stimulated gene factor that binds ifn-stimulated response element (isre) promoter sequences to induce a large antiviral gene cluster. as a consequence, most viruses that are pathogenic in vertebrates have evolved virulence factors both to block ifn-a/b expression and signal transduction downstream of ifn-a/b receptor. human parainfluenza virus type (hpiv ) and human parainfluenza virus type (hpiv ) are important human pathogens that belong to respirovirus genus (paramyxoviridae family; [ ] ). these viruses are responsible for upper respiratory tract infections and colds, but often spread to the lower respiratory tract causing bronchitis, bronchiolitis and pneumonia in young children and immuno-compromised patients. hpiv infection is also suspected to exacerbate chronic airway inflammatory diseases like asthma [ ] . sendai virus and bovine parainfluenza virus type (bpiv ) are animal counterparts of hpiv and hpiv that infect mouse and cattle, respectively. respirovirus genome is a single-strand, negativesense rna molecule that encodes six structural proteins (mononegavirales order). while hemagglutinin-neuraminidase (hn) and fusion (f) are membrane glycoproteins associated with the envelop of hpiv particles, the nucleoprotein (n), the phosphoprotein (p) and the viral polymerase (l) form the ribonucleocapsid complex. the matrix protein (m) is at the interface between glycoprotein tails and ribonucleocapsids. the p gene of respirovirus encodes for p but also for a panel of accessory proteins by site-specific editing of p mrna and usage of overlapping open reading frames (orfs). in all respirovirus except hpiv , the co-transcriptional insertion of one g residue at an editing motif midway of p mrna leads to the expression of a chimeric protein called v. the v proteins of bpiv and sendai virus bind mda and suppress double-stranded rna-stimulated ifn-b production, thereby contributing to the virus evasion of host immune response [ ] . surprisingly in hpiv , multiple stop codons localized downstream of the editing site prevent the normal expression of a full-length v protein. as a result, p mrna molecules edited by the addition of one g residue encode for the amino acid (aa)-long n-terminal residues of p followed by only six additional aa (see materials and methods and [ ] ). but p mrna molecules edited by the addition of five g residues encode for d, a protein exhibiting a large and specific c-terminal domain of unknown function ( figure a ). besides co-transcriptional edition, an overlapping orf embedded in the first half of the p mrna allows the expression of a single c protein (hpiv and bpiv ) or a nested set of four proteins called c , c, y and y (sendai virus and hpiv ). the c proteins of sendai virus and hpiv have a high degree of sequence homology and have been studied in details. they are involved in the regulation of viral rna synthesis [ , ] , the inhibition of innate immune response [ ] and potentially contribute to the budding of viral particles [ ] [ ] [ ] . in particular, the c protein of sendai virus both inhibits ifn-b production [ , ] and blocks interferon signaling downstream of ifn-a/b and ifn-c receptors [ ] [ ] [ ] . the c proteins of hpiv and bpiv only share , % of sequence homology with the c proteins of sendai virus and hpiv , but they have also been shown to target interferon expression and signaling [ , ] . although expression of the c protein of hpiv (hpiv -c) is essential to virulence in vitro and in vivo [ ] and explains hpiv ability to block ifn-a/b signaling [ ] , host proteins that bind hpiv -c remain unknown. (a) organization of the gene p of hpiv that encodes for three proteins: p, d and c. whereas conventional transcription and translation lead to the expression of the phosphoprotein p, co-transcriptional insertion of five g residues at the editing site by the virus rna polymerase leads to the expression of a chimeric protein called d. insertion of one g residue can also occur during transcription but two stop codons immediately downstream of the editing site prevent the expression of the protein v that is specific of paramyxoviridae. the protein c is encoded by an overlapping opened reading frame (orf) embedded in p mrna. (b and c) hek- t cells were transfected with expression vectors encoding gst alone or fused to the c proteins of measles virus (mv-c), hpiv (hpiv -c), or nipah virus (nipah-c), and tested for the interaction with endogenous stat (b) or grb (c). total cell lysates were prepared h post transfection (cell lysate; middle and lower panels), and copurifications of endogenous cellular proteins were assayed by pulldown using glutathione-sepharose beads (gst pull-down; upper panel). gst-tagged c proteins were detected by immunoblotting using anti-gst antibody, while endogenous stat and grb were detected with specific monoclonal antibodies. doi: . /journal.ppat. .g respiroviruses are important pathogens responsible for acute respiratory tract infections associated with severe airway inflammation in children, elderly and immunocompromised individuals. their rna genome encodes for structural proteins that compose viral particles, but also for virulence factors that alter cell biology to enhance virus replication and spreading. among them, the c protein plays a critical role by blocking cellular response to type i interferons, the main antiviral cytokines secreted during virus infections. to provide molecular basis to this activity, we found that the c protein of human parainfluenza virus type (hpiv -c), the most frequent human respirovirus, interacts with stat , a key component of type i interferon receptor complex. but hpiv -c was also found to interact with grb , an adaptor molecule involved in cellular response to epidermal growth factor (egf), and to enhance cell response to this cytokine. this pathway increases protein translation, promotes cell survival and contributes to airway inflammation and mucus secretion. thus, our findings show that hpiv -c not only inhibits the antiviral response but also stimulates cellular response to egf, which benefits virus replication and induces an excessive inflammation of airways during infection. in an attempt to answer this question, we performed a yeast two-hybrid screen and we report here the identification of stat and grb as direct interactors of hpiv -c. although binding to stat accounts for hpiv -c ability to block ifn-a/b signaling, the interaction with grb was unexpected. this adaptor protein bridges growth factor receptor tyrosine kinases (rtks), like epidermal growth factor (egf) receptor, to the mitogenactivated protein kinase/extracellular signal-regulated kinase (mapk/erk) pathway. upon engagement by their ligands, rtks autophosphorylate on tyrosine residues to recruit adaptor proteins containing phosphotyrosine binding (ptb) or src homology (sh ) domains like grb [ ] . once associated to rtks by its sh domain, grb recruits the guanine nucleotidereleasing factor son-of-sevenless (sos) to activate ras. downstream events include mapk/erk kinase (mek / ) activation, which in turn phosphorylates erk / . finally, phosphorylated erk / directly or indirectly activates numerous cellular targets including transcription factors (e.g. elk , sap , sap , c-fos, creb, srf) but also cellular factors that control mrna translation like eukaryotic initiation factor e (eif e) or small ribosomal subunit s protein [ , ] . growth factor binding to rtks regulates a multiplicity of cellular processes including proliferation, differentiation and survival. in the respiratory tract, this signaling cascade has been shown to trigger inflammation and mucus secretion by epithelial cells [ ] [ ] [ ] [ ] , suggesting a critical role in innate immunity [ ] . however, excessive activation of this pathway could benefit to virus replication by inhibiting ifn-a/b signaling [ ] and promoting infected cell survival [ ] . altogether, these data provided a rational to investigate the functional impact of hpiv -c expression on ifn-a/b vs egf receptor and mapk/erk signaling pathways. the c protein of hpiv interacts directly with stat and grb to identify cellular targets of hpiv -c, this viral protein was used as bait in the yeast two-hybrid system to screen a human cdna library. the screen was performed at saturation with a -fold coverage of the library ( . + diploids), and positive yeast colonies growing on selective medium were analyzed by pcr and sequencing to identify binding partners of hpiv -c. stat and grb were the main interactors of hpiv -c identified in the screen with and yeast colonies corresponding to these cellular proteins, respectively. in both cases, cdna clones retrieved from the screen corresponded to full-length stat and grb in frame with the gal -ad transactivation domain. to validate these interactions in human cells, gst-tagged hpiv -c was expressed in hek- t cells and purified with glutathion-sepharose beads. as shown in figure b and c, endogenous stat and grb co-purified with hpiv -c. highly divergent c proteins from measles virus (mv-c) and nipah virus (nipah-c) failed to do so, thereby demonstrating the specificity of identified interactions. binding to stat provides molecular basis to the inhibition of ifn-a/b signaling by hpiv -c [ ] , and parallels the interaction previously identified between sendai virus c protein and mouse stat [ ] . altogether, this suggests that stat is a specific cellular interactor of respirovirus c proteins. in contrast, binding to grb is unexpected and suggests a new function for hpiv -c that we decided to investigate. hpiv -c has opposite effects on ifn-a/b and egf signaling pathways the adaptor protein grb plays a critical role in coupling signal from growth factor receptors to mapk/erk signaling pathway. to address the question of hpiv -c interference with this pathway, we used a trans-reporter gene assay that measures elk activation by erk / . in this system, elk transcription factor is fused to the dna binding domain of gal (gal -db) and binds the promoter sequence of a luciferase reporter gene. upon stimulation with a growth factor like egf, elk is activated as assessed by a significant increase in luciferase expression. surprisingly, we observed a -fold enhancement in this cellular response to egf when flag-tagged hpiv -c was expressed in hek- t cells (figure a ). same results were obtained when using hpiv -c without a tag ( -fold enhancement) or tagged with the red fluorescent protein cherry ( -fold enhancement). in contrast to hpiv -c, neither mv-c nor nipah-c enhanced elk activity upon egf stimulation (figure a ) whereas expression levels of hpiv -c, mv-c and nipah-c were similar in this system ( figure f , left panel). elk activity was also enhanced by hpiv -c expression in vero and hela cells as well as beas- b and a , two epithelial cell lines that originate from the respiratory tract, which is the tissue targeted by hpiv in vivo ( table ). the effect of hpiv -c in these different cell lines was highly significant (see p-values in table ) although relatively modest when compared to hek- t cells. this is probably because our reporter system requires the co-transfection of four plasmids and vero, hela, beas- b and a cells are more difficult to transfect than hek- t. in parallel experiments, cellular response to ifn-a/b was monitored using a cis-reporter gene, of which expression is controlled by five isres. as previously reported [ ] , we found that hpiv -c efficiently blocked ifn-a/b signaling ( figure b ) as opposed to what we observed for the egf pathway. again, mv-c or nipah-c was unable to do so. altogether, these results show that hpiv -c enhances the cellular response to egf in addition to its ability to block ifn-a/b signaling. we also determined if similar effects on the egf pathway were observed in infected cells. hek- t cells were infected with hpiv (moi = ) and then transfected with elk activity reporter plasmids. infection of hek- t cells was confirmed by anti-hpiv hemagglutinin-neuraminidase (hpiv -hn) immunostaining and flow cytometry analysis ( figure e ). like hpiv -c alone, hpiv infection enhanced elk activity upon egf stimulation ( figure c ). interestingly, hpiv infection induced a significant level of elk activity in the absence of egf stimulation. this suggests that in addition to hpiv -c interaction with grb , other mechanisms modulate mapk/erk pathway during hpiv infection. finally, to demonstrate that enhancement of elk activation by hpiv -c is completely dependent on erk / activation, hek- t cells were pre-treated with mek / inhibitor u before stimulation with egf. this molecule targets mek / and totally abrogates downstream phosphorylation and activation of erk / [ ] . as shown in figure d , elk activation was blocked by u , whereas hpiv -c expression was maintained ( figure f , right panel). this demonstrates that hpiv -c is acting through erk / stimulation. altogether, these results support a model where hpiv -c interaction with grb enhances cellular response to growth factors as assessed by an increased activation of mapk/ erk pathway. phosphorylation of erk / , eif e and small ribosomal subunit s protein are stimulated by hpiv -c expression or hpiv infection to further document hpiv -c impact on mapk/erk signaling pathway, we compared the kinetic of erk / phosphorylation in hek- t cells expressing hpiv -c or not. cells were transfected hpiv -c impact on ifn-a/b and egf pathways in addition to these three plasmids, cells were co-transfected with an expression vector encoding flag-tagged mv-c, hpiv -c or nipah-c or the corresponding empty vector pci-neo- flag. h after transfection, cells were starved and h later egf was added at a final concentration of ng/ml. after h, relative luciferase activity was determined. (b) hek- t cells were transfected with pisre-luc, a plasmid containing a luciferase gene of which expression is controlled by five isres, and prl-cmv. in addition to these two plasmids, cells were co-transfected with an expression plasmid encoding flag-tagged mv-c, hpiv -c or nipah-c or the corresponding empty vector pci-neo- flag. h after transfection, iu/ml of recombinant ifn-b were added. after h, relative luciferase activity was determined. (c) hek- t cells were infected with hpiv (moi = ) and then transfected with pfa -elk , pgal -uas-luc, prl-cmv vectors. h later, cells were starved during h and stimulated with egf at a final concentration of ng/ml. after h, relative luciferase activity was determined. (d) same experiment as (a) but mm of mek / specific inhibitor u was added as indicated. (a-d) all experiments were achieved in triplicate, and data represent means sd. (e) hek- t cells were infected as in (c), and hpiv -hn expression determined by immunostaining and flow cytometry analysis. (f) hek- t cells were transfected to express flag-tagged mv-c, hpiv -c or nipah-c as described in (a) and (b), and relative expression levels were determined h later by western blot analysis (left panel). in a parallel experiment, hek- t cells were transfected to express hpiv -c and were cultured with or without egf in the presence or absence of u as described in (d). hpiv -c expression level was determined by western blot analysis (right panel). doi: . /journal.ppat. .g with flag-tagged hpiv -c or a control plasmid and h post transfection, they were starved before stimulation with egf. erk / phosphorylation was determined at , and min after stimulation. as illustrated by one representative experiment in figure a , egf stimulation induced erk / phosphorylation in control cells but signal was markedly and reproducibly increased by hpiv -c expression at maximum phosphorylation time point ( . to . fold increase; p = . ; n = ). we then determined the phosphorylation level of two downstream targets of this pathway that are involved in the control of mrna translation, the translation initiation factor eif e and the ribosomal protein s ( figure b ). before egf stimulation, low levels of phosphorylated eif e and s were detectable in mock-treated cells ( figure b and d). hpiv -c expression had virtually no effects on this background. thus, eif e and s phosphorylation levels were determined at different time-points after egf stimulation. because erk / activation precedes eif e and s phosphorylation, maximal phosphorylation occurs at later time points and was determined at min, h, h and h after stimulation. as observed for erk / , phosphorylation levels of eif e and s were enhanced by hpiv -c expression when stimulating the cells with egf. to validate these observations in infected cells, hek- t cells were infected with hpiv (moi = ) and h later, cells were starved for h before stimulation with egf. like hpiv -c expression alone, hpiv infection enhanced erk / phosphorylation at the peak of induction, i.e. min after adding egf to the cells ( figure c ). interestingly, hpiv infection of a cells also enhanced erk / phosphorylation but the induction profile was different. indeed, erk / phosphorylation was not significantly increased at the peak of induction, but the signal was boosted by hpiv infection at late time points ( figure s ). the same profile was observed when eif e and s phosphorylation levels were analyzed in infected hek- t cells. hpiv infection sustained the phosphorylation of these two translation factors at late time points, but showed no increase at the peak of stimulation, i.e. min after adding egf to the cells ( figure d ). this could relate to the fact that hpiv infection also induces low levels of eif e and s phosphorylation in the absence of egf stimulation ( figure d ). this is reminiscent to what was observed for elk ( figure c ), and suggests that hpiv infection induces a basal activation of mapk/erk pathway leading to the constitutive phosphorylation of downstream targets. altogether, these data demonstrate that hpiv infection or hpiv -c expression alone both enhance mapk/erk pathway activation in egf-stimulated cells. several rna viruses require an activated mapk/erk pathway to produce viral components and replicate properly (for review see [ ] ). to test if the same was true for hpiv , cells were treated for h with mapk/erk pathway inhibitor u and infected with hpiv (moi = ). two days after infection, cell surface expression of hpiv -hn was detected by immunostaining and flow cytometry. u completely blocked the expression of hpiv -hn in hpiv -infected cells (figure ), whereas the same inhibitor had no effect when cells were infected with mv ( figure s ). altogether, this demonstrates that mapk/erk signaling is essential for the expression of hpiv proteins and suggests that hpiv manipulates this pathway to increase replication efficiency. the c-terminal region of hpiv -c binds stat and grb to better understand how hpiv -c targets both the ifn-a/b and egf signaling pathways, we characterized the functional domains of hpiv -c that bind stat and grb . to do so, we generated by pcr a full matrix of hpiv -c overlapping fragments and tested their ability to interact with either stat or grb in the yeast two-hybrid system ( figure and ). both forward and reverse primers were designed every nucleotides along hpiv -c sequence and fused to appropriate tails to allow gap-repair recombination with linearized gal -db yeast two-hybrid vector ( figure a ). all possible combinations of forward and reverse primers were used to amplify hpiv -c fragments. finally, corresponding pcr products were transformed in a yeast strain expressing gal -ad fused to either stat or grb , and growth on selective medium was used to detect potential interactions. a (aa)-long peptide encompassing position to located in the c-terminal half of hpiv -c was sufficient to bind stat ( figure b ) or grb ( figure a ). in an iterative process, we then generated a second, a third and a fourth set of hpiv -c fragments corresponding to one-by-one aa deletions ( figure c -e and figure b -d), allowing to further reduce the stat and grb binding motifs to minimal peptides. a aa peptide encompassing residues to of hpiv -c was sufficient to observe the interaction with stat ( figure e ). the binding region to grb was virtually the same, encompassing aa to ( figure d ). the c-terminal region of hpiv -c required to bind stat and grb in the yeast two-hybrid system is highly conserved among respiroviruses ( figure a ) and suspected to fold into a structured coiled-coil domain [ ] . furthermore, virtually the same c-terminal region of sendai virus c protein (aa - ) was previously reported to mediate the interaction with mouse stat [ ] . to further validate our observations performed in the yeast two-hybrid system, we retested by co-affinity purification the ability of hpiv -c fragment encompassing aa - (hpiv -c - ) to interact with stat and grb in hek- t cells. gst-tagged hpiv -c - was expressed together with -flag-tagged stat or grb , and purified with glutathionsepharose beads. full-length hpiv -c and the n-terminal region encompassing aa - (hpiv -c - ) were used as positive and negative controls, respectively. as shown in figure b and c, hpiv -c - interacted with stat and grb , whereas hpiv -c - did not. although hpiv -c - interacted with grb as efficiently as full-length hpiv -c ( figure c ), interaction with stat was weaker suggesting that more residues contribute to the stabilization of this interaction ( figure b ). altogether, these results confirm that aa - of hpiv- include both stat and grb binding sites. as described in figure , hek- t, hela, vero, a or beas- b cells were transfected with pfa -elk , pgal -uas-luc, prl-cmv to measure the activation level of mapk/erk signaling pathway. cells were co-transfected with plasmids encoding flag-tagged mv-c, hpiv -c or nipah-c or the corresponding empty vector pci-neo- flag. h after transfection, cells were starved and stimulated h later with ng/ml of egf. after h, expression of luciferase was quantified. results were normalized so that reporter activity in cells transfected with a control vector equals . experiments were performed in triplicates and data represent means sd. doi: . /journal.ppat. .t hpiv -c impact on ifn-a/b and egf pathways although stat and grb essentially bind to the same region of hpiv -c as demonstrated above, it remained unclear whether these interactions are mutually exclusive. to answer this question, a competition experiment was designed where gst-tagged hpiv -c was co-expressed with stat in the presence or absence of grb ( figure d ). in this setting, grb expression prevents stat copurification together with gst-tagged hpiv -c. this validates our finding that stat and grb interact with the same region of hpiv -c, and demonstrates that stat and grb compete for hpiv -c binding. interestingly, grb interaction with hpiv -c was not affected by stat expression (figure d and data not shown), suggesting that grb has a higher affinity for hpiv -c than stat . both the n-and c-terminal domains of hpiv -c are required for its activity we finally tested if hpiv -c - was able, like full-length hpiv -c, to block ifn-a/b signaling and enhance cellular response to egf stimulation. first, cells were transfected with flag-tagged hpiv -c, hpiv -c - or hpiv -c - together with the ifn-a/b reporter plasmid, and stimulated h later with recombinant ifn-b. reporter gene expression was determined h post transfection and found to be inhibited exclusively by full-length hpiv -c ( figure a ). although this may reflect the weakness of hpiv -c interaction with stat ( figure b ), this also indicates that both the n-terminal and c-terminal regions of hpiv -c are required to block ifn-a/b signaling, even if only the c-terminal region is required for the binding to stat . the same constructs were tested using the elk activity reporter plasmids ( figure b ). again, only full-length hpiv -c was able to enhance elk activation upon egf stimulation whereas full-length hpiv -c and hpiv -c - were expressed at similar levels in transfected cells ( figure b , upper right panel). because grb binding to hpiv -c and hpiv -c - were essentially equivalent in co-affinity purification experiments, we hypothesized that the n-terminal region of hpiv -c was required for its proper subcellular localization. thus, hpiv -c, hpiv -c - and hpiv -c - were expressed in fusion downstream of the red fluorescent protein cherry. as shown in figure c , cherry alone or fused to hpiv -c - localized both in the nucleus and the cytoplasm of transfected cells. in contrast, full-length hpiv -c essentially accumulated at the cellular membrane whereas hpiv -c - was in the nucleus. although we have no explanation for this unexpected localization of hpiv -c - , these observations show that only full-length hpiv -c is able to target the cellular membrane where both ifn-a/b and egf signaling are triggered. paramyxoviridae have evolved various mechanisms to block ifna/b response, in particular signaling downstream ifnar / ifnar c receptor [ , ] . although members of pneumovirinae subfamily have specific genes to encode inhibitors of ifn-a/b signaling pathway, those expressed by other paramyxoviridae (i.e. paramyxovirinae subfamily) are encoded by overlapping reading frames embedded within the gene p. rubulaviruses express v proteins that target stat and/or stat for ubiquitination and degradation, while morbilliviruses and henipaviruses v proteins essentially impair stat / phosphorylation, activation and nuclear translocation. in addition, morbilliviruses and henipaviruses also encode for c proteins of which role in the inhibition of ifna/b response has been a matter of debates [ ] [ ] [ ] [ ] . recent reports showed that morbillivirus c proteins only have a minor role in the inhibition of ifn-a/b signaling [ ] , but are essential to block ifn-a/b induction [ ] . whether henipavirus c proteins can directly block ifn-a/b or promote viral replication through alternative mechanisms is unclear [ ] . in contrast, it has been clearly established that respirovirus c proteins are potent inhibitors of ifn-a/b signaling [ , , [ ] [ ] [ ] . in this report, we show that hpiv -c, but not mv-c or nipah-c, directly interacts with stat and efficiently inhibits ifn-a/b signaling. in addition, we identified a minimal stat binding domain that encompasses aa - of hpiv -c, a region suspected to fold into a coiled coil. interestingly, this conserved domain is localized within the stat binding region shared by all four isoforms of sendai virus c protein [ ] . together, these results confirm the capacity of hpiv -c to block ifn-a/b signaling pathway [ ] , provide molecular basis to this inhibition and clarify the fact that respirovirus c proteins are functionally distinct from morbillivirus and henipavirus c proteins. in addition to stat , we show that hpiv -c interacts directly with grb and enhances mapk/erk signaling downstream of egf receptor (egfr). our data give the first example of a paramyxoviridae protein that contributes to the stimulation of egfr and mapk/erk pathway and provides molecular basis to this activity. this pathway has been known for decades as a prime target of dna tumor viruses and oncogenic retroviruses, and its activation represents an essential step toward carcinogenesis [ , ] . but recent data demonstrate that non-oncogenic rna viruses also activate this signaling cascade to support viral replication and spreading [ ] . whether it is activated upon egfr engagement or other means, mapk/erk pathway regulates a multiplicity of cellular processes including proliferation, differentiation, development, cell survival and inflammation. as a consequence, how the activation of mapk/erk pathway promotes viral replication is a complex question. interestingly, two non-oncogenic rna viruses associated with acute respiratory tract infections have been recently reported to modulate the egfr pathway. both human respiratory syncytial virus (hrsv), a member of paramyxoviridae like hpiv , and a rhinovirus that belongs to picornaviridae family activate egfr and mapk/erk pathway [ , ] . infection of epithelial cells by these viruses stimulates the processing and activation of egfr ligands by membrane matrix metalloproteinase and subsequent engagement of egfr through autocrine/paracrine mechanisms. experiments performed on rhinovirus show that viral replication and tlr engagement by viral rna are both required to activate the egfr and mapk/erk pathway [ ] . in this report, we show that hpiv infection also activates mapk/erk pathway in the absence of external stimuli, a phenomenon that possibly relies on the engagement of pathogen recognition receptors. although hpiv -c alone is unable to activate this pathway, our data suggest that expression of this virulence factor enhances mapk/erk activation above normal level in infected cells, thereby contributing to viral replication and pathogenesis. induction of mapk/erk pathway by rna viruses has numerous consequences on cell biology. first, it results in increased expression of inflammatory factors, in particular cytokines and chemokines that recruit cellular effectors of immunity [ , [ ] [ ] [ ] [ ] [ ] . mapk/erk pathway was also reported to block the antiviral response induced by ifn-a/b, making its activation beneficial to virus replication [ ] . another consequence of mapk/erk pathway activation is the induction of mucin production by infected epithelial cells [ , ] . although mucin expression is a critical innate defense system, excessive production of mucus results in the obstruction of airways and delays the elimination of pathogens. finally, it has been demonstrated in vitro that upon hrsv infection, activation of egfr and mapk/erk pathway sustains viral replication by retarding the death of infected cells [ ] . altogether, these data suggest that although a moderate activation of mapk/erk pathway contributes to the innate response against viruses, an excessive activation leads to deleterious inflammation, inhibition of ifn-a/b response, airway obstruction and infected cell survival figure . identification of a minimal hpiv -c region interacting with grb . fragments of hpiv -c were generated and tested for their ability to interact with grb following the procedure described in figure a . (a) the first iteration identified a grb binding region of aa. after two (b), three (c) and four additional rounds (d), this domain was finally reduced to a minimal grb binding motif of aa. this binding domain, encompassing position to , is contained in the stat binding region of hpiv -c previously identified in figure . doi: . /journal.ppat. .g hpiv -c impact on ifn-a/b and egf pathways [ ] . therefore, it is tempting to speculate that hpiv -c interaction with grb and egfr pathway participates in such deregulation of airway epithelium homeostasis to promote hpiv replication and spreading. a consequence of such perturbations could be an aggravation of chronic inflammatory airway diseases like asthma or chronic obstructive pulmonary disease as already suggested by epidemiological links with paramyxoviridae infections and in vivo models [ , ] . besides its effects on immune response, activation of mapk/ erk pathway has direct consequences on viral replication as assessed by in vitro experiments. it is now well documented that mapk/erk pathway inhibition with u or pd deeply impairs the replication of numerous rna viruses including hrsv ( [ ] ; and for review see [ ] ). similarly, we show in this report that mapk/erk pathway inhibition prevents hpiv protein expression in infected cells as assessed by hpiv -hn detection. in influenza virus infected cells, membrane accumulation of influenza virus hemagglutinin (ha) induces lipid-rafts clustering that leads to mapk/erk pathway activation and nuclear export of viral ribonucleoprotein complexes to achieve a and b) hpiv -c - and hpiv -c - were tested for their ability to modulate ifn-a/b or egf signaling. (a) as described in figure , hek- t cells were transfected with pisre-luc and prl-cmv to determine the activation level of ifn-a/b signaling pathway. cells were co-transfected with expression plasmids encoding flag-tagged full-length hpiv -c (c fl ) or fragments. h after transfection, iu/ml of recombinant ifn-b were added. after h, relative luciferase activity was determined. experiments were performed in triplicates, and data represent means sd. (b) as described in figure , cells were transfected with pfa -elk , pgal -uas-luc and prl-cmv to determine the activation level of mapk/erk signaling pathway. cells were co-transfected with expression plasmids encoding full-length hpiv -c (c fl ) or fragments. h later, cells were starved during h and stimulated with egf at a final concentration of ng/ml. after h, relative luciferase activity was determined. experiments were performed in triplicate, and data represent means sd. as a control, relative expression levels of c fl , c - and c - were determined by western blot analysis (upper right panel). (c) full-length hpiv -c, hpiv -c - or hpiv -c - was expressed in fusion downstream of the red fluorescent protein cherry to determine subcellular localization in hek- t cells. h after transfection, cells were fixed with pfa, permeabilized and labeled with dapi to stain nuclei. single confocal sections show cherrytagged protein fluorescence in red and dapi staining in blue. doi: . /journal.ppat. .g viral particles assembly [ ] [ ] [ ] . because hpiv replication cycle is only cytoplasmic, mechanisms involved are necessarily distinct. a possible link between mapk/erk pathway and hpiv protein expression lies in the fact that among downstream targets of this pathway are essential factors of cellular translational machinery. we show that hpiv -c expression enhances the phosphorylation of s and eif e. the small ribosomal subunit protein s is the major phosphoprotein of eukaryotic ribosomes with five phosphorylation sites (ser , ser , ser , ser , and ser ). two families of serine/threonine kinases phosphorylate s in vitro: s k / and p ribosomal s kinase (rsk). recently it has been shown that mapk/erk signaling pathway activates rsk family members that contribute to s phosphorylation on ser / thereby stimulating cap-dependent translation [ ] . in addition, eif e that interacts with the cap structure and brings translation initiation factors together with the small ribosomal subunit via the scaffold protein eif g, undergoes regulated phosphorylation on ser upon mapk/erk pathway activation. this phosphorylation event is dependent on eif gassociated mapk signal-integrating kinases, mnk and mnk [ ] . eif e is believed to be the least abundant of all initiation factors and therefore considered as a perfect target to regulate protein synthesis. even though there is no direct link between eif e phosphorylation and the enhanced translation observed, the fraction of phosphorylated eif e dramatically increases following treatment of the cells with growth factors, hormones and mitogens. therefore, eif e phosphorylation has been associated with increased translation rates. hpiv mrnas are capped and polyadenylated like their host counterparts. thus, s and eif e phosphorylation together with a high level of viral gene transcription may contribute to a rapid switch toward viral protein synthesis within infected cells. specific biochemical investigations are still required to decipher how hpiv -c can both inhibit ifn-a/b signaling and enhance egfr and mapk/erk pathway. when searching the literature for viral proteins that target grb , we found specific reports on ns a from hepatitis c virus and orf from hepatitis e virus [ , ] . although ns a inhibits mapk/erk activation induced by exogenous egf, orf was described as an activator of mapk/erk pathway like hpiv -c. both ns a and orf exhibit a proline-rich motif (pxxp) to bind the src homology (sh ) domains of grb , but there is no such motif in hpiv -c suggesting that other mechanisms mediate stat and grb binding. interestingly, these two cellular proteins exhibit sh domains. such domains typically bind a phosphorylated tyrosine residue in the context of a longer peptide motif within a target protein. although there is no evidence that hpiv -c becomes phosphorylated, we have tested hpiv -c interaction with mutant stat and grb exhibiting sh domains disabled for the interaction with phosphotyrosine residues. these mutants were not affected for the interaction with hpiv -c (data not shown). this suggests that hpiv -c either binds distinct regions of stat and grb , or interacts with a region of the sh domain that does not involve the phosphotyrosine binding site. finally, our results also show that full-length hpiv -c is required to modulate ifna/b and mapk/erk pathways since aa - that bind stat and grb are unable to do so when expressed alone. full-length hpiv -c was also required to observe a localization at the cell membrane, suggesting a link with its activity. interestingly, the n-terminal residues of sendai virus c protein act as a membrane targeting signal [ ] . but the n-terminal residues of hpiv -c (aa - ) were unable to do so, and sequence analysis did not show any conservation with the c protein of sendai virus. thus, hpiv -c tertiary structure is apparently required to target this protein at the cell membrane. this specific localization could both sequester stat to prevent the stimulation of ifn-target genes and contribute to the aggregation of grb -sos complexes to enhance mapk/erk signaling [ ] . altogether, this suggests that hpiv -c interaction with stat and grb represents a potential target for the development of antiviral molecules against hpiv and possibly other members of respirovirus genus. plasmid dna constructs p-encoding sequence from hpiv wild-type strain (df ) was amplified by rt-pcr (titan one tube; roche applied science) from total rna purified from infected cells (rneasy kit; qiagen). amplification was performed using the following hpiv -p specific primers flanked with gateway cloning sites: -gggga-caactttgtacaaaaaagttggcatggaaagcgatgctaaaaactatc-aaa and -ggggacaactttgtacaagaaagttggttattggcaattatt-gacatcttcattgaac. pcr products were cloned using topo ta cloning kit (invitrogen) into topo vector. a total of clones were analyzed to establish the sequence of hpiv -p (genbank id: eu ). interestingly, clones were not edited, clones were edited by the addition of one g residue, and clones were edited by the addition of g residues. one of the plasmids containing the unedited sequence of hpiv -p was selected and subsequently used as a template to clone hpiv -c. dna sequences encoding full-length hpiv -c or fragments corresponding to aa - or - were amplified by pcr from p(hpiv -p)-topo and cloned by in vitro recombination into pdonr (gateway system; invitrogen) as previously described [ ] . similarly, mv-c was amplified from p(+)mv that contains the full genome of measles virus wild-type strain ichinose (kindly provided by dr. k. takeuchi, [ ] ). nipah-c was amplified from niv-p plasmid (kindly provided by dr. tf. wild; [ ] ). grb coding sequence was amplified from the human spleen cdna library used to perform the yeast two-hybrid screen (invitrogen). the pdonr plasmid containing stat was previously described [ ] . viral or cellular coding sequences were subsequently transferred by in vitro recombination from pdonr into different gateway-compatible destination vectors (see below) following manufacturer's recommendation (lr cloning reaction, invitrogen). to perform yeast two-hybrid experiments, coding sequences were recombined into ppc (invitrogen) to be expressed in fusion downstream of the activation domain of gal (gal -ad) or into pdest to be expressed in fusion downstream of the dna binding domain of gal (gal -db). in mammalian cells, gst-tag and flag-tag fusions were achieved using pdest (invitrogen) or pci-neo- flag vector, respectively [ ] . we also used pci-neo (promega) and pmcherry-c (clontech) to express proteins without a tag or in fusion downstream of cherry, respectively. these two plasmids were made gateway-compatible using the gateway vector conversion system (invitrogen). hek- t, hela and vero cells were maintained in dulbecco's modified eagle's medium (dmem; gibco-invitrogen) containing % fetal bovine serum, penicillin, and streptomycin at uc and % co . a and beas- b cells were maintained in f- k medium (gibco-invitrogen) containing % fetal bovine serum, penicillin, and streptomycin at uc and % co . hpiv (strain c ) was amplified and titrated on vero cells following recommendations of atcc (american type culture collection). recombinant mv-egfp virus used in figure s has been hpiv -c impact on ifn-a/b and egf pathways previously described [ ] . infections were performed for h at uc in optimem (gibco-invitrogen). later on, cells were washed and incubated in fresh culture medium for or h. to detect viral replication, cells were recovered and incubated in pbsparaformaldehyde . % for min. after extensive washing with pbs, cells were permeabilized with pbs-triton . % for min, and then incubated with a monoclonal antibody specific to hpiv -hn (m , abcam). cells were washed and incubated in the presence of an anti-mouse cy -conjugated antibody (jackson immunoresearch). after extensive washing, cellular immunostaining was analyzed using a facscalibur flow cytometer (bd). when specified, cells were pre-treated with mek / specific inhibitor u ( mm final; promega) for h before, during and after infection to study the impact on hpiv infection. to perform co-affinity purification experiments, cloned orfs were transferred from pdonr to pdest expression vector (invitrogen) to achieve gst fusion, and to pci-neo- flag vector [ ] for flag-fusion. cell transfections were performed using lipofectamine (invitrogen). unless specified otherwise, hek- t cells were dispensed in each well of a -well plate, and transfected h later with ng of each plasmid dna per well. two days post transfection, hek- t cells were washed in pbs, then resuspended in lysis buffer ( . % nonidet p- , mm tris-hcl at ph , mm nacl and mm edta) supplemented with complete protease inhibitor cocktail (roche). cell lysates were incubated on ice for min, and then clarified by centrifugation at , g for min. for pull-down analysis, mg of protein extracts were incubated for h at uc with ml of glutathione-sepharose beads (amersham biosciences) to purify gst-tagged proteins. beads were then washed times in ice-cold lysis buffer and proteins were recovered by boiling in denaturing loading buffer (invitrogen). purified complexes and protein extracts were resolved by sdspolyacrylamide gel electrophoresis (sds-page) on - % nupage bis-tris gels with mops running buffer (invitrogen), and transferred to a nitrocellulose membrane. proteins were detected using standard immunoblotting techniques. flagand gst-tagged proteins were detected with a mouse monoclonal hrp-conjugated anti- flag antibody (m ; sigma-aldrich) and a rabbit polyclonal anti-gst antibody (sigma-aldrich), respectively. specific antibodies were used to detect endogenous stat (clone- ; bd biosciences), grb (clone- ; bd biosciences), phospho-erk / (clone- d ; upstate), erk / (ct; upstate), phospho-eif e (ser ; cell signaling), eif e (cell signaling), phospho-s - (ser / ; cell signaling), s (clone- d ; cell signaling) and hpiv -hn (m ; abcam). secondary anti-mouse and anti-rabbit hrp-conjugated antibodies were from ge-healthcare. densitometric analysis of the gels was performed using a specific module of photoshop cs extended (adobe systems inc.). yeast two-hybrid screening and gap-repair procedure our yeast two-hybrid protocols have been described in details elsewhere [ ] . briefly, pdest plasmid encoding gal -db fused to hpiv -c was transformed in ah yeast strain (clontech), and used to screen by mating a human spleen cdna library cloned in the gal -ad ppc vector (invitrogen) and previously established in y yeast strain (clontech). yeast cells were plated on a selective medium lacking histidine and supplemented with mm -amino-triazole ( -at; sigma-aldrich) to select for interaction-dependent transactivation of his reporter gene. ad-cdnas from [his+] colonies were amplified by pcr and sequenced to identify the host proteins interacting with hpiv -c. the gap-repair procedure was used to map the minimal portion of hpiv -c interacting with stat and grb . as previously described [ ] , both forward and reverse pcr primers were designed along the sequence of hpiv -c and fused to specific tails allowing yeast-based recombination in gal -db two-hybrid vector. matrix combinations of forward and reverse primers were used to amplify fragments of hpiv -c by pcr. ah yeast cells expressing ad-fused stat or grb were co-transformed with ml of each pcr product in the presence ng of linearized pdest vector to achieve recombinatorial cloning by gaprepair. fragments of hpiv -c fused to gal -db were then tested for interaction with ad-stat or ad-grb by plating yeast cells on selective medium lacking histidine and supplemented with mm of -at. luciferase reporter gene assay hek- t, hela or vero cells were plated in -well plates ( cells per well). one day later, cells were transfected with either pisre-luc ( . mg/well; stratagene) or pfa -elk ( . mg/well; stratagene) and pgal -uas-luc plasmids ( . mg/ well; provided by dr. y. jacob) together with prl-cmv reference plasmid ( . mg/well; promega). cells were simultaneously cotransfected with . mg/well of pci-neo- flag, pci-neo or pmcherryc expression vectors encoding viral proteins as specified. h after transfection, cells were stimulated with ifnb (biosource) at iu/ml or starved for h then stimulated with egf (upstate) at ng/ml. h post transfection, cells were lysed, and both firefly and renilla luciferase activities in the lysate were determined using the dual-luciferase reporter assay system (promega). reporter activity was calculated as the ratio of firefly luciferase activity to reference renilla luciferase activity, and normalized so that positive control activity equals . when indicated, cells were treated with u (promega) at mm final concentration upon egf stimulation. subcellular localization of cherry-tagged hpiv -c fl , c - and c -well plates containing coverslips were seeded with hek- t cells ( cells per well). one day later, cells were transfected with pmcherryc expression vector alone or encoding hpiv -c fl , hpiv -c - or hpiv -c - . h after transfection, cells were incubated with pbs-pfa % for min at rt, then treated with pbs-triton . % for min at rt to permeabilize the cells. finally, cells were incubated for min at rt in a pbs-pfa % solution containing dapi ( - -diamidino- -phenylindole) at mg/ml. preparations were mounted using fluoromount-g (southernbiotech), and imaging performed using a sp confocal miscroscope (leica). figure s erk / phosphorylation is enhanced by hpiv infection in a cells. a cells were infected with hpiv (moi = ) and after h, cells were starved for h before stimulation with ng/ml of egf. phosphorylation of erk / was determined by western blot analysis at min, min and h post stimulation. hpiv infection was confirmed by anti-hpiv hemagglutinin-neuraminidase (hpiv -hn) immunoblotting. found at: doi: . /journal.ppat. .s ( . mb tif) hpiv -c impact on ifn-a/b and egf pathways figure s mek / inhibitor u has no effect on mv protein synthesis. hek- t cells were left untreated (a) or treated with mm of u for h (b). then, cells were mocktreated or infected with a recombinant mv strain expressing egfp (moi = ) and cultured with or without u (a and b, respectively). h after infection, egfp expression was quantified by flow cytometry analysis. found at: doi: . /journal.ppat. .s ( . mb tif) pathogenic viruses: smart manipulators of the interferon system how cells respond to interferons ) fields' virology naturally occurring parainfluenza virus infection in adults induces mild exacerbation of asthma associated with increased sputum concentrations of cysteinyl leukotrienes bovine parainfluenza virus type accessory proteins that suppress beta interferon production rna editing in the phosphoprotein gene of the human parainfluenza virus type sendai virus wild-type and mutant c proteins show a direct correlation between l polymerase binding and inhibition of viral rna synthesis paramyxovirus sendai virus c proteins are essential for maintenance of negative-sense rna genome in virus particles sendai virus c protein plays a role in restricting pkr activation by limiting the generation of intracellular double-stranded rna aip /alix is a binding partner of sendai virus c protein and facilitates virus budding sendai virus budding in the course of an infection does not require alix and vps a host factors recruitment of alix/aip to the plasma membrane by sendai virus c protein facilitates budding of virus-like particles c and v proteins of sendai virus target signaling pathways leading to irf- activation for the negative regulation of interferon-beta production activation of the beta interferon promoter by unnatural sendai virus infection requires rig-i and is inhibited by viral c proteins sendai virus c protein physically associates with stat all four sendai virus c proteins bind stat , but only the larger forms also induce its mono-ubiquitination and degradation importance of the anti-interferon capacity of sendai virus c protein for pathogenicity in mice inhibition of stat phosphorylation by human parainfluenza virus type c protein mutations in the c, d, and v open reading frames of human parainfluenza virus type attenuate replication in rodents and primates paramyxoviridae use distinct virus-specific mechanisms to circumvent the interferon response specificity in signal transduction: from phosphotyrosine-sh domain interactions to complex cellular systems phosphorylation of the cap-binding protein eif e by the mapk-activated protein kinase mnk ras/erk signaling promotes site-specific ribosomal protein s phosphorylation via rsk and stimulates cap-dependent translation epidermal growth factor system regulates mucin production in airways activation of the epidermal growth factor receptor by respiratory syncytial virus results in increased inflammation and delayed apoptosis multiple tlrs activate egfr via a signaling cascade to produce innate immune responses in airway epithelium rhinovirus-induced major airway mucin production involves a novel tlr -egfr-dependent pathway epidermal growth factor receptor-mediated innate immune responses and their roles in airway diseases negative regulation of the alpha interferon-induced antiviral response by the ras/raf/mek pathway identification of a novel inhibitor of mitogen-activated protein kinase kinase rna viruses and the mitogenic raf/mek/erk signal transduction cascade the stat activation process is a crucial target of sendai virus c protein for the blockade of alpha interferon signaling inhibition of interferon induction and signaling by paramyxoviruses the c protein of measles virus inhibits the type i interferon response newcastle disease virus (ndv)-based assay demonstrates interferon-antagonist activity for the ndv v protein and the nipah virus v, w, and c proteins stringent requirement for the c protein of wild-type measles virus for growth both in vitro and in macaques regulation of interferon signaling by the c and v proteins from attenuated and wild-type strains of measles virus the rinderpest virus non-structural c protein blocks the induction of type interferon sendai virus c proteins counteract the interferon-mediated induction of an antiviral state knockout of the sendai virus c gene eliminates the viral ability to prevent the interferonalpha/beta-mediated responses human parainfluenza virus type but not sendai virus replicates in human respiratory cells despite ifn treatment the egfr as a target for viral oncoproteins mapk activation is involved in posttranscriptional regulation of rsv-induced rantes gene expression role of mitogen-activated protein kinases in influenza virus induction of prostaglandin e from arachidonic acid in bronchial epithelial cells ebola virus-like particle-induced activation of nf-kappab and erk signaling in human dendritic cells requires the glycoprotein mucin domain japanese encephalitis virus infection stimulates src tyrosine kinase in neuron/glia spike protein of sars-cov stimulates cyclooxygenase- expression via both calcium-dependent and calcium-independent protein kinase c pathways the causal direction in the association between respiratory syncytial virus hospitalization and asthma persistent activation of an innate immune response translates respiratory viral infection into chronic lung disease erk- / activity is required for efficient rsv infection influenza virus propagation is impaired by inhibition of the raf/mek/erk signalling cascade membrane accumulation of influenza a virus hemagglutinin triggers nuclear export of the viral genome via protein kinase calpha-mediated activation of erk signaling clustering of raftassociated proteins in the external membrane leaflet modulates internal leaflet h-ras diffusion and signaling the orf protein of hepatitis e virus binds to src homology domains and activates mapk ns a, a nonstructural protein of hepatitis c virus, binds growth factor receptor-bound protein adaptor protein in a src homology domain/ligand-dependent manner and perturbs mitogenic signaling targeting of the sendai virus c protein to the plasma membrane via a peptide-only membrane anchor aggregation of membrane proteins by cytosolic cross-linkers: theory and simulation of the lat-grb -sos system measles virus v protein blocks jak -mediated phosphorylation of stat to escape ifn-alpha/beta signaling recovery of pathogenic measles virus from cloned cdna establishment of a nipah virus rescue system human papillomavirus type e oncoprotein represses the transforming growth factor beta signaling pathway by binding to smad a molecularly cloned schwarz strain of measles virus vaccine induces strong immune responses in macaques and transgenic mice inhibition of ifn-alpha/beta signaling by two discrete peptides within measles virus v protein that specifically bind stat and stat we would like to thank dr. k. takeuchi and dr. tf. wild for kindly providing p(+)mv and niv-p plasmids, respectively. we thank all members of pf -pasteur genopole sequencing core facility. we thank members of the infection-mapping project i-map for fruitful discussions. we thank dr. m. mesel-lemoine and dr. j. pothlichet for their technical support. we thank miss r. parker for proofreading the manuscript. key: cord- - datxv j authors: gronvall, gigi kwik; waldhorn, richard e; henderson, d. a title: the scientific response to a pandemic date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: datxv j nan t omorrow, the world could face a pandemic. it could be due to h n avian influenza-experts warn that it is just a matter of time-or a different influenza strain, an unknown pathogen, or a bioterrorist attack. in all cases, biological scientists face the challenge of characterizing the pathogen and determining how to control it. their scientific judgments and public statements will shape the global pandemic response. during the sars outbreak in , scientists generated information that influenced everything from medical treatments to travel restrictions, trade policy, and political decisions. given the importance of getting scientific information out into the world, scientists should consider now how they will respond and communicate in the setting of the next pandemic. in hopes of sparking that discussion, the center for biosecurity at the university of pittsburgh medical center organized an international conference on biosafety and biorisks (http://upmc-biosecurity.org/pages/events/biosafety/ report.html) in collaboration with the world health organization (who) communicable disease surveillance and response office in . more than scientists and public health practitioners from countries gathered in lyon, france, to hear speakers from the who, the european commission, scientific journals, interpol, and public health networks-many of the institutions and individuals who will likely play key roles in the global response to the next pandemic. by discussing the biosafety and biosecurity challenges presented by past epidemics such as sars, participants recognized the importance of scientific and public health collaboration in combating disease-and the need to plan. each epidemic is different, and will have its own scientific and political dimensions that make planning difficult. however, based on past epidemics, there are some likely patterns. researchers will need to share biological samples between laboratories, sometimes internationally; decision makers and journalists will want the latest information, which may not be peer reviewed; and researchers will risk contracting the disease they research, which could then spread outside of the laboratory. scientists need to harmonize and modernize standards and training in these areas, to help make their response to a pandemic prompt, accurate, and safe. scientists need access to samples from patients and laboratories in order to conduct research and public health surveillance, as well as to develop diagnostic tests. not every researcher who requests a strain for research in the midst of a pandemic is likely to get it, but international standards for documenting, referencing, tracking, and shipping samples would save valuable time in a pandemic. for example, during the sars outbreak in , laboratories with access to samples were not always willing to share with other, potentially competing, laboratories. shipping samples was also a problem. in one report, ''it took eight weeks to determine the protease structure [of the sars coronavirus]. but half that time was taken in obtaining the dna for the viral proteins from collaborators. . .and getting it through customs'' [ ] . sharing samples can be difficult even within the same research institution. many clinical laboratories cannot release patient samples without preapproved protocols from an institutional review board. universities should consider how to make it easier for their researchers to comply with institutional review board regulations [ ] , including developing emergency institutional review board protocols. h n -sample access problems have already begun due to slow responses for shipping samples to international reference laboratories. noted virologists from universities outside of the who laboratory network have also had difficulty getting access to samples and thus contributing their expertise [ ] . bioterrorism problem, as japan and hong kong require permits for handling highly pathogenic viruses, including h n [ ] . in the us, h n is a select agent, so the department of agriculture must certify laboratories before they can receive the virus [ ] . however, improved standards for tracking samples, if correctly applied, could bring benefits to biosecurity and also help researchers. according to a who official, ''during the [sars] outbreaks, lots of samples were taken...and we don't know where they all are'' [ ] . sars may no longer be infecting people naturally, but there is risk that a laboratory sample could end up in improperly trained or malevolent hands. h n could be similarly dangerous. in a time when one can track a package from warehouse to mailbox, the current methods for keeping track of vital biological samples are grossly out of date. scientists, journal editorial boards, and other scientific professional organizations should consider creating standards for communicating scientific results, so decision makers can make use of the information and scientists can get professional recognition for their work. traditionally, scientists communicate results through conferences and peer-reviewed publications. however, during the sars epidemic, it became common to announce results in a press conference. sharing results with the press may save time, but there are problems with this approach: the reports are not peer reviewed and newspapers do not usually give in-depth technical reports of experimental results, which limit the usefulness of news reports to researchers. publication in scientific journals also poses problems. while journals provide peer review and professional recognition for the authors, publication is often slow. in the absence of hard data, some reporters cover the reactions of people who have seen papers under review-for example, ''researchers who have seen [the] data say they are convinced'' [ ] -rather than the facts themselves. one way to speed dissemination of results is for scientific journals to agree to peer review articles of importance within hours, before web posting. reviewers should also have strict deadlines so that they do not delay important information being made public. accidents happen, and working with infectious agents will never be risk free. however, a lack of training, mentoring, and formal standards for biosafety leaves researchers at unnecessary risk of infection, and has had tragic effects outside of the laboratory. there are no international standards for biosafety, but there are guidelines from the who and the centers for disease control and prevention that could be developed into formal training for all researchers. the consequences of not adhering to laboratory biosafety guidelines can be dire. some experts believe that the dominant strain of flu in originated from a laboratory [ ] . during the sars outbreak, there were four documented laboratory accidents in three laboratories-singapore, taiwan, and china [ ] . in each case, the laboratories had the best equipment, but training and experience with pathogens were lacking. the public may not easily forgive the scientific community at large for an epidemic that starts in a laboratory. the scientific community has a responsibility to take reasonable precautions to prevent accidents and to train researchers in safety. accidents should be documented, and the lessons learned from those accidents should be incorporated into safety training. if scientists do not promote biosafety, regulations could be imposed on them. the strict safety regulations proposed for laboratories in boston, for example, directly result from three laboratory-acquired cases of tularemia [ ] . in the event of an avian flu pandemic, it is likely that the who will coordinate the international response, as they did with sars. however, no one organization has the resources and expertise to deal with all of the scientific and medical complexities that a pandemic will present. the who relies on a variety of information networks and laboratories, such as the global public health intelligence network, which gathers reports of disease outbreaks in seven languages, and promed-mail, which is an open-source electronic reporting system for disease outbreaks. a similar set of open-source electronic systems for scientists to convey information and collaborate could also amplify who scientific expertise and the availability of hard data for decision making in a pandemic. scientific information fuels and directs the response to epidemics. public health professionals, clinicians, politicians, journalists, and members of the public will make critical decisions based on what is known about a disease as an outbreak unfolds. scientists can take this opportunity, before doi: . /journal.ppat. .g transmission electron micrography ( , ) reveals two avian influenza a (h n ) virions (image: c. goldsmith, j. katz, cdc) the next pandemic, to plan to give accurate information to those who need it as fast and as safely as possible. " company says it mapped part of sars virus researchers break the rules in frustration at review boards avian influenza. who controls the samples? safeguarding the united states from highly-pathogenic avian influenza (hpai): usda actions, plans, and capabilities for addressing the bird flu threat sars case in lab worker: taiwan man working in military lab contracted the virus in early december. the scientist tracking the roots of a killer influenza: a developing crisis infectious diseases. second lab accident fuels fears about sars regulations readied for research labs the authors would like to thank bradford a. kay, gué naë l r. rodier, and the world health organization communicable disease surveillance and response office in lyon, france, for collaborating on the conference, as well as the nuclear threat initiative for their support.funding. the authors received no specific funding for this article.competing interests. the authors have declared that no competing interests exist. key: cord- -qaogpxy authors: too, issac horng khit; bonne, isabelle; tan, eng lee; chu, justin jang hann; alonso, sylvie title: prohibitin plays a critical role in enterovirus neuropathogenesis date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: qaogpxy a close relative of poliovirus, enterovirus (ev ) is regarded as an important neurotropic virus of serious public health concern. ev causes hand, foot and mouth disease and has been associated with neurological complications in young children. our limited understanding of the mechanisms involved in its neuropathogenesis has hampered the development of effective therapeutic options. here, using a two-dimensional proteomics approach combined with mass spectrometry, we have identified a unique panel of host proteins that were differentially and dynamically modulated during ev infection of motor-neuron nsc- cells, which are found at the neuromuscular junctions where ev is believed to enter the central nervous system. meta-analysis with previously published proteomics studies in neuroblastoma or muscle cell lines revealed minimal overlapping which suggests unique host-pathogen interactions in nsc- cells. among the candidate proteins, we focused our attention on prohibitin (phb), a protein that is involved in multiple cellular functions and the target of anti-cancer drug rocaglamide (roc-a). we demonstrated that cell surface-expressed phb is involved in ev entry into neuronal cells specifically, while membrane-bound mitochondrial phb associates with the virus replication complex and facilitates viral replication. furthermore, roc-a treatment of ev -infected neuronal cells reduced significantly virus yields. however, the inhibitory effect of roc-a on phb in nsc- cells was not through blocking the craf/mek/erk pathway as previously reported. instead, roc-a treated nsc- cells had lower mitochondria-associated phb and lower atp levels that correlated with impaired mitochondria integrity. in vivo, ev -infected mice treated with roc-a survived longer than the vehicle-treated animals and had significantly lower virus loads in their spinal cord and brain, whereas virus titers in their limb muscles were comparable to controls. together, this study uncovers phb as the first host factor that is specifically involved in ev neuropathogenesis and a potential drug target to limit neurological complications. a a a a a enterovirus (ev ) is a non-enveloped, positive-sense, single-stranded rna virus, and causes hand, foot and mouth disease (hfmd) in humans. being a close relative of poliovirus, ev is deemed as an important neurotropic virus worldwide [ ] . since its first isolation in california in , several major outbreaks have been reported in china, singapore, korea, and japan [ ] [ ] [ ] [ ] . although the clinical manifestations are generally mild and self-limiting, including hfmd and herpangina, severe neurological complications have been consistently reported with ev -associated infections, causing brainstem encephalitis, acute flaccid paralysis, pulmonary edema and cardiopulmonary failure [ , ] . in addition, some patients who have recovered from severe disease have been reported to develop long term neurologic and psychiatric disorders [ ] . there are currently no effective prophylactic or therapeutic agents against ev . although several vaccines have completed phase iii clinical trials [ ] , regulatory issues may limit their widespread utilization. in addition, as these vaccine candidates consist of inactivated virus from a single ev genotype (c ), cross-protection against other genotypes may be limited [ , ] . the increasing awareness of life-threatening ev infections has boosted research in recent years to further understand virus-host interactions and develop effective antiviral strategies [ ] [ ] [ ] [ ] [ ] [ ] . however, the neuropathogenesis of ev is still poorly understood. infection occurs when the virus enters the body upon ingestion and/or inhalation. the virus multiplies initially in the alimentary tract mucosa and rapidly reaches the deep cervical and mesenteric lymph nodes via the tonsils and peyer's patches [ ] . after a short transient systemic dissemination phase, the virus accumulates and actively replicates in muscles where it is believed to infect motor neurons at the neuromuscular junctions. experimental evidence supports that ev migrates to the brainstem via retrograde axonal transport as previously described for its close relative poliovirus [ , , [ ] [ ] [ ] . however, the molecular mechanisms involved in ev infection of motor neurons to access the central nervous system (cns) have not been studied. indeed in vitro studies aiming at studying ev neurovirulence have employed neuroblastoma cell lines that may not reflect accurately infection in motor neurons. to address this gap, we have recently reported a novel in vitro model of ev infection in the murine motor neuron cell line nsc- [ ] . nsc- cells originate from the fusion between murine neuroblastoma and spinal cord cells, and possess motor neuron-like properties, such as generation of action potentials and production of acetylcholine [ ] , therefore making it a relevant model to study the mechanism of ev neuropathogenesis. we demonstrated that nsc- cells are permissive to ev clinical isolates and found that, unlike any other mammalian cell types so far reported, ev -infected nsc- cells do not undergo apoptosis and lysis. instead we showed that the virus exits the cells via a non-lytic mode, a phenomenon that has also been previously described for poliovirus [ , , ] . these unique features thus suggested that the infection cycle of ev in nsc- cells involves host pathways and partners that are likely to be different from those previously identified in other mammalian cell types such as muscle cells and neuroblastoma cells. in this work, using a proteomics approach coupled with mass spectrometry, we have identified a panel of cellular proteins that were dynamically regulated during ev infection of nsc- cells. among the host protein candidates that were up-regulated, we focused our attention on prohibitin (phb) and characterized its role during ev infection in nsc- cells. we also demonstrated the importance of phb during ev infection in a symptomatic mouse model of ev infection. to identify the host proteins involved in ev infection cycle in nsc- cells, a de proteomic approach was undertaken. nsc- cells were infected with ev at m.o.i. , and the cell lysates were harvested at , , and hours for downstream proteomic analysis in which a range of - spots were resolved. by using pdquest -d analysis software (biorad), a total of protein spots (fig a) that displayed at least . -fold differential expression (p< . , two-tailed student's t-test) compared to uninfected controls, were excised for in-gel digestion and maldi-tof ms analysis. the peptide fingerprints were then searched against ncbinr mouse genome database for protein identification using mascot program (http://www.matrixscience.com/). the protein candidates were then categorized based on their primary functional class indicated in uni-protkb database (s table) . to illustrate the dynamic regulation of host proteins during the viral infection, a heat map was generated using multiexperiment viewer (mev), with the distance between proteins represented by euclidean average linkage clustering (fig b) . this clustering analysis revealed that proteins that were up-regulated (fig c) during infection are mainly involved in motility ( %) and catalytic processes ( %), while proteins that participate in rna processing ( %) and energy biosynthesis ( %) generally displayed a down-regulation trend during the course of infection (fig d) . functional interactions among the selected host proteins were analyzed by string (search tool for the retrieval of interacting genes/proteins). this platform allows establish proteinprotein interactions based on published literature, online databases, predicted functional associations using genomic information or observations made with other organisms [ ] . the protein network obtained was significantly enriched with the p value of less than . , suggesting that the interactions are highly associated and unbiased (fig ; s table) . furthermore, some of the selected host proteins appear to have strong associations among each other as indicated by the thickness of connecting lines which reflects the confidence level of the interactions [ ] . using go annotations for biological processes, molecular functions, cellular compartments and protein classes, the protein candidates were localized within the cytoplasm ( . %), organelles ( . %) and macromolecular complexes ( . %) (s a fig). in addition, they were found to be involved in various biological processes including mitochondrial biogenesis, proteolytic activity, cytoskeletal machinery and rna processing (s a fig) , consistent with the protein clusters observed in the string network (fig ) . finally, molecular function analysis indicates that majority of these proteins contribute to nucleic acid binding transcription factor activity ( . %), structural molecule activity ( %) or binding ( . %) (s a fig). a meta-analysis with other selected proteomic studies of ev -infected muscle and neuronal cells [ ] [ ] [ ] , [ ] [ ] [ ] revealed minimal overlap between nsc- , rd and other neuronal cell types with protein candidates only, namely actb, tubb, pdia and eno , suggesting that the host-pathogen interactions in nsc- cells are unique (s b fig). the limited overlap may also be partly explained by differential proteomics approaches. act and tubb are involved in maintaining cytoskeletal structure, and they are found highly modulated during viral infection to facilitate virus internalization and transportation [ ] [ ] [ ] . eno has been shown previously to interact with cytoskeletal proteins in intermediate filaments framework rearrangement [ ] . on the other hand, pdi functions in catalyzing reduction and oxidation processes and protein folding [ ] . it has also been demonstrated to be involved in humoral immune response [ ] or viral replication (denv) [ ] and entry (hiv) [ ] . not surprisingly, greater overlap was seen between motor-neuron nsc- and other neuronal cells ( shared hits) than between nsc- and rd cells ( shared hits). importantly, neuro-specific proteins such as prph and uchl were only identified from profiling studies in nsc- and other neuronal cells, thus validating our de proteomic approach. seven protein candidates namely, alpha-enolase (eno ), dep domain-containing mtorinteracting protein (deptor), peripherin (prph), phosphatidylethanolamine-binding protein (pebp ), prohibitin (phb), stomatin-like protein (stoml ) and protein disulfideisomerase a (pdia ) were selected for validation of the proteomic findings by gene knockdown. these host proteins have been previously shown to be associated with various steps in the life cycle of viruses, such as entry [ ] [ ] [ ] [ ] and replication [ , , ] , or to be involved in autophagy [ ] [ ] [ ] and axonal transport [ ] [ ] [ ] . silencing of each selected gene target was achieved by reverse transfecting the on-targetplus sirna smartpool into nsc- cells, prior to viral infection. sirna smartpools consist of four highly potent gene-specific sirna molecules which have been modified to minimize off-target activity and enhance gene specificity [ ] . cytotoxicity of the sirnas smartpools was first established. apart from the sirna pool targeting pdia , no significant cytotoxicity was observed with the other sirna pools at and nm with cell viabilities greater than the % viability threshold (fig a) . the pdia -specific sirna pool concentrations were lowered to and nm to avoid cytotoxicity (fig a) . virus titers in the culture supernatants of sirna-transfected cells were then determined at h.p.i. results indicated that silencing of stoml , prph, phb and deptor led to significantly lower virus titers, whereas pdia-, pebp -and eno -knocked down resulted in increased viral titers in the supernatants of ev -infected nsc- cells compared to control (fig b) . importantly, both trends were dose-dependent. therefore, these results validate the d-proteomics approach as a powerful way to identify host proteins that play a role during ev infection in nsc- cells. prohibitins belong to a highly conserved protein family present in unicellular and multicellular eukaryotes [ ] . prohibitin (phb; bap- ) and prohibitin (phb , rea, bap- ) are two highly homologous members of this family and are ubiquitously expressed in multiple cellular compartments including the mitochondria, nucleus, and the plasma membrane. prohibitins have been involved in multiple cellular functions including cell proliferation and maintenance of the functional integrity of the mitochondria [ ] . in addition, phb specifically has been previously reported to be involved in the entry step of alphavirus chikungunya (chikv) [ ] , and flaviviruses dengue (denv) [ ] and hepatitis c (hcv) [ ] , and to interact with envelope proteins from white spot syndrome virus to prevent infection [ ] . however, there has been no report so far on the role of phb during ev infection. first, modulation of phb expression during ev infection in nsc- cells was confirmed by western blot and showed an overall up-regulation of phb during the course of infection compared to uninfected control (s fig). next, the impact of phb gene silencing on virus production was further analyzed using a wider range of sirna pool concentrations including , , and nm. efficacy of the gene silencing was assessed by western blot and showed a dose-dependent decrease of phb expression in nsc- cell lysates (fig c) . this dose-dependent phb knockdown correlated well with a dose-dependent reduction in the viral titers measured in the culture supernatant (fig d) , therefore supporting the role for phb in ev infection cycle. to address the possibility of false positive or off-target effects of the sirna pool, phb gene silencing was performed with the individual sirnas species from the pool. western blot showed that each individual sirna was capable of silencing phb expression significantly (s a . the viral titers in the culture supernatants were determined by plaque assay at h.p.i.. cell viability of the transfected cells was assessed using alamarblue assay. statistical analysis was performed using two-tailed student's t-test ( Ãà p< . , ÃÃà p< . ). relative band quantification (below western blot) was determined by imagej, by normalizing to loading control, β-actin. error bars represent mean ± standard deviation. one representative of two biological repeats is shown. to determine if phb mediates entry of ev into nsc- cells, a competition assay was performed using commercially available anti-phb antibody. incubation of nsc- cells with anti-phb antibody prior to infection led to significant reduction of the viral titer in a dosedependent manner (fig a) . to demonstrate a physical interaction between cell surfaceexpressed phb and ev , a proximity ligation assay (pla) was performed. in this assay, phb and ev are recognized by specific primary antibodies raised in two different species, which are in turn recognized by species-specific secondary antibodies conjugated to a probe and a target, respectively. should ev and phb be in close proximity, the probe and the target ligate, amplify and result in emission of a fluorescent signal. scarb- which has been previously demonstrated as the main receptor for ev in rd cells [ ] was used as positive control and a red fluorescent signal was readily detected in ev -infected rd cells (fig b) . a positive signal was also detected with nsc- cells incubated with anti-phb and anti-ev antibodies thus supporting the close proximity between ev virus particles and surfaceexpressed phb (fig b) . in contrast, and expectedly, no signal was detected with mouse scarb- (mscarb ) and ev antibodies (fig b) , since we have shown previously that entry of ev into nsc- cells is not mediated by mscarb- [ ] . the physical interaction between ev and cell surface-expressed phb was further assessed by performing a co-immunoprecipitation experiment. nsc- cells were incubated with ev for hours at ˚c to allow viral adsorption onto the cell surface but no internalization. the total cell lysate was obtained and a pulldown was carried out using antibody specific to phb or an isotype igg antibody control. the immunoprecipitates were then analyzed by western blot using anti-ev primary antibody. a discrete band at the expected size was obtained when pulldown was performed with the anti-phb antibody whereas no band was seen when pulldown was done with the igg isotype (fig c) . these findings thus support that ev physically interacts with cell surface-expressed phb, and suggest that phb may serve as a receptor for ev entry into nsc- cells. in addition to its association to the plasma membrane at the cell surface, phb is also present intracellularly [ ] . to study the role of intracellular phb in ev infection cycle, phbknocked down nsc- cells were transfected with ev rna genome and the viral titers were determined at , , and hours post-transfection, in order to assess virus production within a single cell infection cycle. transfection of viral genome was meant to bypass the virus entry step which we have shown involves cell surface-expressed phb. no viral titer was obtained at h.p.t. in the phb-knocked down and control cells (fig a) . from h.p.t onwards the viral titers detected in the culture supernatant from phb-knocked down cells were consistently lower than those measured in sintc or non-treated cells (fig a) . this result thus demonstrates that phb plays a role in the intracellular virus infection cycle. to further confirm this hypothesis, a luciferase ev (lucev ) replicon transfection assay was performed. in this replicon, the viral structural genes have been replaced with a luciferaseencoding gene while the other parts of the viral genome are retained (fig b) . upon transfection, the replicon undergoes a single replication cycle with no production of virus progeny since it is deficient in viral structural proteins. the luminescence measured from the cell culture is proportional to the amount of luciferase produced inside the cell thereby reflecting the replication activity of the replicon. here, a significant reduction in the luminescence signal was observed in phb-knocked down nsc- cells compared to sintc-treated and nontreated cells (fig c) . thus, together the data support that intracellular phb is involved in ev viral replication. ) were incubated with ev at ˚c for hours to allow viral adsorption, prior to pla staining. scarb -stained nsc- and rd cells served as negative and positive controls, respectively. scale bar represents μm. a representative experiment of two independent repeats is shown. (c) co-immunoprecipitation of ev and surface-expressed phb. ev and nsc- cells were co-incubated for hours at ˚c. the cell lysate was pulled down with anti-phb or igg isotype control antibodies prior to immunoblotting using anti-vp primary antibody. mock-infected cells and igg isotype served as control. a representative experiment of two independent repeats is shown. ip, immunoprecipitation; ib, immunoblot. to further investigate the role of intracellular phb during ev infection cycle, immunostaining was performed on ev -infected nsc- cells probing for phb, ev capsid proteins vp /vp and the viral replication intermediate dsrna. co-localization between phb and dsrna, and between phb and ev capsid proteins was readily observed (fig a) , supporting that intracellular phb could be involved in the viral replication and/or assembly processes. to further study the role of intracellular phb in viral replication, co-immunoprecipitation was carried out using antibody against phb. pull down with anti-phb antibody followed by western blot using anti-ev d/ cd antibody led to the detection of a kd band that corresponds to the ev cd protein complex and a kda band ( d polymerase) which comigrated with igg heavy chain (fig b) . taken together, these data support a physical interaction between intracellular phb and ev non-structural proteins d and cd, indicating that intracellular phb is likely involved in viral replication. previous studies have shown that the main replication sites of picornavirus are located at the golgi apparatus and endoplasmic reticulum (er) [ ] . we have demonstrated that intracellular phb co-localizes with dsrna and is closely associated with the ev d polymerase. given that intracellular phb is abundantly and mainly expressed on mitochondria [ ] , we speculated that in nsc- cells mitochondria may be exploited by ev as replication site. consistently, co-localization of phb and ev with mitochondria was observed by ifa (fig a and b) . furthermore, co-localization of phb, dsrna and mitochondria was also readily detected, thus indicating that mitochondrial phb is associated with the viral replication (fig c) . to exclude the possibility that the replication complexes detected were actually associated to the er, which is in close proximity to mitochondria, the mitochondrial fraction was prepared from ev -infected nsc- cells and western blot analysis revealed the presence of viral capsid protein vp ( kd), d ( kd) and cd ( kd) proteins (fig d) . furthermore, the mitochondrial fraction was shown to be free of cytoplasmic contamination, as evidenced by the presence of mitochondrial marker (atpb, kd) and lack of er marker (calreticulin, kd). similar observation was made with the mitochondrial fraction prepared from ev -infected rd cells (fig d) , suggesting that ev is able to exploit various cellular organelles as replication scaffolds in various mammalian cell types from different species. this finding is consistent with a previous study where ev vp was found to be associated with mitochondria in hela cells [ ] . to further support the close proximity and likely interactions between the viral replication complexes and mitochondria, transmission electron microscopy was performed on ev -infected nsc- cells. clustering of mitochondria surrounding viral replication complexes could be seen (electron-dense like structures) in the infected cells, and examination at a higher magnification indicated a close association between mitochondria membrane and virus complex/virus particle (fig e) . collectively, the data strongly indicate that mitochondria in nsc- cells are exploited by ev as a replication scaffold and that mitochondria-associated phb plays a role in this process. recent studies have shown that phb activity is inhibited by a group of phytochemicals called rocaglamides, which are derived from the traditional chinese medicinal plants aglaia [ , [ ] [ ] [ ] . we therefore investigated whether roc-a could interfere with ev infection cycle by blocking phb activity in nsc- cells. incubation of roc-a with virus prior to nsc- cell infection (co-treatment) did not result in any significant reduction in viral titer (s a fig) . when cells were pre-treated with roc-a prior to ev infection (pre-treatment), less than log pfu/ml of decrease in viral titer was observed at the highest drug concentration only ( nm) (s b fig). in contrast, a dose-dependent decrease in the viral titer was seen when roc-a treatment was applied after infection (post-treatment) at concentrations ranging between - nm (fig a) . western blot analysis of the cell lysates further confirmed the dose-dependent reduction of intracellular viral capsid protein and phb (fig b) . taken together, the data suggest that the antiviral effect of roc-a on ev -infected nsc- cells targets the viral replication step and not the entry step, in contrast to a previous study with hcv [ ] . prior studies focusing on cancer have reported that the mechanism by which roc-a targets and inhibits phb activity involves blocking the craf/mek/erk pathway [ , ] , and this was also described in the hcv study [ ] . to investigate whether the craf/mek/erk to decipher the mode of action of roc-a in nsc- cells, immunostaining of roc-a treated nsc- cells was performed. decreased signals for phb and mitochondria were observed with increasing concentrations of roc-a (fig c) , suggesting that roc-a might affect mitochondrial integrity. we thus assessed the mitotoxicity and cytotoxicity of roc-a using the mitochondrial toxglo assay (promega). while cytotoxicity remained generally minimal over the range of roc-a concentrations tested, the intracellular atp levels were significantly reduced in a dose-dependent manner, thus indicating functional impairment of the mitochondria in roc-a-treated nsc- cells (fig d) . consistently, using the membrane-permeant jc- dye as an indicator of mitochondrial membrane potential, roc-a-treated nsc- cells displayed marked and dose-dependent mitochondrial depolarization as evidenced by the decrease of red fluorescent j-aggregates, compared to untreated cells (s fig). together, these observations suggest that roc-a treatment in nsc- cells results in reduced levels of phb, leading to mitochondrial destabilization and lower atp production. one could thus speculate that the lack of intact mitochondria and reduced intracellular atp levels might eventually impact negatively on ev replication efficacy. the role of phb in ev infection cycle was also studied in human muscle (rd) and neuronal (sk-n-sh) cell lines. as human (gi ) and murine (gi ) phb display high similarity in their amino acid composition, most of the anti-phb antibodies commercially available demonstrate good cross reactivity with cell lines of both species. we first showed by flow cytometry comparable levels of surface expression of phb on rd, sk-n-sh and nsc- cells (s fig). however, both phb gene silencing and phb receptor blocking experiments performed in human muscle rd cells did not impact the viral titer (s a and s b fig) . on the contrary, phb silencing in the human neuroblastoma cells sk-n-sh led to a significant dosedependent reduction in viral titer in the culture supernatant (fig a) . in addition, reduced virus titers were observed with sk-n-sh cells pre-treated with anti-phb antibodies, thus supporting that cell surface-expressed phb is involved in ev entry into this human neuroblastoma cell line (fig b) . to investigate if intracellular phb is also involved in viral replication in sk-n-sh cells, transfection of lucev replicon into phb-silenced sk-n-sh cells was performed. results showed a significant reduction in the luminescence signal compared to controls (fig c) . finally, the effectiveness of roc-a treatment in ev -infected sk-n-sh cells was also assessed. similar to our observations with nsc- cells, a significant dose-dependent decline in viral titers was observed (fig d) . taken together, these findings thus strongly indicate the specific involvement of phb in both viral entry and replication of ev in neuronal cells from both human and murine origins. concentrations of roc-a for hours before assessment of cytotoxicity (fluorescence) and mitotoxicity (luminescence) using mitochondrial toxglo assay. statistical analysis was performed using one-way anova with dunnett's post-test ( à , p< . ; Ãà , p< . ; ÃÃà , p< . ; ÃÃÃà , p< . ). one representative from two independent experiments is shown. https://doi.org/ . /journal.ppat. .g i. the culture supernatant was collected for viral titer determination by plaque assay, and the cell lysate was harvested for western blot analysis. statistical analysis was performed using one-way anova with dunnett's post-test ( Ãà , p< . ). relative band quantification (below western blot) was determined by imagej, by normalizing to loading control, β-actin. error bars represent mean ± standard deviation. cellular cytotoxicity was assessed using alamarblue assay. one representative from two independent experiments is shown. https://doi.org/ . /journal.ppat. .g the role of phb was further investigated in vivo, using the mouse model of ev infection that we established previously where -week old ag mice (deficient in type i&ii ifn pathways) infected with ev display progressive limb paralysis and spatio-temporal virus accumulation in the limb muscles, spinal cord and brainstem [ ] . here, immunohistochemical analysis showed that phb was readily detected in the limb muscles, brainstem and spinal cord at day p.i. (fig a) . furthermore, some co-localization with ev was observed (fig a) . next, the in vivo anti-ev efficacy of roc-a was assessed by treating therapeutically ev -infected mice with roc-a at day and p.i. the development of clinical manifestations was clearly delayed in the roc-a-treated mouse group which resulted in increased survival time compared to the untreated or vehicle-treated control groups (fig b) . in addition, the viral loads in limb muscles, spinal cord and brain in both roc-a-treated and vehicle-treated mice were determined. comparable viral loads were detected in the limb muscles from both groups (fig c) . in contrast, viral titers in the spinal cord and brain from the roc-a-treated animals were significantly lower compared to the vehicle-treated group (fig c) , thus supporting that roc-a treatment specifically impairs ev neuropathogenesis. these findings correlate well with our in vitro data showing that the role of phb during ev infection cycle is specific to neuronal cells. together, the in vivo data support that phb plays a critical role in ev neurovirulence, and that roc-a represents a potential therapeutic strategy to limit ev neuropathogenesis, thereby minimizing neurological manifestations and complications. the re-emergence of neurotropic enteroviruses in recent years has motivated investigations into ev transmission in the neuronal system. understanding the interplay between virus and host proteins is likely to result in the identification of potential novel drug targets and development of novel antiviral strategies. here, using a proteomics approach, we have identified a panel of host factors that displayed dynamic regulation during the course of ev infection in the motor neuron nsc- cells. the host protein candidates are mainly involved in cytoskeletal structure maintenance, rna processing and mitochondrial biogenesis. by employing a sirna gene silencing approach, we have shown that some of these host factors either facilitate or limit ev productive infection. among these host factors, phb was found to exert a pro-viral effect as evidenced by the reduced viral titers measured in the culture supernatant of nsc- cells when the expression of phb was down-regulated, and by an increased viral titer in phb over-expressing cells. phb is mainly localized on plasma membrane, mitochondria and nucleus, and has been involved in multiple signaling pathways regulated by growth factors, immune response, mitochondrial biogenesis, cell migration, proliferation and survival [ , ] . interestingly, knockdown in nsc- cells of stoml , which was shown to interact with phb and participate to mitochondria biogenesis [ ] , resulted in significant reduction of viral titer, similar to that seen with phb-knocked down cells. this further supports the involvement of mitochondrial proteins during ev infection cycle in nsc- cells. in addition, previous studies have reported the association of phb with internalization of several viruses, including hcv [ ] , chikv [ ] , denv [ ] , and coronavirus (sars-cov) [ ] . furthermore, phb was shown to promote hiv replication by interacting with the hiv- glycoprotein [ ] . using various experimental approaches, we have demonstrated that cell surface-expressed phb is physically associated with ev and is involved in the entry of the virus into nsc- cells. on the other hand, by employing a lucev replicon, we have shown that intracellular (mitochondrial) phb plays a role in ev replication activity. this was further supported by (a) two week-old ag mice (n = ) were infected i.p. with ev ( pfu) and the limb muscles, spinal cord and brainstem were harvested for immunohistochemical analysis at day p.i. scale bars denote μm ( × magnification) and μm ( × magnification). (b) ev -infected ag mice (n = ) were treated i.p. with roc-a at . mg/kg at day and p.i. and were monitored for survival and clinical manifestations. clinical scores were defined as follows: , healthy; , ruffled hair and hunched back; , limb weakness; , one limb paralysis; , both limbs paralysis at which point the animals were euthanized. statistical analysis of survival curve was performed using logrank (mantel-cox) test ( Ãà , p< . ). (c) limb muscles, spinal cord and brain were harvested at day p.i. for viral load determination by plaque assay (n = / ). dotted line represents the limit of detection. statistical analysis was performed using mann-whitney u test ( à , p< . ). error bars represent mean ± sem. one representative of two biological repeats is shown. https://doi.org/ . /journal.ppat. .g role of prohibitin in ev neuropathogenesis the observation that mitochondrial phb co-localizes with the replicating viral genome (dsrna) and the non-structural proteins d polymerase and cd complex. co-immunoprecipitation and tem approaches also confirmed the physical proximity and likely interactions between viral complexes/viral particles and mitochondria. these data thus led us to propose that mitochondria could serve as replication site for ev in nsc- cells. the association of ev with mitochondria was reported in a previous study where it was proposed that ev could potentially interact with some mitochondrial signaling proteins to evade host anti-viral innate immunity [ ] . previous studies have reported that membrane-bound phb binds to ras in a gtp-dependent manner, which in turn activates craf kinase and eventually triggers the mapk pathway [ ] . similar to other flavaglines, rocaglamide (roc-a) is a natural product that displays insecticidal, anti-fungal, anti-inflammatory and anti-cancer activities [ ] . mechanistically, roc-a was found to inhibit craf-phb interactions in tumor cells [ ] [ ] [ ] , and in an in vitro model of hcv infection [ ] . in ev -infected nsc- cells, incubation with nm concentrations of roc-a resulted in a dose-dependent reduction in virus titers. however, the mechanism by which roc-a exerts its antiviral effect against ev in nsc- cells does not seem to be mediated by blocking the craf/mek/erk pathway, given that phosphorylated erk proteins could not be detected in uninfected nsc- cells, suggesting that this pathway is not functional in these cells. instead, we found that roc-a-treated cells displayed reduced expression of mitochondrial phb and lower levels of intracellular atp, which suggests that mitochondria integrity/functionality is impaired in roc-a treated nsc- cells. since we showed that mitochondrial phb is involved in ev replication and that mitochondria serve as replication site for this virus, the impact of roc-a on phb expression and mitochondria integrity could represent the basis of its antiviral activity. further investigation is necessary to decipher the molecular mechanisms by which roc-a affects the expression of mitochondrial phb. interestingly, we found that the role of phb in ev entry and replication was limited to cells of neuronal origin, thus supporting a role of phb specifically in ev neuropathogenesis. this neuro-specific phenotype was also observed in vivo where roc-a-treatment resulted in reduced virus loads in the cns (spinal cord and brain) only but not in the limb muscles from infected mice, although phb was readily detected in the muscle cells as well. the cell typedependent involvement of phb during ev infection likely reflects differential intracellular events with different host factors being engaged during ev intracellular life cycle. since ev is known to be able to use multiple receptors to enter host cells, one could speculate that the host factors that are engaged during ev infection depend on the entry receptor that is being used by the virus. further study is necessary to explore this idea. in conclusion, our work has uncovered a novel host factor that is specifically involved in ev neurovirulence. in addition, our data support that roc-a, a previously established anticancer drug that targets phb, could represent a therapeutic approach to limit ev neuropathogenesis, and thus prevent or limit associated neurological complications. given the current attrition in effective antiviral drugs against ev , roc-a repurposing is worth considering seriously. all the animal experiments were carried out under the guidelines of the national advisory committee for laboratory animal research (naclar) in the aaalac-accredited nus animal facilities. the animal experiments described in this work were approved under the nus institutional animal care and use committee (iacuc) protocol number - . non-terminal procedures were performed under anesthesia, and all efforts were made to minimize suffering. murine motor neuron nsc- cells (cellutions biosystems, clu ), human rhabdomyosarcoma (rd) cells (atcc ccl- ) and human neuroblastoma sk-n-sh cells (atcc htb- ) were used in this study. all cell lines were cultured in dulbecco's modified eagle's medium (dmem) (gibco) containing % fetal bovine serum (fbs) (gibco) at ˚c with % co . non-mouse-adapted ev s ( /sin/ , accession no.: af ), kindly provided by prof. chow v. t. k. at national university of singapore, was isolated from the lymph node of a ev -infected patient who died of encephalitis and pulmonary edema in singapore [ ] . the virus stocks were made in rd cells and the viral titers were determined by plaque assay on rd cells. total proteins extract from infected cells was prepared using proteoextract complete mammalian proteome extraction kit (millipore). briefly, the cell pellet was thawed by resuspension in ice-cold resuspension buffer and the proteins were extracted with extraction buffer at room temperature (rt). benzonase and reducing agent were added during protein extraction to minimize nucleic acid contamination and to remove disulphide bonds, respectively. the solubilized protein suspension was subjected to centrifugation at , ×g for minutes at ˚c to remove the remaining insoluble material. the recovered cell extract was stored at - ˚c until further analysis. the protein samples ( μg, quantified using rc dc bradford assay, biorad) were loaded onto individual lanes of the isoelectronic focusing (ief) tray with pre-wetted electrode wicks. passive rehydration was performed for each protein sample using cm ph - readystrip ipg strips (biorad) for hours at rt with gel side down configuration. after rehydration, the protein sample was subjected to ief on protean ief cell i (biorad) according to the following conditions: v for minutes with linear ramp, , v for . hours with linear ramp and , v for , v-hours with rapid ramping. ipg strips equilibration was achieved by incubating the strips with pre-warmed dtt equilibration buffer i (biorad) followed by iodoacetamide-supplemented equilibration buffer ii (biorad) for minutes each on orbital shaker. equilibrated ipg strips were then transferred onto . % tris-hcl criterion gel (biorad) and overlaid with readyprep overlay agarose (biorad). electrophoresis was run at v for minutes. after electrophoresis, the gels were stained with instantblue (expedeon) for hour and submerged in milliq water overnight to remove background signal. gels were scanned using gs- calibrated densitometer (biorad). gel images were further processed using pdquest -d analysis software (biorad), whereby the different gel images from three independent experiments were matched and the intensities of detected spots were measured. protein spots that showed at least . -fold change in spot intensity (p< . , two-tailed student's t-test), compared to the uninfected control sample, were excised for maldi-tof ms. the fold change was calculated using the equation: mean of spot expression in infected samples for each time point. in-gel digestion and maldi-tof ms of the excised protein spots were done by protein and proteomics centre, national university of singapore (singapore). the data was search against the murine and viruses national centre for biotechnology information non-redundant (ncbinr) database using a mascot program (http://www.matrixscience.com). no threshold was applied to the ms/ms fragment ions intensities. data mining of the identified proteins was done by searching in panther (http://www. pantherdb.org/) and swiss-prot/trembl (http://www.uniprot.org/) databases. the enrichment analysis of protein-protein interactions was performed using string network analysis version (http://string-db.org/). hierarchical clustering and classification were performed using multiexperiment viewer version . (http://mev.tm .org/#/welcome). on rd cells ( cells/well) were seeded onto wells plate. culture supernatant from ev -infected samples was serially diluted ( -fold) with dmem containing % fbs prior to infection. the cell monolayer was incubated with μl of the diluted viral suspension for hour at ˚c. the cells were then washed twice with pbs and replaced with ml dmem containing % fbs and % carboxymethyl cellulose (cmc, sigma aldrich). after days incubation at ˚c, the infected monolayers were fixed and stained with % paraformaldehyde/ . % crystal violet solution (sigma aldrich). the number of plaques was scored visually and viral titers were expressed as plaque-forming units (pfu) per milliliter (pfu/ml). drug treated or sirna-transfected cells ( . × nsc- or × sk-n-sh cells/well) were washed twice with pbs and × alamarblue reagent (invitrogen) diluted with dmem containing % fbs was added. after hours incubation at ˚c, the fluorescence signals were determined using microplate reader (infinite , tecan) at ex nm and em nm . percentage of viable cells was calculated using non-treated cells as control. table) for mins at rt, the protein complexes were pulled down and subjected to western blot analysis (s table) . briefly, the protein extracts were incubated with the conjugated magnetic beads and further incubated for hour at ˚c with constant rotating, before eluting using laemmli buffer. isotype antibody pull-down and uninfected cells were used as controls. table) . ) . briefly, μl of × cytotoxicity reagent were added into each well and incubated at ˚c for minutes. cytotoxicity was measured using fluorescence at ex nm and em nm . after equilibrating the assay plate to rt for minutes, μl of atp detection reagent were added into each well and mitotoxicity was measured by luminescence. all readings were normalized against non-treated cells. sodium azide (s , sigma aldrich) and staurosporine (s , sigma aldrich) were used as mitochondrial toxin and cytotoxin positive controls, respectively. nsc- ( cells/well) cells were seeded onto -wells chamber slides (ibidi) and incubated overnight. the cells were treated with various concentrations of roc-a (diluted with % dmem) for hours. jc- dye (invitrogen) ( μg/ml in % dmem) was added into each well and incubated for another mins, prior to imaging. nsc- cells incubated with sodium azide nan at μm in % dmem (s , sigma aldrich) was used as positive control. cell lysate was prepared using m-per mammalian protein extraction reagent containing % of halt protease inhibitor cocktail and % of . m edta (thermoscientific). protein quantification was performed using quick start bradford protein assay (biorad). denatured proteins ( μg) were resolved in % sds-page gel and transferred electrophoretically onto nitrocellulose membrane using trans-blot turbo transfer system (biorad). after blocking with % (w/v) milk in tbst (tbs buffer with . % tween ) for hour at rt, the membrane was probed using specific primary antibodies and relevant secondary antibodies (s table) . the chemiluminescence signal was visualized using clarity western ecl substrate (biorad) on x-ray films. densitometric quantification was performed using imagej and the relative band intensity was normalized against β-actin. nsc table) . the nucleus was revealed using nucblue live readyprobes reagent (molecular probes). fluorescence images were captured using olympus ix microscope and further processed using imagej. limb muscles, spinal cord and brainstem from ev -infected ag mice were harvested at day p.i. after systemic perfusion. the organs were fixed in % pfa overnight prior immersing in % and % sucrose solution, and embedded in tissue-tek oct (vwr) solution. the organ samples were then frozen at - ˚, sectioned ( μm) using a cryostat (leica) and mounted on a glass slide prior to blocking and staining as described above. samples were fixed for h at rt with . % glutaraldehyde containing % tannic acid in . m cacodylate buffer (ph . ), then washed three times for min (each time) in . m cacodylate buffer and post-fixed for h at rt with % osmium tetroxide in the same buffer. samples were then dehydrated in a graded series of ethanol and embedded in spurr. thin sections were stained with % uranyl acetate and lead citrate and observations were performed by transmission electron microscopy using a fei tecnai spirit g at kv. image were taken using a fei eagle k ccd camera. nsc- , sk-n-sh ( × cells) and rd ( × cells) cells were seeded onto -well plate overnight. the cells were dislodged by incubating them in mm edta/pbs in ˚c for mins and spun down to collect the cell pellet. cells were then blocked with either human or murine fc-blocker ( or , bd pharmigen; : ) for min at rt, and subsequently stained with anti-phb antibody (ab , abcam; : ) and/or anti-rabbit af antibody (a , thermoscientific; : ) for min at ˚c. stained cells were then fixed with % pfa. flow cytometry analysis was carried out using the becton-dickinson fortessa flow cytometer and analysed using flowjo v . two-week old ag mice (b&k universal) were bred and housed under specific pathogenfree conditions in individual ventilated cages. infection was performed by injecting intraperitoneally (i.p.) pfu of s (in μl) per mouse. at day and post-infection (p.i.), mice were injected i.p. with . mg/kg of roc-a (in . % dmso in sterile olive oil). the control group was inoculated with . % dmso in olive oil. clinical manifestations were observed for a period of days. clinical score was graded as follows: , healthy; , ruffled hair and hunched back; , limb weakness; , one limb paralysis; , two limbs paralysis; and , death. two limbs paralysis was used as criterion for early euthanasia. for virus titer determination, infected mice were euthanized at day p.i. and perfused with ml of sterile pbs systemically. the fore and hind limb muscles, spinal cord and brain were harvested and weighed before mechanical homogenization in ml of serum-free dmem. the homogenates were spun down at , rpm for minutes at ˚c, and clarified using a . μm filter before serial dilution was carried out for plaque assay. viral titers were expressed as pfu per gram of tissue. supporting information s relative band quantification (below western blot) was determined by imagej, by normalizing to loading control, β-actin. statistical analysis was performed using two-tailed student's t-test ( Ãà , p< . ). one representative from two independent experiments is shown. (pdf) (a) for co-treatment assay, virus was incubated with roc-a for hour before adding the mixture onto the cells. after one hour of incubation on cell monolayer, the mixture was then removed and replaced with fresh % dmem. (b) for pre-treatment assay, the cells were pre-treated with roc-a for hours, prior to viral infection. culture supernatants were harvested at h.p.i. for viral titer determination by plaque assay. cell viability was assessed using alamarblue viability assay. statistical analysis was performed using one-way anova with dunnett's post-test ( Ãà , p< . ). one representative from two independent experiments is shown. (pdf) . viral titer in the culture supernatant was determined by plaque assay at h.p.i. statistical analysis was performed using two-tailed student's t-test ( à p< . , Ãà p< . , ÃÃà p< . , ÃÃÃà p< . ). error bars represent mean ± standard deviation. relative band quantification (below western blot) was determined by imagej, by normalizing to loading control, β-actin. non-targeting sirna (ntc) served as control. (b) rd cells were pre-incubated with anti-phb antibody or isotype control antibody for hour before infection. culture supernatant was harvested at h.p.i. for viral titer determination. cell viability was determined using alamarblue cytotoxicity assay. one representative of two biological repeats is shown. (pdf) neurotropic enterovirus infections in the central nervous system epidemiological analysis, detection, and comparison of space-time patterns of beijing hand-foot-mouth disease enterovirus infection with central nervous system involvement the epidemiology of hand, foot and mouth disease in asia: a systematic review and analysis virology, epidemiology, pathogenesis, and control of enterovirus . the lancet infectious diseases outbreaks of hand, foot, and mouth disease by enterovirus . high incidence of complication disorders of central nervous system ev vaccine, an invaluable gift for children ev vaccine: protection from a previously neglected disease status of research and development of vaccines for enterovirus proteomic analysis of extremely severe hand, foot and mouth disease infected by enterovirus proteomics analysis of ev -infected cells reveals the involvement of host protein nedd l in ev replication comparative proteome analyses of host protein expression in response to enterovirus and coxsackievirus a infections transcriptomic and proteomic analyses of rhabdomyosarcoma cells reveal differential cellular gene expression in response to enterovirus infection human genome-wide rnai screen reveals host factors required for enterovirus replication global quantitative proteomic analysis of human glioma cells profiled host protein expression in response to enterovirus type infection a practical guide to clinical virology retrograde axonal transport: a major transmission route of enterovirus in mice virus infections in the nervous system & feuer r. enterovirus infections of the central nervous system enterovirus infection of motor neuron-like nsc- cells undergoes a non-lytic exit pathway neuroblastoma x spinal cord (nsc) hybrid cell lines resemble developing motor neurons nonlytic viral spread enhanced by autophagy components topology of double-membraned vesicles and the opportunity for nonlytic release of cytoplasm string v : protein-protein interaction networks, integrated over the tree of life cellular proteome alterations in response to enterovirus and coxsackievirus a infections in neuronal and intestinal cell lines beta-actin variant is necessary for enterovirus replication proteomic analysis of human brain microvascular endothelial cells reveals differential protein expression in response to enterovirus infection requirement for an intact t-cell actin and tubulin cytoskeleton for efficient assembly and spread of human immunodeficiency virus type tubulin: a factor necessary for the synthesis of both sendai virus and vesicular stomatitis virus rnas subversion of the actin cytoskeleton during viral infection multifunctional alpha-enolase: its role in diseases protein disulfide isomerase in redox cell signaling and homeostasis comparing the primary and recall immune response induced by a new ev vaccine using systems biology approaches dengue virus infection induces upregulation of hn rnp-h and pdia for its multiplication in the host cell galectin- binding to cell surface protein disulfide isomerase regulates the redox environment to enhance t-cell migration and hiv entry identification and characterization of prohibitin as a receptor protein mediating denv- entry into insect cells a novel class of small molecule compounds that inhibit hepatitis c virus infection by targeting the prohibitin-craf pathway identification of prohibitin as a chikungunya virus receptor protein interaction of stomatin with hepatitis c virus rna polymerase stabilizes the viral rna replicase complexes on detergent-resistant membranes network based analysis of hepatitis c virus core and ns b protein interactions pebp , a raf kinase inhibitory protein, negatively regulates starvation-induced autophagy by direct interaction with lc epigenetic regulation of autophagy by the methyltransferase ezh through an mtordependent pathway knockdown of deptor induces apoptosis, increases chemosensitivity to doxorubicin and suppresses autophagy in rpmi- human multiple myeloma cells in vitro defective axonal transport of neurofilament proteins in neurons overexpressing peripherin intermediate filaments in peripheral nervous system: their expression, dysfunction and diseases neuronal subtype and satellite cell tropism are determinants of varicella-zoster virus virulence in human dorsal root ganglia xenografts in vivo prohibitins role in cellular survival through ras-raf-mek-erk pathway prohibitin interacts with envelope proteins of white spot syndrome virus and prevents infection in the red swamp crayfish, procambarus clarkii human scarb -dependent infection by coxsackievirus a , a , and a and enterovirus viral and host proteins involved in picornavirus life cycle the mitochondrial phb complex: roles in mitochondrial respiratory complex assembly, ageing and degenerative disease enterovirus protease apro targets mavs to inhibit anti-viral type i interferon responses inhibition of the craf/prohibitin interaction reverses craf-dependent resistance to vemurafenib the natural anticancer compounds rocaglamides inhibit the raf-mek-erk pathway by targeting prohibitin and prohibitin ligands in cell death and survival: mode of action and therapeutic potential a non-mouse-adapted enterovirus (ev ) strain exhibits neurotropism, causing neurological manifestations in a novel mouse model of ev infection stomatin-like protein binds cardiolipin and regulates mitochondrial biogenesis and function severe acute respiratory syndrome coronavirus nonstructural protein interacts with a host protein complex involved in mitochondrial biogenesis and intracellular signaling identification of the cellular prohibitin /prohibitin heterodimer as an interaction partner of the c-terminal cytoplasmic domain of the hiv- glycoprotein rocaglamide, silvestrol and structurally related bioactive compounds from aglaia species expression of a mutant prohibitin from the ap gene promoter leads to obesity-linked tumor development in insulin resistance-dependent manner & chellappan s. prohibitin induces the transcriptional activity of p and is exported from the nucleus upon apoptotic signaling prohibitin: a potential target for new therapeutics complete sequence analyses of enterovirus strains from fatal and non-fatal cases of the hand, foot and mouth disease outbreak in we would like to thank a/p vincent chow from the department of microbiology & immunology, nus, for sharing the s strain and the electron microscopy unit core facility from yong loo lin school of medicine, nus. conceptualization: issac horng khit too, sylvie alonso. formal analysis: issac horng khit too, isabelle bonne. key: cord- -qxkwjkif authors: geisbert, thomas w.; daddario-dicaprio, kathleen m.; lewis, mark g.; geisbert, joan b.; grolla, allen; leung, anders; paragas, jason; matthias, lennox; smith, mark a.; jones, steven m.; hensley, lisa e.; feldmann, heinz; jahrling, peter b. title: vesicular stomatitis virus-based ebola vaccine is well-tolerated and protects immunocompromised nonhuman primates date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: qxkwjkif ebola virus (ebov) is a significant human pathogen that presents a public health concern as an emerging/re-emerging virus and as a potential biological weapon. substantial progress has been made over the last decade in developing candidate preventive vaccines that can protect nonhuman primates against ebov. among these prospects, a vaccine based on recombinant vesicular stomatitis virus (vsv) is particularly robust, as it can also confer protection when administered as a postexposure treatment. a concern that has been raised regarding the replication-competent vsv vectors that express ebov glycoproteins is how these vectors would be tolerated by individuals with altered or compromised immune systems such as patients infected with hiv. this is especially important as all ebov outbreaks to date have occurred in areas of central and western africa with high hiv incidence rates in the population. in order to address this concern, we evaluated the safety of the recombinant vsv vector expressing the zaire ebolavirus glycoprotein (vsvΔg/zebovgp) in six rhesus macaques infected with simian-human immunodeficiency virus (shiv). all six animals showed no evidence of illness associated with the vsvΔg/zebovgp vaccine, suggesting that this vaccine may be safe in immunocompromised populations. while one goal of the study was to evaluate the safety of the candidate vaccine platform, it was also of interest to determine if altered immune status would affect vaccine efficacy. the vaccine protected of shiv-infected macaques from death following zebov challenge. evaluation of cd + t cells in all animals showed that the animals that succumbed to lethal zebov challenge had the lowest cd + counts, suggesting that cd + t cells may play a role in mediating protection against zebov. ebola virus (ebov) has been associated with sporadic episodes of hemorrhagic fever (hf) that produce severe disease in infected patients. mortality rates in outbreaks have ranged from % for sudan ebolavirus (sebov) to up to % for zaire ebolavirus (zebov) (reviewed in [ ] ). a recent outbreak caused by an apparently new species of ebov in uganda appears to be less pathogenic than sebov or zebov with a preliminary case fatality rate of about % [ ] . ebov is also considered to have potential as a biological weapon and is categorized as a category a bioterrorism agent by the centers for disease control and prevention [ ] [ ] [ ] . while there are no vaccines or postexposure treatment modalities available for preventing or managing ebov infections there are at least four different vaccine systems that have shown promise in completely protecting nonhuman primates against a lethal ebov challenge [ ] [ ] [ ] [ ] [ ] [ ] [ ] . of these prospective ebov vaccines two systems, one based on a replication-defective adenovirus and the other based on a replication-competent vesicular stomatitis virus (vsv), were shown to provide complete protection when administered as a single injection vaccine [ ] [ ] [ ] . most intriguingly, the vsv-based vaccine is the only vaccine which has shown any utility when administered as a postexposure treatment [ , ] . of these two leading ebov vaccine candidates that can confer protection as single injection vaccines each has advantages and disadvantages. adenovirus vectors are highly immunogenic as documented by clinical trials evaluating gene transfer efficacy and immune responses. because they are replication-defective adenovirus vectors are also perceived to be safer for human use than a replication-competent vaccine. the most significant challenge for the adenovirus-based vaccines is the concern that a significant portion of the global population has pre-existing antibodies against the adenovirus vector which may affect efficacy [ ] [ ] [ ] and has performed poorly as a vaccine vector in recent clinical trials [ ] [ ] . in contrast, pre-existing immunity against vsv in human populations is negligible [ ] and efficacy is likely greater with replication-competent vectors. the main concern with the vsv vaccine vector is that replication-competent vectors may present more significant safety challenges in humans particularly those with altered immune status. because ebov outbreaks in man have occurred exclusively in central and western africa, the populations in this region are among those that may benefit from the development and availability of an ebov vaccine. however, populations in this region are among the most medically disadvantaged in the world. in particular, the prevalence of individuals with a compromised immune system is high and hiv infections rates range up to % or more in this area [ ] . while the vsv vaccine vector has been enormously successful in protecting healthy immunocompetent animals against ebov [ , , ] , we are uncertain as to how these vectors would behave in individuals with altered or compromised immune systems. therefore, we conducted a study to assess the pathogenicity and protective efficacy of the recombinant vsvbased zebov vaccine vector in rhesus macaques that were infected with simian-human immunodeficiency virus (shiv) which is known to deplete the populations of naive cd + t cells, naive cd + t cells, and memory cd + t cells in these animals [ , ] . in order to take into account the degree or severity of compromised immune function animals were selected with varying degrees of cd + t cell loss. the recombinant vsv expressing the glycoprotein (gp) of zebov (strain mayinga) (vsvdg/zebovgp) was generated as described recently using the infectious clone for the vsv, indiana serotype [ ] . zebov (strain kikwit) was isolated from a patient of the zebov outbreak in kikwit in [ ] . nine filovirus-seronegative adult rhesus macaques (macaca mulatta) ( - kg) were used for these studies. the macaques were infected three months prior to the current study with shiv p (kindly provided by dr. ranajit pal, advanced bioscience laboratories, inc., kensington, md). these animals all had clinical laboratory evidence of shiv infection as evidenced by reduced cd + t cell counts, decreased ratios of cd +/cd + t cells (table ) and the presence of shiv in plasma of four out of nine animals ( table ). six of the nine shiv-infected animals were vaccinated by i.m. injection with , ^ recombinant vsvdg/zebovgp. three animals served as placebo controls and were injected in parallel with saline. all six vsvdg/ zebovgp-vaccinated animals and two of the three control ebola virus is among the most lethal microbes known to man, with case fatality rates often exceeding %. since its discovery in , outbreaks have been sporadic and geographically restricted, primarily to areas of central africa. however, concern about the natural or unnatural introduction of ebola outside of the endemic areas has dramatically increased both research interest and public awareness. a number of candidate vaccines have been developed to combat ebola virus, and these vaccines have shown varying degrees of success in nonhuman primate models. safety is a significant concern for any vaccine and in particular for vaccines that replicate in the host. here, we evaluated the safety of our replication-competent vesicular stomatitus virus (vsv)-based ebola vaccine in shiv-infected rhesus monkeys. we found that the vaccine caused no evidence of overt illness in any of these immunocompromised animals. we also demonstrated that this vaccine partially protected the shiv-infected monkeys against a lethal ebola challenge and that there appears to be an association with levels of cd + lymphocytes and survival. our study suggests that the vsv-based ebola vaccine will be safe in immunocompromised populations and supports further study and development of this promising vaccine platform for its use in humans. animals were challenged days after the single dose immunization with pfu of zebov (strain kikwit). the monkeys were challenged with the heterologous kikwit strain of zebov as our macaque models have been developed and characterized using this strain [ , ] . animals were closely monitored for evidence of clinical illness (e.g., temperature, weight loss, changes in complete blood count, and blood chemistry) during both the vaccination and zebov challenge portions of the study. in addition, vsvdg/zebov and zebov viremia and shedding were analyzed after vaccination and challenge, respectively. animals were given physical exams and blood and swabs (nasal, oral, rectal) were collected at , , , , , , , and days after vaccination and on days , , , , and after zebov challenge. the vaccination portion of the study was conducted at bioqual and was approved by niaid, bioqual, and usamriid laboratory animal care and use committees. the zebov challenge was performed in bsl- biocontainment at usamriid and was approved by the usamriid laboratory animal use committee. animal research was conducted in compliance with the animal welfare act and other federal statues and regulations relating to animals and experiments involving animals and adheres to the principles stated in the guide for the care and use of laboratory animals, national research council, . both facilities used are fully accredited by the association for assessment and accreditation of laboratory animal care international. total white blood cell counts, white blood cell differentials, red blood cell counts, platelet counts, hematocrit values, total hemoglobin, mean cell volume, mean corpuscular volume, and mean corpuscular hemoglobin concentration were determined from blood samples collected in tubes containing edta, by using a laser-based hematologic analyzer (coulter electronics, hialeah, fl, usa). the white blood cell differentials were performed manually on wright-stained blood smears. serum samples were tested for concentrations of albumin (alb), amylase (amy), alanine aminotransferase (alt), aspartate aminotransferase (ast), alkaline phosphatase (alp), gamma-glutamyltransferase (ggt), glucose (glu), cholesterol (chol), total protein (tp), total bilirubin (tbil), blood urea nitrogen (bun), and creatinine (cre) by using a piccolo point-of-care blood analyzer (abaxis, sunnyvale, ca, usa). flow cytometry for circulating cell populations ul of whole blood was added to a tube and incubate with the antibodies for minutes at room temperature. the samples was then lysed and fixed in % paraformaldehyde and washed three times in pbs. samples were analyzed on a becton dickinson facs calibur (becton dickinson, san jose, ca). all antibodies were purchased from becton dickinson; clones used were cd -sp , cd -l , cd -rpa-t and cd - h . for measurement of plasma siv rna levels, a quantitative taqman rna reverse transcription-pcr (rt-pcr) assay (applied biosystems, foster city, ca) was used, which targets a conserved region of siv gag and has an accurate detection limit as low as rna copies/ml. briefly, isolated plasma viral rna was used to generate cdna using one-step rt-pcr master mix (applied biosystems). the samples were then amplified as previously described [ ] with the following pcr primer/probes: siv-f agtatgggcagcaaatgaat (forward primer), siv-r ttc tcttctgcgtg aatgc (reverse primer), siv-p fam-agatttggattagcagaaagcctgttg ga-tamra (taqman probe) in a sequence detection system ( cycles of uc, seconds, and uc, minute). the signal was then compared to a standard curve of known concentrations to determine the viral copies present in each sample. the assay lower limit was copies/ml. rna was isolated from blood and swabs using tripure reagent (invitrogen, grand island, new york). for the detection of vsv we used a q-rt-pcr assay targeting the matrix gene (nt position - , am ). zebov rna was detected using a q-rt-pcr assay targeting the l gene (nt position - , ay ). the low detection limit for this zebov assay is . pfu/ml of plasma. virus titration was performed by plaque assay on vero e cells from all blood and selected organ (liver, spleen, lung, kidney, adrenal gland, pancreas, axillary lymph node, inguinal lymph node, mesenteric lymph node, ovary or testis, and brain) and swab samples. briefly, increasing -fold dilutions of the samples were adsorbed to vero e monolayers in duplicate wells ( . ml per well); thus, the limit for detection was pfu/ml. igg antibodies against zebov were detected with an enzyme-linked immunosorbent assay (elisa) using purified virus particles as an antigen source as previously described [ , ] . necropsies were performed on each animal and selected tissues were collected for histological analysis. histology and immunohistochemistry were performed as previously described for zebov-infected monkeys [ ] . we employed nine shiv-infected rhesus macaques, of which six animals were vaccinated by i.m. injection with a single dose of vsvdg/zebovgp (subjects # - ) and the remaining three animals (controls # - ) received sterile saline. the animals were monitored closely for clinical symptoms and shedding of recombinant vsvs. none of the animals vaccinated with vsvdg/zebovgp or treated with saline showed overt fever or any evidence of clinical illness during the day vaccination period. importantly, no evidence of reaction at the vaccine injection site was noted among any of the vsvdg/zebovgpvaccinated animals nor was any change noted in activity or behavior during the vaccination phase of the study (day to day after vaccination). in addition, no changes were detected in hematology or clinical chemistry following vaccination. a mild vsvdg/zebovgp viremia (, pfu/ml) was detected only on day after vaccination by virus isolation (figure ) and rt-pcr (data not shown) in four of the six vsvdg/zebovgp-immunized macaques (subjects # , , , ) . surprisingly, the two animals with the lowest cd + counts (subjects # , ) never showed any detectable level of vsv viremia. vsvdg/zebovgp was undetectable in all analyzed swab samples (data not shown). thus, vaccination led to a transient viremia from virus replication at as yet undetermined sites but no virus shedding of the vaccine virus. following successful completion of the safety portion of the study all six of the vsvdg/zebovgp-vaccinated shivinfected monkeys and two of the three placebo control shivinfected monkeys were challenged days after the single immunization by i.m. injection with pfu of zebov (strain kikwit). four of the six vsvdg/zebovgp-vaccinated shivinfected monkeys and both of the placebo control animals started to show clinical signs of disease on day after challenge including fever (subject # , and control # , ) and lymphopenia and thrombocytopenia (subject # , , and control # , ) ( table ) . disease progressed in two of the vsvdg/zebovgp-vaccinated shiv-infected monkeys (subject # and ) and both of the placebo control animals with the development of additional evidence of clinical illness including increased levels of serum enzymes associated with liver function, depression, anorexia, and the appearance of macular rashes (table ). all four of these animals succumbed to the zebov challenge with the two vsvdg/ zebovgp-vaccinated monkeys expiring on days (subject # ) and (subject # ) and the placebo controls succumbing on days (control # ) and (control # ) after zebov challenge (figure ). disease did not progress in the two vsvdg/zebovgp-vaccinated shiv-infected monkeys that were febrile (subjects # , ) and had changes in hematology values on day (subjects # ) and both of these animals remained healthy and survived the zebov challenge ( figure ). the remaining vsvdg/zebovgp-vaccinated macaques (subject # , ) never showed any evidence of clinical illness and survived ( figure ) . interestingly, the vsvdg/zebovgpvaccinated macaques that succumbed were the two animals with the most significant reduction in cd + t cells ( %, %) (table ) , the lowest total cd + t cell counts ( , ) (table ) , the highest shiv viremia (table ) , and no evidence for vsv viremia (figure ) suggesting that cd + t cells may play a role in protection. blood samples were analyzed after challenge for evidence of zebov replication by plaque assay and rt-pcr. by day , both of the placebo control animals developed high zebov titers in plasma as detected by plaque assay (. . log pfu/ml) ( table ). in comparison, only one of the vsvdg/zebovgp-vaccinated monkeys (subject # ) showed a zebov viremia at day by plaque assay (, log pfu/ml) ( table ). zebov was detected in a second vsvdg/zebovgp-vaccinated monkey (subject # ) by day (, . log pfu/ml). rt-pcr was more sensitive and showed evidence of zebov in plasma of this animal (subject # ) at day . in addition, rt-pcr was more sensitive in detecting zebov in swabs which were positive on a number of samples derived from subject # at day and day ( table ). in contrast, no zebov was detected in the plasma by virus isolation or rt-pcr in the four vsvdg/zebovgp-vaccinated monkeys that survived zebov challenge. moreover, no evidence for reactivation of vsvdg/ zebovgp was detected from any blood or swab sample from any animal after zebov challenge (data not shown). although we failed to detect zebov viremia in the two surviving animals that were clinically ill (subject # and ) at days , , , and after zebov challenge we cannot exclude the possibility that these animals had low levels of circulating zebov at time points not evaluated. the four surviving vsvdg/zebovgp-vaccinated macaques (subjects # , , , ) were euthanized days after the zebov challenge to perform a virological and pathological examination of tissues. organ infectivity titration from these four animals showed no evidence of zebov in any of the tissues examined. in comparison, zebov was recovered from tissues of both vsvdg/zebovgp-vaccinated animals that succumbed (subject # , ) and both shiv-infected control animals. organ titers of infectious zebov were consistent with values previously reported for immunocompetent zebov-infected rhesus macaques [ , ] . vsvdg/zebovgp was not recovered in any of the tissues examined from any animal on this study. pathological and immunohistochemical evaluation of tissues from the four vsvdg/zebovgp-vaccinated animals (subjects # , , , ) that survived zebov challenge showed no evidence of zebov antigen. in contrast, zebov antigen was readily detected in typical target organs (e.g., liver, spleen, adrenal gland, lymph nodes) of the two vsvdg/zebovgp-vaccinated animals that succumbed to zebov challenge (subject # , ) ( figure ) and the two placebo controls. lesions and distribution of zebov antigen in these macaques was consistent with results reported in other studies [ , ] . while cellular immune responses against zebov gp in macaques vaccinated with vsvdg/zebovgp vectors have been difficult to detect before challenge in previous studies [ ] , humoral immune responses have been more robust and consistent ( [ ] ; tw geisbert, unpublished observations). therefore, we measured the antibody responses of the rhesus macaques vaccinated with vsvdg/zebovgp before vaccination (day ), after vaccination (day and day ), and after zebov challenge (day and day after vaccination) by igg elisa. none of the six vsvdg/zebovgp-vaccinated macaques developed igg antibody titers against the zebov gp by the day of zebov challenge ( figure ). two animals (subjects # , ) developed modest igg antibody titers against zebov by day after zebov challenge (day after vaccination) while a third animal developed a titer by day after zebov challenge (day after vaccination) (figure ). an often raised concern regarding the use of the recombinant vsv vaccine platform in humans is related to the fact that this is a replication-competent vaccine, and thus demonstration of safety is of paramount importance. taking into account our previous work it is not surprising that the vsvdg/zebovgp was tolerated well in our shiv-infected macaques. specifically, we failed to observe evidence of any adverse events in a large cohort of over macaques receiving vsv vectors expressing different gps from viral hf agents ( cynomolgus macaques and rhesus macaques vaccinated with vsvdg/zebovgp; cynomolgus macaques table . clinical findings in shiv-infected rhesus monkeys challenged with zebov. day - day - day - subject fever ( ), lymphopenia ( ), thrombocytopenia ( ) thrombocytopenia ( ) anorexia ( ), depression ( ), moderate rash ( ), epistaxis ( ), thrombocytopenia ( ), alpqqq ( ), altqqq ( ), astqqq ( ), bunqqq ( ), creqqq ( ), ggtqqq ( ), gluq ( ) day control fever ( ), anorexia ( ), depression ( ), lymphopenia ( ), thrombocytopenia ( ), alpqq ( ), astq ( ) anorexia ( ), depression ( ), moderate rash ( ) day fever is defined as a temperature more than . uf over baseline or at least . uf over baseline and $ . uf. mild rash: focal areas of petechiae covering less than % of the skin; moderate rash: areas of petechiae covering between % and % of the skin; severe rash: areas of petechiae and/or echymosis covering more than % of the skin. [ ] . while transient vsv viremia in this study was only observed in surviving macaques but not in animals that had succumbed to zebov challenge ( figure ), viremia data from previous studies [ , , ] do not support any correlation between vsv viremia and survival. in addition, no evidence for vaccine vector shedding was detected in this study supporting previous results [ , , ] with no compelling evidence to suggest that occasional virus shedding (only detected by rt-pcr; negative on virus isolation) would lead to vaccine vector transmission. the vsv glycoprotein exchange vector that we employed in this study has also shown promise as a preventive vaccine and postexposure treatment against marburg hf [ , ] and as a preventive vaccine against lassa fever in nonhuman primates [ ] . similar recombinant vsv vectors have been evaluated in animal models as vaccine candidates for a number of viruses that cause disease in humans including hiv- , influenza virus, respiratory syncytial virus, measles virus, herpes simplex virus type , hepatitis c virus, and severe acute respiratory syndrome coronavirus [ ] [ ] [ ] [ ] [ ] [ ] [ ] . many of these studies have employed vsv vectors that maintained either the entire vsv glycoprotein (g) or the transmembrane and/or cytoplamic domains of this protein to facilitate more efficient incorporation of the foreign antigen. it is known that vsv g is an important vsv protein associated with pathogenicity [ , ] . it has been shown that truncation of the cytoplasmic tail has greatly reduced vector pathogenicity in mice following intranasal inoculation indicating the importance of this domain for pathogenicity [ ] . in this regard, a vsv vector including portions of the vsv g and expressing hiv genes was found to be insufficiently attenuated for clinical evaluation when assessed for neurovirulence in nonhuman primates [ ] . these investigators subsequently showed that safety and immunogenicity can be improved by genetic manipulation of the vsv genome but it remained unclear whether neurovirulence was associated with the vsv g or other genome manipulations [ ] . nevertheless, our zebov vaccine is a g-deficient vsv vector [ ] and thus lacks g-associated pathogenicity [ ] as well as the target for vsvspecific neutralizing antibodies [ ] . aside from g, the vsv matrix (m) protein has been associated with cytopathic effects in vitro including the inhibition of host gene expression, induction of cell rounding and induction of apoptosis [ , ] . it is largely unclear to what extent m alone contributes to pathogenicity, but inoculation studies with the vsv-based vaccines in different animal species (as described above) do not suggest a major pathogenic effect of the m protein in vivo [ , , ] . currently, the mechanism by which any filovirus vaccine confers protection in nonhuman primates is not well understood. nearly all studies have detected modest to good humoral immune responses. for the vsvdg/zebovgp vaccine a humoral response is detected in macaques by day after vaccination ( [ ] ; tw geisbert, unpublished observations). however, in the current study and consistent with an impaired immune system, our shiv-infected macaques did not develop a humoral immune response by the time of zebov challenge. three animals developed modest anti-zebov igg titers to days after zebov challenge. we are uncertain as to why four of the six vsvdg/zebovgp-vaccinated macaques survived zebov challenge. regardless of any humoral immune response elicited in these animals it is unlikely that antibody alone confers protection. specifically, passive antibody studies in nonhuman primates using a variety of anti-zebov immune reagents including polyclonal equine immune globulin [ ] , a recombinant human monoclonal antibody [ ] , and convalescent monkey blood [ ] have uniformly failed to provide protection and more importantly have failed to provide any beneficial effect. a number of studies have evaluated the cellular immune response in nonhuman primates vaccinated against ebov and the results have been mixed with some studies showing a modest cellular response and other studies showing weak and/or no cellular immune responses [ , , ] . however, it is likely that the intracellular cytokine assays that have been employed in some of these studies are not sensitive or thorough enough to detect a cellular immune response against zebov. indeed, it has been reported that the inability to demonstrate a robust cellular response may illustrate the limitation of the evaluation of cellular immune responses using small numbers of functional measurements (such as interferon-gamma) [ ] . one interesting finding in the current study may begin to shed some light on the mechanism of protection elicited by the vsvdg/zebovgp. notably, the two rhesus macaques that grouped together with the most severe loss of cd + t cells were the only animals that failed to survive zebov challenge. this suggests that cd + t cells may play a role in mediating protective immunity in ebov infections. cd + t cells have been shown to be depleted in nonhuman primate following zebov infections [ , ] and in vitro zebov infection of human peripheral blood mononuclear cells causes massive bystander death of cd + t cells by apoptosis [ ] . while rodents do not appear to faithfully reproduce zebov infection of humans and nonhuman primates [ ] studies have suggested that cd + t cells are required for protection of rodents against zebov. specifically, in a study using liposome-encapsulated zebov antigens, rao and colleagues showed that treatment of mice with anti-cd antibodies before or during vaccination abolished protection, while treatment with anti-cd antibodies had no effect, thus indicating a requirement for cd + t lymphocytes for successful immunization [ ] . similarly, depletion of cd + t cells did not compromise protection in mice indicating that cd + cytotoxic t cells are not a requirement for protection [ ] . in conclusion, our results show that the vsv-based zebov vaccine (vsvdg/zebovgp) did not cause any illness in immunocompromised shiv-infected rhesus macaques and resulted in sufficient protective efficacy in all but the most severely compromised animals against a lethal zebov challenge. protection in the immunocompromised macaques appeared to be dependent on cd + t cells rather than the development of ebov-specific antibodies. this provides strong support for the safety of the vsv-based vectors and further development of this promising vaccine platform for its use in humans. while these data are very encouraging, as the number of shiv-infected macaques in the current study was small, additional safety studies will be needed in order to determine whether vaccines based on attenuated vsv will ultimately prove safe in immunocompromised humans. filoviridae: marburg and ebola viruses ebola outbreak in uganda ''atypical'', say experts hemorrhagic fever viruses as biological weapons: medical and public health management centers for disease control and prevention. bioterrorism agents/diseases exotic emerging viral diseases: progress and challenges successful topical respiratory tract immunization of primates against ebola virus live attenuated recombinant vaccine platform protects non-human primates against lethal challenge with either ebola virus or marburg virus immune protection of nonhuman primates against ebola virus with single lowdose adenovirus vectors encoding modified gps accelerated vaccination for ebola virus haemorrhagic fever in non-human primates development of a preventive vaccine for ebola virus infection in primates complete protection of nonhuman primates against multi-strain ebola and marburg virus infections. clin vaccine immunol ebola virus-like particle-based vaccine protects nonhuman primates against lethal ebola virus challenge effective post-exposure treatment of ebola infection recombinant vesicular stomatitis virus vector mediates postexposure protection against sudan ebola hemorrhagic fever in nonhuman primates infections in , infants and children in a controlled study of respiratory tract disease. i. adenovirus pathogenicity in relation to serologic type and illness syndrome incidence and prevalence of neutralizing antibodies to the common adenoviruses in children with cystic fibrosis: implication for gene therapy with adenovirus vectors established immunity precludes adenovirus-mediated gene transfer in rat carotid arteries. potential for immunosuppression and vector engineering to overcome barriers of immunity aids research. did merck's failed hiv vaccine cause harm? the failed hiv merck vaccine study: a step back or a launching point for future vaccine development? rhabdoviridae: the viruses and their replication unaids ( ) report on the global aids epidemic hiv- prophylactic vaccine comprised of topical dermavir prime and protein boost elicits cellular immune responses and controls pathogenic r shiv p ccr or cxcr use influences the relationship between cd cell depletion, nkp l expression and nk cytotoxicity in shiv-infected macaques properties of replication-competent vesicular stomatitis virus vectors expressing glycoproteins of filoviruses and arenaviruses evaluation of immune globulin and recombinant interferon a- b for treatment of experimental ebola virus infections pathogenesis of ebola hemorrhagic fever in cynomolgus macaques: evidence that dendritic cells are early and sustained targets of infection plasma siv rna viral load determination by real-time quantification of product generation in reverse transcriptase-polymerase chain reaction pathologic findings associated with delayed death in nonhuman primates experimentally infected with zaire ebola virus lethal experimental infection of rhesus monkeys with ebola-zaire (mayinga) virus by the oral and conjunctival route of exposure cross-protection against marburg virus strains using a live, attenuated recombinant vaccine development of a new vaccine for the prevention of lassa fever assessment of a vesicular stomatitis virus-based vaccine by use of the mouse model of ebola virus hemorrhagic fever postexposure protection against marburg haemorrhagic fever with recombinant vesicular stomatitis virus vectors in non-human primates: an efficacy assessment immunogenicity of attenuated vesicular stomatitis virus vectors expressing hiv type env and siv gag proteins: comparison of intranasal and intramuscular vaccination routes replicationcompetent or attenuated, nonpropagating vesicular stomatitis viruses expressing respiratory syncytial virus (rsv) antigens protect mice against rsv challenge longterm protection from sars coronavirus infection conferred by a single immunization with an attenuated vsv-based vaccine recombinant vesicular stomatitis virus vectors expressing herpes simplex virus type gd elicit robust cd + th immune responses and are protective in mouse and guinea pig models of vaginal challenge vaccination with a recombinant vesicular stomatitis virus expressing an influenza virus hemagglutinin provides complete protection from influenza virus challenge an effective aids vaccine based on live attenuated vesicular stomatitis virus recombinants successful vaccine-induced seroconversion by single-dose immunization in the presence of measles virus-specific maternal antibodies vesicular stomatitis virus glycoprotein is a determinant of pathogenesis in swine, a natural host attenuated vesicular stomatitis viruses as vaccine vectors neurovirulence properties of recombinant vesicular stomatitis virus vectors in non-human primates attenuation of recombinant vesicular stomatitis virus-human immunodeficiency virus type vaccine vectors by gene translocations and g gene truncation reduces neurovirulence and enhances immunogenicity in mice the glycoprotein of vesicular stomatitis virus is the antigen that gives rise to and reacts with neutralizing antibody the cell-rounding activity of the vesicular stomatitis virus matrix protein is due to the induction of cell death matrix protein and another viral component contribute to induction of apoptosis in cells infected with vesicular stomatitis virus neutralizing antibody fails to impact the course of ebola virus infection in monkeys ebola hemorrhagic fever: evaluation of passive immunotherapy in nonhuman primates vaccination in humans generates broad t cell cytokine responses depletion of peripheral blood t lymphocytes and nk cells during the course of ebola hemorrhagic fever in cynomolgus macaques implication of a retrovirus-like glycoprotein peptide in the immunopathogenesis of ebola and marburg viruses evaluation in nonhuman primates of vaccines against ebola virus induction of immune responses in mice and monkeys to ebola virus after immunization with liposome-encapsulated irradiated ebola virus: protection in mice requires cd (+) t cells from usamriid, the authors thank john crampton and carlton rice for animal care and anna honko for assistance with flow cytometry. from the national microbiology laboratory (nml) of the public health agency of canada (phac), the authors thank friederike feldmann and jason gren for technical assistance. we are grateful to john rose (yale university) for kindly providing us with the vesicular stomatitis virus reverse genetics system. we thank howard young for helpful discussions. key: cord- -je ar authors: wang, david title: challenges in understanding the role of the virome in health and disease date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: je ar nan comprehensive census of the bacterial and fungal microbiome can be achieved through consensus pcr approaches that target the s rrna and internal transcribed spacer (its) loci, respectively. by contrast, there is no such analogous conserved sequence present in all viruses. the lack of a consensus sequence poses a significant challenge for efforts to systematically define the set of viruses present in a given specimen. rather than simply amplifying and sequencing a signature target locus, metagenomic sequencing of nucleic acid in a sample is required for virus sequences to be represented in a sequencing library. the increased sequencing depth necessary dramatically increases the cost compared to s or its sequencing. moreover, the relative abundance of viral to nonviral nucleic acids is an important parameter that drives sensitivity. to mitigate this problem, physical enrichment for viral particles by filtration and/or nuclease treatment is often necessary. another consequence of the requirement for metagenomic sequencing is that the subsequent bioinformatic analysis is also much more complex; the metagenomic sequences must be aligned at both nucleotide and amino acid levels to large reference databases of viral sequences (not just to a database of reference amplicon nucleic acid sequences). thus, the experimental sequencing depth, computational infrastructure requirements, and computing costs are significantly higher for virome analyses. the precise experimental and bioinformatic analysis steps utilized can contribute significantly to variability and, in some instances, potentially incorrect conclusions. however, a detailed discussion of both of these is beyond the scope of this review. currently, classification of sequence reads as being viral in origin relies primarily upon detectable primary sequence alignment to reference viruses. one challenge is that, among a a a a a those that are alignable to reference viruses, sequences can be misclassified due to the remoteness of the similarity and/or low stringency. an even more significant problem is that in most virome studies, more than % of the sequences in virus-enriched preparations have no detectable sequence similarity to any known reference sequences; these unalignable sequences are referred to as viral "dark matter" [ ] and may include novel, highly divergent viruses that are unrecognizable. thus, most current virome studies are presenting only a partial view of the virome because the dark matter sequences are typically ignored. the dark matter clearly harbors hidden treasures because mining of the dark matter has led to discoveries such as crassphage, the most abundant bacteriophage in human enteric viromes [ ] . to computationally address the dark matter, new and improved data mining strategies are needed. one approach is to apply methods superior at detection of remote homologies, such as hidden markov models customized for viral proteins [ ] . other machine learning strategies may include development of primary sequence-alignment-independent classification approaches [ ] or development of artificial neural networks [ ] . experimentally, as described below, expanding the set of culturable viruses will provide additional reference sequences that should also decrease the amount of viral dark matter. one bias in many virome studies to date is a focus only on sequencing of dna. this is due in part to the bacterial microbiome being the primary driver of many studies, and thus at the time of sampling, experimental measures to extract dna were employed without regard to trying to either preserve or recover the rna. in addition, some studies utilize dna-specific amplification strategies such as phi -mediated whole-genome amplification. these studies are unable to assess the rna component of the virome. yet the majority of the known eukaryotic viral denizens of the human enteric tract are rna viruses, such as enteroviruses, rotaviruses, noroviruses, and astroviruses. on the phage side, while it is true that the vast majority of known phages have dna genomes, the bias of sequencing exclusively dna has served as a self-reinforcing positive feedback loop; however, recent studies examining samples that have sequenced rna have demonstrated that there is a much greater abundance and diversity of rna phages in the world [ ] . thus, in order achieve a more complete view of the virome, it is critical that virome studies be designed such that rna viruses are preserved in the specimens and that the rna fraction is incorporated into the sequencing and analysis. the advent of metagenomics has greatly enhanced our ability to detect known and novel viral sequences in unbiased fashion and to establish novel associations of these sequences with various disease [ ] [ ] [ ] [ ] [ ] [ ] [ ] . however, it is not known whether the virome plays a causative role or not. koch's postulates remain the gold standard for microbial disease causality, and thus the first step is to establish culture systems for viruses associated with the disease of interest. the lack of culture systems for viruses identified in virome studies, for both eukaryotic viruses and phages, is pronounced. as an example, although dozens of novel eukaryotic viruses have been identified in the mammalian enteric tract by metagenomic sequencing, culture systems for only a very limited number have been described to date [ , ] . likewise, for phages, genomic sequences of thousands of novel phages can be identified in a single study [ , ] , but very few have been isolated [ ] . thus, while (in the past) discovery of novel viruses was rate limiting, today the rate-limiting step has shifted to development of culture systems for the viruses that have been molecularly identified. how can this be addressed? to some extent, this is simply a matter of effort. in all likelihood, no effort has been made to culture the vast majority of the novel viruses identified in virome studies, and some will surely succumb to standard culture conditions once applied. limitations in quantity or quality of primary specimens containing viable virus can contribute to the problem. in part, it is challenging for labs to obtain funding to culture novel viruses, particularly in the absence of any strong disease association. moreover, the risk of a negative result, exemplified by many decades of unsuccessful attempts to culture human norovirus, is substantial. nonetheless, given that the lack of ability to culture a virus is perhaps the most fundamental barrier to progress in the study of that virus, dedicated efforts to develop culture systems are absolutely necessary. for some of the more recently discovered eukaryotic viruses, the advent of primary, differentiated culture systems has been key to unlocking this riddle. for example, the respiratory viruses bocavirus and coronavirus hku can be propagated using primary airway epithelial cells [ , ] . in the enteric tract, the development of enteroids and organoids and recognition of the need for additional enteric tract components have opened the frontier for propagation of human norovirus [ , ] . broader application of these systems will undoubtedly lead to increased success in culture of some eukaryotic viruses. on the phage front, one significant challenge is that many of the bacterial host species are themselves unculturable using standard bacterial-growth media. thus, efforts to propagate novel phages will also entail improving systems for bacterial in vitro growth. some approaches toward this include development of bioreactors [ , ] and other systems that better mimic the natural environment [ ] , but additional new and innovative strategies are needed. for the majority of complex diseases, in vitro systems are not adequate to assess viral contributions to pathogenicity, and thus appropriate animal-disease models are required that also reflect the potentially complex interactions with the host, bacteria, and eukaryotic microbes that may be present. in bacterial microbiome studies, a powerful approach has been the ability to colonize mice with either a single defined bacteria of interest or a consortia of bacteria. by analogy, it would be ideal if the virome could be functionally interrogated in similar fashion. however, a prerequisite for eukaryotic viruses is that one must first be able to establish infection in the relevant animal model. as with establishing cell-culture systems, the paucity of animal-infection models for novel viruses stems in part from lack of effort to develop such systems. moreover, the challenge is exacerbated by the multitude of potential infection routes that need to be evaluated, need to overcome the immune response, cross-species transmission barriers, and (even within a species) potential impact of varying genetic backgrounds. use of transgenic animals that are immunodeficient [ ] or that have been partially humanized [ ] has enabled development of murine models for some viruses. in an ideal world, animal models for all eukaryotic viruses identified in virome studies would be established, enabling the roles of any single virus (or cocktail of viruses) to be functionally evaluated. likewise, having an extensive library of cultivated phages (see above) will allow them to be combined in defined proportions along with eukaryotic viruses for experimental studies. for phages, presuming they act by modulating the bacterial community, the animal model must harbor the relevant bacterial host species, and thus the bacterial microbiome may need to be manipulated as well. notably, some recent studies have suggested that phages may also interact directly with eukaryotic host cells [ , ] . overall, significant effort and resources must be expended to establish robust animal-infection models suitable to define the role of the virome. this is a step that is absolutely necessary in order to move virome studies beyond the realm of mere association studies. a barrier to progress in the virome field is the division between virologists who study viruses infecting eukaryotes versus those that infect bacteria. very few scientists today have expertise in both. this balkanization of virology leads to challenges when an eukaryotic virologist discovers in a virome study that the strongest disease association is with a phage (or vice versa). lack of familiarity with the field and relevant experimental approaches often limits further investigation. these communities have been largely segregated, often holding separate conferences and distinct grant-review panels. to illustrate this point, the united states national institutes of health (nih) virology study sections address only "non-bacteriophage viral genetics, infection and replication, cellular and host responses to viral infections, and mechanisms of viral disease pathogenesis." thus, there is a great need to bring these disparate communities together in order to collectively attack questions associated with the virome, especially as more complex trans-kingdom interactions are identified linking phages, bacteria, eukaryotic viruses, and eukaryotic cells. in conclusion, the coming years will undoubtedly be witness to many more studies demonstrating associations of the virome with various diseases. hopefully, there will be commensurate development of new computational approaches that significantly decrease the fraction of viral dark matter and an increase in the fraction of studies that holistically evaluate the virome. with new cell-culture systems and animal models for novel viruses, there will ideally be studies that attribute causal roles for some of the associations. finally, it may be that virome studies will serve as a catalyst to help integrate the eukaryotic viral and phage communities. pathogenic simian immunodeficiency virus infection is associated with expansion of the enteric virome altered virome and bacterial microbiome in human immunodeficiency virus-associated acquired immunodeficiency syndrome disease-specific alterations in the enteric virome in inflammatory bowel disease gut dna viromes of malawian twins discordant for severe acute malnutrition the eukaryotic gut virome in hematopoietic stem cell transplantation: new clues in enteric graft-versus-host disease distinct gut virome profile of pregnant women with type diabetes in the endia study. open forum infectious diseases intestinal virome changes precede autoimmunity in type i diabetes-susceptible children origins and challenges of viral dark matter a highly abundant bacteriophage discovered in the unknown sequences of human faecal metagenomes profile hidden markov models for the detection of viruses within metagenomic sequence data virfinder: a novel k-mer based tool for identifying viral sequences from assembled metagenomic data artificial neural networks trained to detect viral and phage structural proteins hyperexpansion of rna bacteriophage diversity cultivation and serological characterization of a human theiler's-like cardiovirus associated with diarrheal disease propagation of astrovirus va , a neurotropic human astrovirus, in cell culture marine dna viral macro-and microdiversity from pole to pole viral dark matter and virus-host interactions resolved from publicly available microbial genomes phicrass represents the most abundant bacteriophage family in the human gut and infects bacteroides intestinalis human bocavirus can be cultured in differentiated human airway epithelial cells culturing the unculturable: human coronavirus hku infects, replicates, and produces progeny virions in human ciliated airway epithelial cell cultures replication of human noroviruses in stem cell-derived human enteroids enteric bacteria promote human and mouse norovirus infection of b cells cultivation of stable, reproducible microbial communities from different fecal donors using minibioreactor arrays (mbras) chemostat culture systems support diverse bacteriophage communities from human feces a new antibiotic kills pathogens without detectable resistance a mouse model of zika virus pathogenesis precision mouse models with expanded tropism for human pathogens expansion of bacteriophages is linked to aggravated intestinal inflammation and colitis bacteriophage trigger antiviral immunity and prevent clearance of bacterial infection key: cord- - kpkhz o authors: lam, tommy tsan-yuk; hon, chung-chau; pybus, oliver g.; kosakovsky pond, sergei l.; wong, raymond tze-yeung; yip, chi-wai; zeng, fanya; leung, frederick chi-ching title: evolutionary and transmission dynamics of reassortant h n influenza virus in indonesia date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: kpkhz o h n highly pathogenic avian influenza (hpai) viruses have seriously affected the asian poultry industry since their recurrence in . the viruses pose a threat of emergence of a global pandemic influenza through point mutation or reassortment leading to a strain that can effectively transmit among humans. in this study, we present phylogenetic evidences for the interlineage reassortment among h n hpai viruses isolated from humans, cats, and birds in indonesia, and identify the potential genetic parents of the reassorted genome segments. parsimony analyses of viral phylogeography suggest that the reassortant viruses may have originated from greater jakarta and surroundings, and subsequently spread to other regions in the west java province. in addition, bayesian methods were used to elucidate the genetic diversity dynamics of the reassortant strain and one of its genetic parents, which revealed a more rapid initial growth of genetic diversity in the reassortant viruses relative to their genetic parent. these results demonstrate that interlineage exchange of genetic information may play a pivotal role in determining viral genetic diversity in a focal population. moreover, our study also revealed significantly stronger diversifying selection on the m and pb genes in the lineages preceding and subsequent to the emergence of the reassortant viruses, respectively. we discuss how the corresponding mutations might drive the adaptation and onward transmission of the newly formed reassortant viruses. the h n highly pathogenic avian influenza (hpai) virus was originally isolated from a farmed goose in guangdong province of china in [ ] , and soon spread to live-poultry markets in hong kong [ ] , resulting in cases of human infection in , of which were fatal [ , ] . the first wave of h n infection ceased after the depopulation of all poultry in hong kong, although the h n virus was later found to circulate continuously in southern china without causing apparent disease symptoms among infected poultry [ ] . h n outbreaks recurred in , persistently affecting poultry farms in many southeast asia countries, such as china, thailand, vietnam, indonesia and cambodia. the viruses also spread outside asia, including to some european countries. more importantly, occasional zoonotic transmissions to humans occurred in most of the affected asian countries and the virus continued to pose a serious threat to global public health [ ] . h n outbreaks in indonesia were initially detected in poultry farms in december [ ] . it was suggested that the h n virus was first introduced to java and subsequently spread to other parts of the country [ ] . the virus rapidly became endemic in indonesia [ , ] , and continued to cause sporadic zoonotic transmissions to humans beginning in july [ ] . three clusters of h n transmission among family members were identified in , raising concerns of possible human-to-human transmission of the virus [ , ] . as of april , , indonesia had confirmed human cases with deaths [ ] , the largest number of deaths among all affected countries. previous studies have shown that several h n genotypes have emerged in asia through reassortment between h n viruses and other subtypes [ , ] . one of these genotypes, z, predominated the h n outbreaks throughout - , causing most h n outbreaks in asian countries, including indonesia [ ] . moreover, a variety of antigenically distinct sublineages of z genotype virus have been established [ ] . unlike vietnam and thailand, indonesia was invaded by only a single sublineage of genotype z virus. previous phylogenetic analyses suggested that hunan province of china may be the source of the initial h n outbreak in indonesia [ ] , and classified the indonesian h n hpai viruses into three groups [ ] ; however, further statistical analysis is necessary to characterize and compare different aspects of their evolutionary histories. in this study, we examined molecular phylogeny of the most recent indonesian h n viruses isolated from avian and mammal hosts. a group of putative reassortant viruses was discovered and their genetic parents were identified. in addition, we investigated the evolutionary behaviors (including spatial migration, growth of genetic diversity, and evolutionary drift and selection) of the reassortant viruses and compare with those of the parental strain, thereby providing insights into the nature and impact of this emerging reassortant strain. phylogenetic trees of indonesian h n viruses were reconstructed from separate gene datasets (table s ) , using a maximum likelihood (ml) approach with bootstrapping analyses to assess clade robustness (figures , s -s ; computer files of dendrogram are available as dataset s ). in all the phylogenies, viruses sampled from avian species during earlier years of outbreaks (predominantly - ) tended to cluster near the root as expected, but with a poorly resolved branching structure that is likely due to relatively low sequence divergence. in contrast, viruses sampled from recent infections ( ) ( ) ( ) from avian and mammalian hosts formed three well-supported lineages with bootstrap support (or posterior probabilities) over (or . ) under neighbor-joining (nj), ml and bayesian markov chain monte carlo (bmcmc) methods. we denote these lineages as groups , , and in the hemagglutinin (ha) and neuraminidase (na) phylogenies ( figures a and s c ). this structure was preserved in the phylogenies of other genes for which sufficient sequence data were available (viruses from group were missing sequence data for the np, ns, ns , ns , and pb genes). the group lineage in the mp, m , m , and pb phylogenies was only represented by the a/indonesia/ / strain. it is important to recognize that our phylogenetic groupings (groups , , and ) of indonesian h n viruses ( figure and table s ) are slightly different to those by smith and coworkers [ ] who did not require the same level of clustering support for each group, leading to the inclusion of earlier viruses (predominantly - ) . we chose to be conservative, and did not include poorly supported branches (e.g., earlier viruses) in our viral group definition. therefore, we did not define a group corresponding to the group b of smith et al., because most of group b taxa are earlier viruses. groups and in this study correspond to groups c and a defined by smith et al. respectively, plus some more recent viruses. smith did not report group , because the sequences were unavailable at that time. we found previously unrecognized phylogenetic discordance between gene trees involving human and cat isolates (n = , denoted in red in figures , s -s )-the main focus of our study-suggesting that they are reassortant viruses descending from group and lineages. in addition, the placement of two avian viruses isolates from java (ck/idn/semerang - / and ck/idn/magelang - / , shown in blue in figures a and s c ) differed between ha and na phylogenies, suggesting another reassortment event. to further investigate the putative reassortant human and cat viruses, a selected dataset (n = ) of manually concatenated full genomes (figure a ; see methods) of indonesian h n hpai viruses were analyzed using more sophisticated analysis methods, including similarity plots, bootscan analyses and gard analyses (genetic algorithm for recombination detection). in the similarity and bootscan plots ( figure b and c), the putative reassortants (represented by a consensus sequence) showed a high degree of sequence similarity and phylogenetic clustering with the group strain a/indonesia/ / in the mp and pb segments, but not in other genomic regions, where they were more similar to the consensus sequence of group viruses ( figure b and c). moreover, gard detected two well-supported breakpoints near the boundaries of mp and pb segments in the concatenated genomes ( figure d ), suggesting that the phylogenetic incongruence was significant between the three regions. in summary, all three analyses agreed that the newly reassortant strains had arisen from acquiring pb and mp genome segments from the group lineage and the remaining segments from the group lineage. based on the ha phylogeny ( figure a ), we further classified the reassortant viruses into three subgroups (r , r , and r ) with bootstrap support of % or better, as shown in the phylogenies containing only reassortant viruses ( figure s ). similar groupings were observed in the na phylogeny ( figures s c and s b ), although here subgroup r clustered with subgroup r , and two reassortant viruses isolated in (idn/cdc / , idn/ cdc / ) moved to a different subgroup. these inferred clustering patterns can be explained by multiple reassortment events, or by a single reassortment followed by divergence due to mutation and selection in different populations. we note that some group viruses also cluster inside the reassortant subgroups ( figures a and s c ) and may indicate more reassortment events; however, most of them formed polytomies close to the most recent common ancestor (mrca) of the reassortant subgroups and had poor bootstrap support for their exact placement. as the divergence between the reassortant subgroups and other intercalating group viruses are low, the three subgroups may actually be linked uninterruptedly, implying a single origin. therefore, the times and number of reassortment events that generated the putative mosaic reassortant viruses remains elusive. we examine both the single and multiple origin hypotheses in subsequent analyses, excluding the intercalating group viruses from the reassortant group. to estimate the times of the reassortment events that generated the putative reassortant viruses, the times of the mrca (tmrca) of the three reassortant subgroups were estimated using bmcmc methods [ , ] . bayes factors (bf) [ ] were used to select author summary h n highly pathogenic avian influenza (hpai) virus emerged in china in , and has spread beyond asia since . following the first outbreak reported in indonesian poultry farms in december , the virus spilled over to indonesian provinces by june , and became endemic in the country. in the following years, repeated sporadic human infections in indonesia had been attributed to h n hpai viruses. nonetheless, the viral evolution and transmission have not been fully understood. here, we report phylogenetic evidence of a group of interlineage reassortant viruses isolated from human and cats in java. our comparative study of the reassortant viruses and one group of genetic parents found that although their rates of evolution were similar and both of their phylogenies were not geographically structured within mainland java, the growths of genetic diversity were different. we also detected significant positive selection on the viral matrix and polymerase genes preceding and subsequent to the emergence of the reassortant viruses, which might correspond to viral adaptation. based on our findings, we discuss the possibility of host switching in facilitating the emergence of the reassortant strain, and call for more extensive viral surveillances in the non-avian population in indonesia. among strict and relaxed clock models of evolution [ ] . the uncorrelated exponentially-distributed clock model (uced) significantly outperformed the other models (lnbf. ) for most datasets, except for the na gene of the reassortant viruses, for which the strict clock model was not rejected (lnbf, ; table s ). the results of tmrca estimation are summarized in figure c and d. in addition, sequence isolation dates were plotted against their genetic distance (units of substitutions/site) to their mrca, to graphically show the accumulation of mutations through time ( figure a and b). the tmrca of all reassortant viruses (all- were isolated from central and east jakarta (table ) . parsimony reconstruction (see methods) of binary ancestral geographical states (either greater jakarta or west java) upon the ha and na ml phylogenies suggested that the mrca of all reassortants (and the mrcas of each reassortant subgroup) likely originated from greater jakarta and surroundings (table s ; result robust to random resolution of polytomies; see methods). the mean numbers of observed geographical state changes (gsc) of the reassortant and of the group parental strains were estimated independently and compared with the null distribution of gsc values under the null hypothesis of completely unrestricted migration (i.e. panmixis; figure s ) [ ] . for the reassortant strain, the observed gsc value was not significantly lower than the gsc value expected under panmixis (slatkin-maddison test: p. . ). therefore the observed geographic structure is not significantly different to that expected by chance alone. for group viruses, the observed gsc value for all geographical state pairs is significantly (p, . ) lower than the null value. however, the observed value of gsc within java (i.e., migrations between greater jakarta and the rest of java) and between the three islands (i.e. migrations between mainland java, sumatera and sulawesi selatan including papua) are insignificantly (p. . ) and significantly (p, . ) lower than the corresponding null values respectively, suggesting that the phylogeny of group viruses is not geographically structured within java, but is subdivided by island-to-island migrations. however, we could not address whether the viral migrations inside sumatera and sulawesi selatan including papua are panmictic or structured due to limited operative localities in our dataset to distinguish between different regions inside these islands. we also found that the migration of group viruses from greater jakarta and surroundings to sumatera and sulawesi selatan including papua was more frequent than expected under the null hypothesis, and there is relatively little viral migration from the rest of java to sumatera and sulawesi selatan including papua (table s ). this observation suggests greater jakarta played a more salient role in dispersing group viruses to other indonesian islands than other parts of java did. there is another family member (brother) who was confirmed with h n infection (index # , fatal); however, virus sequences are not available. f numbers in parentheses are the unique references to the localities shown in figure . numbers - were assigned to greater jakarta and surroundings; numbers - were assigned to west java. n/a = information not available. doi: . /journal.ppat. .t we used the bayesian skyline plot (bsp) [ ] to estimate the change of relative genetic diversity of the reassortant viruses and of the group parental strain over time, as shown in figure e - h. for both the ha and na datasets, the group viruses consistently show a slow growth in relative genetic diversity over time which appears to follow a constant size or exponential growth model, whereas the reassortant viruses initially exhibited an abrupt rise in relative genetic diversity followed by stabilization, which visually resembles a logistic growth curve with two phases [ , ] ( figure e and f) . when the bsps are superimposed upon the demographic results obtained under parametric growth models (i.e., constant, exponential and logistic growth; figure s ), then a similar observation can also be made. however, bf tests (table s ) indicate there is insufficient statistical power to discriminate between the three parametric growth models (lnbf, . ), suggesting a lack of strong demographic signal in these data. when the parametric demographic models were fitted to the data, the median estimates of growth rates for the reassortant datasets are generally higher than those estimated for the datasets of group viruses (table s ). however, the confidence intervals of some growth rate estimates are fairly large and overlapped among the reassortant and group viral datasets. diversifying selection in the pb and m genes using the random effects likelihood (rel) method [ ] we found sites under positive selection in the pb gene (codons , , , and ) and the pa gene (codon ) of the reassortant viruses. the fixed effects likelihood (fel) method [ ] was more conservative and only identified pb codon as being positively selected. for the group viruses, ha codon (starting from ha ) and m gene codon were the only selected sites identified by the fel and rel methods, respectively. using a lineage-specific selection model (see methods), we identified elevated rates of diversifying selection, measured by the ratio of non-synonymous to synonymous substitutions (dn/ds), on the m gene in the lineage leading to the mrca of the group viruses and preceding the emergence of the reassortant viruses (highlighted in figure b ). the dn/ds values for the m gene in this lineage (which we call the preemergence lineage) was estimated to be . ( % ci: . - . ; see table s ), significantly higher (lrt p, . ) than the mean estimates for other lineages (dn/ds = . ) in the indonesian clade and for lineages in other h n hpai clades (e.g., fujian, qinghai, thailand and vietnam clades which have dn/ds ranging from . to . ). this lineage-specific elevation of dn/ds was not significant (lrt p. . ) for other genes (i.e. ha, na, m , pb ; see table s ). four amino acid changes in m occurred along the pre-emergence lineage, including threonine to alanine at reside , arginine to lysine at reside , threonine to alanine at reside , glutamine to histidine at reside . three (residue , , and ) of them are located close to the electrostatic positive surface of the n-terminal domain of the m protein molecule ( figure s ), and one (residue ) is located in the remaining c-terminal fragment. this study classified h n hpai viruses in indonesia into three distinct viral lineages (groups , , and ) and discovered a group of naturally occurring reassortant viruses that represent a newly emergent h n hpai strain in java in . several phylogenetic methods concurred that two (mp and pb ) of the reassortant viruses' genome segments descended from the group ancestral viruses, and the remaining six (pb , pa, ha, np, na, ns) segments descended from the group ancestral viruses. although the majority of reassortant viruses ( / ) are human isolates, few of the associated human infections are epidemiologically linked (table ) , suggesting multiple sporadic zoonotic transmissions from birds. the phylogeographic results indicate that the parental viruses of the reassortants have been co-circulating in java since . despite the identification of parental lineages, the exact number of reassortment events remains difficult to assess. although the three fairly consistent phylogenetic subgroups (subgroups r , r , and r in figure s ) formed by the reassortant viruses suggest three independent reassortments, the underlying uncertainty in our estimated phylogenies means that we cannot rule out the possibility of a single origin. the hypothesis of three reassortments implies that the viruses have acquired exactly the same genome segments from the same group of parental viruses, which seems unlikely to occur by chance (probability = . , assuming panmixis and that exactly two genomic segments are swapped out). this probability might be increased if reassortments confer a selective advantage. we did detect a significantly stronger selection pressure on the m protein in the pre-emergence lineage of group parental strain that led to the reassortant viruses (table s ) . previous reports suggested a few amino acid changes in m of influenza a and b viruses can confer a growth advantage in mouse lungs [ ] [ ] [ ] . although the m mutations identified in this pre-emergence lineage have not been functional characterized elsewhere in the authors' knowledge, one (residue , tra) of them is close to a previously characterized mutation (residue , tra) which controls the virulence in mouse model [ , ] . three of the inferred residue changes are located close to the electropositive surface of n-terminal domain of m protein ( figure s ) that acts to bind viral rna [ , ] . the m matrix protein mediates encapsulation of viral rnanucleoproteins into membrane envelope during packaging [ ] , and has close contact with other viral proteins inside the viral particle. it seems possible that some of these changes may be involved in the adaptation of reassortant viruses, through promotion of structural interactions among viral proteins. according to our analyses, the common ancestor of the reassortant viruses is dated to july (hpd: april-october), approximately months prior to the first case of human infection caused by the reassortant virus (index case # defined by who; see table ). our analysis of virus phylogeography suggests the ancestors of these reassortant viruses first arose in greater jakarta and surroundings, which agrees with the observation that the first two cases of human infection by the reassortant viruses occurred in central and east jakarta (index cases # and # ). the molecular dating and phylogenetic analyses suggest that nascent reassortant viruses might take several months to spread and expand their diversity in the local bird population, eventually leading to the exposure of human population. the subsequent spread of the reassortant strain seems to become more rapid and extensive, as human cases were reported outside greater jakarta one month later, and the reassortant virus spread to as far as the south and east of west java in the following six months (table and figure ) . commercial poultry transportation, as well as carriage by migratory birds, may facilitate the viral migration, but their tangible contributions need further studies. our results suggest that the circulations of reassortant viruses and their genetic parent (group ) were not restricted by geography within java. the viral migration back to greater jakarta could be driven by the inter-province transfer of infected poultry, in particular the importation of live poultry or fresh poultry products to the densely human populated jakarta from the remote provinces engaged in poultry-farming. future studies on economic and social geography (e.g., addressing the modes of inter-provincial poultry transport) in indonesia might help to further elucidate the effect on the viral dispersal by human, agricultural and industrial activities. in this study, we opted for a lower geographical resolution (i.e., four widely ranged geographical states instead of distinct geographical coordinate for each viral isolate) in our phylogeographic analyses because of the varying precision of the geographical data we have. therefore, more complex hypotheses of viral origin and migration trajectory cannot be investigated here, but can be explored when more high-quality geographical data of indonesian h n viral samples is available. the bsp analyses ( figure e- h) indicate that the reassortant viruses follow a logistic-like growth curve, which is typical for virus invasion and maintenance, especially in a structured population [ , ] . in contrast, the group viruses followed a more continuous and relatively slow growth in diversity. there was insufficient data in our samples to definitively discriminate between alternative population growth models and provide narrow confidence intervals for parameter estimates, but our results are suggestive and future sequencing will add to the needed statistical power. what factors have contributed to the apparent difference in the growth of genetic diversity? rates of molecular evolution between the two groups were similar ( figure s ) and therefore are not likely to be the cause. since our analyses could not resolve the temporal dynamics of population subdivision by geography, we cannot directly investigate how viral genetic variation is affected by the population structure. we expect future development of analysis methods will help to shed more light on the interaction between viral migration and genetic diversity. analysis of clinical records (table ) found that the mean duration from onset to death in those fatal human cases caused by indonesian reassortant h n viruses is . days (standard deviation [sd] = . ; n = ) and those caused by other indonesian h n viruses is . days (sd = . ; n = ), and their means are not significantly different (student t-test, p. . , two-tails). therefore, based on the clinical records, the reassortant viruses did not kill human faster than other indonesian h n viruses did. however, we would recommend more experimental studies addressing the virulence, pathogenicity and immunogenicity of the reassortant viruses and the parent strains to verify this claim in the future. mechanisms of viral transmissions are sometimes correlated with genetic diversity dynamics. for example, hepatitis c viruses transmitted by drug injection or blood products have a faster rate of spread than endemic strains circulating in asia and africa [ ] . it has also been suggested that mosquito susceptibility may affect the growth of dengue viruses [ ] . therefore, it is possible that a change of host species could generate the difference in the viral dynamics we observe. in our study, the majority of the reassortant viruses ( / ) were isolated from humans, whereas only a minority of the group viruses were isolated from humans ( / and / in the ha and na datasets, respectively). it has been previously shown that the receptor binding specificity of hemagglutinin [ ] and mutations in the viral polymerase (e.g., lysine at residue of pb ) [ ] [ ] [ ] can determine viral transmissibility and replication in different host species. none of the aforementioned ha mutations which confer recognition to human-type host cell receptors [ , ] were found in the indonesian reassortant viruses; however, our detection of positively selected sites in the pb gene of the reassortant viruses could potentially reflect adaptation to mammalian hosts, and requires further investigation. in particular, amino acid changes on two positively selected sites (threonine to methionine at reside , glutamic acid to glycine and alanine at reside ) were found on the internal branches of the reassortant lineage, corresponding to molecular changes during sustainable transmissions. however, some of these positively selected changes may also result from the compensatory evolution as the mix of genome segments from different strains might alter their epistatic physiochemistry [ ] . although most of the human isolates in our datasets were epidemiologically unlinked, such linkage is theoretically possible if many asymptotic or mildly manifested human infections are not reported. recently, some evidence of subclinical or asymptotic h n infection in humans has been put forward [ , ] ; however, the ability of the viruses to transmit from these infected individuals to other susceptible individuals remains unknown. the possible role of other animal host species in the transmissions of reassortant viruses in indonesia should not be neglected. in particular, one of the reassortant viruses was isolated from a dead cat in jakarta, where h n outbreaks in poultry and sporadic human infections have been reported [ ] . moreover, unusual high mortality of cats in the vicinity of h n hpai outbreaks has been reported [ ] . an unofficial report also detected h n hpai sero-positivity in around % of blood samples taken from stray cats near poultry markets in java and sumatera [ ] . in addition to small cats in germany [ ] , iran [ ] , and indonesia [ ] , dogs and zoo tigers were also found infected with h n hpai viruses in thailand [ , ] . furthermore, previous experimental studies have demonstrated that cats can be infected with h n hpai virus [ , ] , and that cat-to-cat transmission is possible [ , ] . could cats, or other non-avian species, have played a role in spreading the reassortant viruses in java? similarly, could cats act as amplifying hosts facilitating viral expansion and cross-species transmission, as civets did in the sars outbreaks [ ] ? future experimental studies on these reassortant viruses, that assess viral transmissibility between species, together with epidemiological studies, such as viral monitoring within indonesian animal populations using serological tests and pcr detection, would give more clues to these questions. h n hpai viruses have been endemic and evolved into different genetic lineages that have spread across indonesia. areas where more than one lineage of virus is co-circulating, such as jakarta, are most likely to generate novel viruses by inter-lineage reassortment. these reassortant viruses have distinctive evolutionary and transmission dynamics, as shown in this study. we suggest that more intensive and timely field surveillance and analysis of influenza viruses, including h n hpai and human h n , h n , and h n epidemic strains, should be employed, so that bio-security can be undertaken promptly and appropriate strains can be selected for vaccine production whenever a novel reassortant strain emerges. the reassortant viruses reported in this study should be also added to the watch list for the future epidemiological surveillance. sequence data collection and alignment h n influenza viruses isolated from avian and mammalian hosts in indonesia during - were studied. their genomic sequences (n = ) were extracted from the influenza virus resource [ ] and the influenza sequence database [ ] in september , and aligned using muscle version . [ ] . columns with gaps were removed from the alignments, and sequences from the same virus strain (duplicated submission in the two databases) were filtered such that one copy was retained. eight genome segment alignment datasets (pb , pb , pa, ha, np, na, mp, and ns), as well as four coding sequences (m , m , ns , and ns ), were generated. full details of our datasets can be found in table s and s . phylogenetic trees of alignment datasets were reconstructed using the ml approach implemented in phyml . [ ] . the robustness of the ml tree topology was assessed by comparing the ml topology with the topologies sampled in the bmcmc analysis performed in mrbayes version . . [ ] , and with bootstrapping analyses of , pseudo-replicate datasets. ml and nj trees were estimated from the bootstrap datasets using phyml [ ] and paup* version beta [ ] , respectively. a general-time-reversal (gtr) substitution model with gamma distributed rate heterogeneity of rate categories (c ) and a proportion of invariable sites were used in all tree reconstruction methods. phylogenies were rooted with the h n hpai strain a/ck/hk/yu / , which is genetically close to the newly reported hunan strains [ ] , and shares comparable genetic proximity to indonesian clade. homologous recombination within each gene segment among indonesian h n isolates was extensively searched using recombination detection program version (rdp ) [ ] , and the datasets are found to be free of homologous recombination. putative reassortant viruses were preliminarily identified by their topological incongruity across the phylogenies of different gene segments. this was further investigated using a smaller set of indonesian h n virus isolates with full genome sequences, which included sequences of early viruses (n = ), group - lineages (n = ) and putative reassortant viruses (n = ). the eight gene segment alignments were manually concatenated in the order of their length to generate a single alignment of complete genome sequences, and was further analyzed using ) similarity plots and ) bootscan analyses [ ] implemented in simplot version . . [ ] , and ) gard [ ] available via the datamonkey website [ ] . the hypothesis of reassortment was supported if the recombinant breakpoints were detected near the junctions where the genome segments were manually concatenated. the geographic locations of virus isolation were either obtained from the sequence databases, or obtained through personal communication with catherine smith (from disease control and prevention, atlanta, usa), or inferred from their strain names (tables and s ). the locations of isolates were indicated on the map of main island of java in indonesia (figure ). due to the limit of our geographical data, the localities of the isolates shown in the map (figure ) should be regarded as arbitrary within the province which is the highest precision level shared by all viral samples. each of the reassortant viruses was assigned with a state of either greater jakarta (surroundings included) or west java depending on its place of origin ( table ). the migratory history of the reassortant viruses (n = ) between these geographical states were inferred based on the refined ml phylogeny of ha and na ( figure s ) independently using two parsimony optimization methods, called acctran (accelerated transformation) and deltran (delayed transformation) implemented in paup* software. the geographical states of all ancestral nodes in the tree were estimated to achieve minimum state changes in overall, and therefore the number of state changes and state of the mrca of the reassortant was obtained. polytomies were randomly resolved , times, and state changes were estimated separately for each resolution. the mean number of state changes was then calculated. to test against the null hypothesis of completely unrestricted migration between geographical states (panmixis), the mean number of observed state changes was compared with the frequency distribution of the mean number of expected state changes under the null hypotheses. the null distribution and critical values were generated by randomly shuffling the states of isolates , times (the slatkin-maddison test [ , , ] ). the migratory history of group viruses was also studied using the ha gene in a similar manner, while each group virus was assigned to either of four widely ranged geographical states: greater jakarta and surroundings, the rest of java, sumatra, and sulawesi selatan, including papua. this assignment scheme is comparable to that of reassortant viruses, as west java is part of java. parameters of codon-partitioned substitution rates, demographic functions, tmrca and tree topologies were co-estimated from ha and na gene datasets of reassortant and group viruses separately in a bmcmc framework [ ] using beast version . . [ ] . substitution model hky+c with invariable site portion was used. isolation dates were used to calibrate the molecular clock. three clock models including strict clock, ucen and ucln relaxed clocks [ ] were attempted independently, and the best-fit clock model was selected by comparing the bf calculated from their posterior distributions [ ] . the bayesian skyline plot [ ] was used to estimate population dynamics, in terms of relative genetic diversity. less complex parametric demographic models (constant size, exponential growth and logistic growth) were applied independently, and the best-fit models selected by bf tests were used to quantitatively estimate the growth rate and other demographic parameters. the bmcmc analyses contained states, with sampling every , states, and the first % of each chain was discarded as burn-in. convergences and effective sample sizes of the estimates were checked using tracer v . [ ] . positively selected sites were detected using random effect likelihood (rel) and fixed effect likelihood (fel) methods [ ] via the datamonkey website [ ] . bayes factors larger than and pvalues smaller than . were used as thresholds for strong evidence of selection in rel and fel, respectively. to test lineage-specific positive selection, the two-ratio branch model was used, which pre-specifies a single rate of synonymous substitution (ds) for the whole phylogeny and two rates of non-synonymous substitution (dn and dn ). the dn was specified for the pre-emergence lineage (indicated as the ancestral branch connecting the group mrca; see figure b ) for the group viruses (including the reassortant viruses for m , m , and pb genes). the dn was specified for other lineages across the phylogenies. the ml estimates of these rate parameters were performed in hyphy version . [ ] . the resulting likelihood score of the two-ratio model was then compared with that of the one-ratio model, which assumes the same dn and ds across the phylogeny, using the likelihood ratio test (lrt, with degree of freedom = ). the substitution model mg xgtr+c was used. the ancestral nucleotide sequences of all internal nodes were reconstructed using joint ml method [ ] implemented in hyphy. amino acid changes along the pre-emergence lineage were determined, and were then mapped onto the three-dimensional structure of the n-terminal domain of m matrix protein molecule [ ] available (pdb-id: ea ) in rcsb protein data bank. figure s substitution rates of ha and na genes from reassortant and group parental strains. % higher probability densities (hpds) are indicated by the error bars. st, nd, rd, and c denote the rate for the st codon position, nd codon position, rd codon position, and whole sequence (non-partitioned), respectively. substitution rate units for codon partitioned and non-partitioned sequences are substitution/codon/year and substitution/site/year, respectively. table s estimations of dn/ds using -ratio and -ratio lineage-specific selection models. these estimations were performed in hyphy software. gene datasets other than pb , ha, na, m , and m were not analyzed because group is represented by the single virus idn/ / . found at: doi: . /journal.ppat. .s ( . mb doc) information and phylogenetic groupings of sequences used in this study. , , , and x denotes groups , , , and unclassified (early viruses; see main text for explanation). empty entries indicate the unavailability (e.g., no sequence found, too short, too many ambiguous codes, and too many gaps) of the sequence. genetic characterization of the pathogenic influenza a/goose/guangdong/ / (h n ) virus: similarity of its hemagglutinin gene to those of h n viruses from the outbreaks in hong kong characterization of avian h n influenza viruses from poultry in hong kong characterization of an avian influenza a (h n ) virus isolated from a child with a fatal respiratory illness clinical features and rapid viral diagnosis of human disease associated with avian influenza a h n virus characterization of h n influenza viruses that continue to circulate in geese in southeastern h n influenza-continuing evolution and spread highly pathogenic avian influenza in indonesia. jakarta, indonesia. in: agriculture do avian influenza: the indonesian experience epidemiology of cases of h n virus infection in indonesia evolution and adaptation of h n influenza virus in avian and human hosts in indonesia and vietnam three indonesian clusters of h n virus infection in human transmission but no pandemic in indonesia cumulative number of confirmed human cases of avian influenza a (h n ) h n influenza: a protean pandemic threat genesis of a highly pathogenic and potentially pandemic h n influenza virus in eastern asia establishment of multiple sublineages of h n influenza virus in asia: implications for pandemic control identification of the progenitors of indonesian and vietnamese avian influenza a (h n ) viruses from southern china estimating mutation parameters, population history and genealogy simultaneously from temporally spaced sequence data evolutionary analyses of european h n swine influenza a virus by placing timestamps on the multiple reassortment events bayesian selection of continuoustime markov chain evolutionary models relaxed phylogenetics and dating with confidence a cladistic measure of gene flow inferred from the phylogenies of alleles bayesian coalescent inference of past population dynamics from molecular sequences invasion and maintenance of dengue virus type and type in the americas genetic analysis reveals the complex structure of hiv- transmission within defined risk groups not so different after all: a comparison of methods for detecting amino acid sites under selection pattern of mutation in the genome of influenza a virus on adaptation to increased virulence in the mouse lung: identification of functional themes a single amino acid change in the c-terminal domain of the matrix protein m of influenza b virus confers mouse adaptation and virulence the influenza virus variant a/fm/ / -ma possesses single amino acid replacements in the hemagglutinin, controlling virulence, and in the matrix protein, controlling virulence as well as growth combined results from solution studies on intact influenza virus m protein and from a new crystal form of its n-terminal domain show that m is an elongated monomer structure of a bifunctional membrane-rna binding protein, influenza virus matrix protein m the epidemiology and iatrogenic transmission of hepatitis c virus in egypt: a bayesian coalescent approach differential susceptibility of aedes aegypti to infection by the american and southeast asian genotypes of dengue type virus haemagglutinin mutations responsible for the binding of h n influenza a viruses to human-type receptors molecular basis for high virulence of hong kong h n influenza a viruses molecular basis of replication of duck h n influenza viruses in a mammalian mouse model pb amino acid at position affects replicative efficiency, but not cell tropism, of hong kong h n influenza a viruses in mice structure and receptor specificity of the hemagglutinin from an h n influenza virus the genomic and epidemiological dynamics of human influenza a virus safety and immunogenicity of an inactivated adjuvanted whole-virion influenza a (h n ) vaccine: a phase i randomised controlled trial confronting potential influenza a (h n ) pandemic with better vaccines can cats spread avian flu? animal health special report deadly h n may be brewing in cats new scientist molecular analysis of highly pathogenic avian influenza virus of subtype h n isolated from wild birds and mammals in northern germany qinghai-like h n from domestic cats, northern iraq genetic analysis of influenza a virus (h n ) derived from domestic cat and dog in thailand avian influenza h n in tigers and leopards avian h n influenza in cats influenza a virus (h n ) infection in cats causes systemic disease with potential novel routes of virus spread within and between hosts probable tiger-to-tiger transmission of avian influenza h n isolation and characterization of viruses related to the sars coronavirus from animals in southern china the influenza virus resource at the national center for biotechnology information the value of a database in surveillance and vaccine selection muscle: multiple sequence alignment with high accuracy and high throughput a simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood mrbayes : bayesian phylogenetic inference under mixed models phylogenetic analysis using parsimony rdp: detection of recombination amongst aligned sequences identification of breakpoints in intergenotypic recombinants of hiv type by bootscanning full-length human immunodeficiency virus type genomes from subtype cinfected seroconverters in india, with evidence of intersubtype recombination gard: a genetic algorithm for recombination detection datamonkey: rapid detection of selective pressure on individual sites of codon alignments a statistical phylogeography of influenza a h n beast: bayesian evolutionary analysis by sampling trees hyphy: hypothesis testing using phylogenies a fast algorithm for joint reconstruction of ancestral amino acid sequences we gratefully acknowledge the sharing of virus samples by ministry of health, indonesia, and thank the scientists in h n reference laboratories for genome sequences. we thank catherine smith for her help in refining the geographical data used in this study. we also thank a.m. vandamme, philippe lemey, andrew rambaut, alexei drummond and the anonymous reviewers for their helpful advices. we gratefully acknowledge the support of biosupport and hpcpower projects for providing bioinformatic and computational services from computer centre in the university of hong kong. we also thank w. k. kwan and frankie cheung for their computational assistance. conceived and designed the experiments: ttyl fccl. analyzed the data: slkp rtyw. contributed reagents/materials/analysis tools: rtyw. wrote the paper: slkp. collected and organized the sequence data: ttyl rtyw cwy fz. performed the research experiments and analyses: ttyl. analyzed the data and wrote the manuscript: ttyl cch ogp slkp fccl. key: cord- - sdg ll authors: guo, sheng; yang, chengying; diao, bo; huang, xiaoyong; jin, meihua; chen, lili; yan, weiming; ning, qin; zheng, lixin; wu, yuzhang; chen, yongwen title: the nlrp inflammasome and il- β accelerate immunologically mediated pathology in experimental viral fulminant hepatitis date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: sdg ll viral fulminant hepatitis (fh) is a severe disease with high mortality resulting from excessive inflammation in the infected liver. clinical interventions have been inefficient due to the lack of knowledge for inflammatory pathogenesis in the virus-infected liver. we show that wild-type mice infected with murine hepatitis virus strain- (mhv- ), a model for viral fh, manifest with severe disease and high mortality in association with a significant elevation in il- β expression in the serum and liver. whereas, the viral infection in il- β receptor-i deficient (il- r (-/-)) or il- r antagonist (il- ra) treated mice, show reductions in virus replication, disease progress and mortality. il- r deficiency appears to debilitate the virus-induced fibrinogen-like protein- (fgl ) production in macrophages and cd (+)gr- (high) neutrophil infiltration in the liver. the quick release of reactive oxygen species (ros) by the infected macrophages suggests a plausible viral initiation of nlrp inflammasome activation. further experiments show that mice deficient of p (phox), a nicotinamide adenine dinucleotide phosphate (nadph) oxidase subunit that controls acute ros production, present with reductions in nlrp inflammasome activation and subsequent il- β secretion during viral infection, which appears to be responsible for acquiring resilience to viral fh. moreover, viral infected animals in deficiencies of nlrp and caspase- , two essential components of the inflammasome complex, also have reduced il- β induction along with ameliorated hepatitis. our results demonstrate that the ros/nlrp /il- β axis institutes an essential signaling pathway, which is over activated and directly causes the severe liver disease during viral infection, which sheds light on development of efficient treatments for human viral fh and other severe inflammatory diseases. viral fulminant hepatitis (fh) is a clinical syndrome characterized by massive necrosis of hepatocytes along with hepatic encephalopathy during the infections [ ] . despite advances in the development of antiviral drugs, a poor understanding of the immune mechanisms underlying viral fh has largely stalled the identification of effective clinical interventions. fortunately, the recent development of an animal model of fh using murine hepatitis virus strain- (mhv- ) infection has provided insights in understanding the pathogenesis and developing novel therapeutics for the disease [ ] . mhv- is a single-stranded, positive-sense rna virus belonging to the coronavirus family [ ] . the hallmarks of mhv- -induced fh in susceptible balb/cj and c bl/ mice include the appearance of liver sinusoidal thrombosis and hepatocellular necrosis, resulting from over expression of a virus-induced, monocyte/macrophage-specific procoagulant, fibrinogen-like protein- (fgl ). liver accumulation of fgl directly activates the coagulation cascades, a phenomenon known as virus induced procoagulant activity [ ] . mhv- -induced fh exhibits a syndrome that is very similar to the clinical manifestations of patients with viral fh, making it a good animal model for exploring mechanisms underlying the pathogenesis of human viral fh. in addition to fgl , pro-inflammatory mediators such as tnf-α, ifn-γ and complement c a have been proposed to accelerate viral fh pathogenesis [ , ] . nevertheless, the mechanisms on how the inflammatory signaling events that regulate the disease progression are not well understood. recently, it has been shown that dysregulated nlrp (also known as nalp and cryopyrin) inflammasome in macrophages causes the pathogenesis of inflammatory diseases, which highlights the importance of inflammasome in regulating immune-mediated tissue damages [ ] . the generation of biologically active il- β requires cleavage of the inactive precursor proil- β by the nlrp inflammasome, a protein-scaffolding complex consisting of nlrp , caspase- , and the adaptor molecule asc (apoptosis-associated peck-like protein with card domain, pycard) [ , ] . nlrp inflammasome and il- β mediate the host protection against pathogen invasions, whereas, the hyperactivation of nlrp inflammasome contributes to the pathogenesis of certain inflammatory syndromes, including liver injuries such as nonalcoholic/alcoholic steatohepatitis [ , ] , liver fibrosis [ ] , and immune mediated liver injuries [ ] . however, the role of nlrp inflammasome signaling pathway participates in the pathogenesis of viral fh is still unclear. a variety of danger-associated molecular patterns (damps) and pathogen-associated molecular patterns (pamps), including virus rna, nigericin, atp, silica crystals, mitochondrial dna, and aluminum hydroxide, appear to be capable of activating the nlrp inflammasome [ ] . nevertheless, the reactive oxygen species (ros) generated by nicotinamide adenine dinucleotide phosphate (nadph) oxidase are considered to be one of the major factors that activate nlrp inflammasome [ ] . it has been shown that pharmacological inhibition of the nadph oxidase complex (nox) or the down regulation of the nox subunit p phox eliminates nlrp inflammasome activation by preventing ros secretion [ , ] . however, recent studies have also illustrated that mitochondria-originated ros (mitosox) rather than noxderived ros drive nlrp inflammasome activation [ , ] . various stress condition, including increased metabolic rates, hypoxia, or membrane damage, all significantly induce mitosox secretion [ ] . conversely, it remains uncertain for which of the nox-derived ros or mito-sox is responsible for causing nlrp inflammasome-dependent pathology in viral fh development. here, we showed that c bl/ wild type (wt) mice infected with mhv- manifest with high levels of il- β in the serum and liver. conversely, the virus infected il- r -/mice present with much attenuated pathologies, showing with a significant reduction in macrophagederived fgl expression and less liver infiltration of cd + gr- high neutrophils. furthermore, we showed that the in vivo bioactivation of proil- β during mhv- infection is mediated by nlrp inflammasome activation, thereafter, both the nlrp -/mice and the caspase- -/mice display substantial resistance to mhv- -induced il- β production. mechanistically, mhv- infection triggers an acute release of nox-derived ros. blocking ros with diphenyleneiodonium chloride (dpi) inhibits caspase- activation and il- β maturation in vitro. furthermore, nox subunit p phox -deficient mice also exhibited a delayed and moderate viral pathogenesis due to reduction in nlrp inflammasome activation in vivo. these results reveal that the ros/nlrp /il- β axis is a critical signaling pathway leading to the pathogenesis of viral fh. to examine the status of il- β activation in macrophages in response to mhv- infection, primary peritoneal exudative macrophages (pems) and the macrophage line-raw . cells were infected with the virus in vitro. a time course data showed a significant induction of the activated form of il- β (il- β p ) within hours, sustaining to h (fig a) . assessment of the pems isolated from the h of virus infected c bl/ wt mice also revealed a significant increase in proil- β mrna expression ( fig b) . moreover, proil- β mrna expression in the infected livers appeared to be markedly augmented at h (p = . ), sustaining to h (p = . , fig b) . in accordance, western-blotting showed with increases in proil- β and il- β p protein expression at corresponding time points in the infected livers ( fig c) . flow cytometry further validated the patterns of proil- β protein induction in the pems isolated from the virus-infected mice ( fig d) . in agreement, the infected mice also showed significant accumulation of serum il- β during the infection (fig e) . in contrast, serum il- α concentration exhibited little change in mhv- infected mice ( fig f) . these results suggest that il- β significantly elevate in the liver and periphery during viral fh. intervention of il- β signaling reduces mhv- -mediated hepatitis il- β amplifies the pro-inflammatory response via the type-i of il- receptor (il- r ) [ ] . to further investigate whether il- β signaling affects the pathogenesis of viral fh, we infected il- r -/mice with mhv- ( pfu) via intraperitoneal (i.p.) injection. interestingly, il- r -/mice displayed with a significant increase in survival rate with % staying alive for days, as compared to a % death of the wt littermates within days of the viral infection (fig a) . il- r -/mice manifested a significant reduction in hepatocellular damage and a decrease in serum alt/ast levels during the infection (fig b) . the expression of biliary glycoprotein- (bgp ), the receptor for mhv- [ ] , appeared to be significantly lower in the virus infected il- r -/livers comparing to that in the wt controls (fig c) , concurring with the plaque assay data showing with limited virus entrance and amplification in the livers h post-infection ( fig d) . in support, the mhv- infection efficiency in il- r -/-pems dropped more significantly than in the wt counterparts in vivo ( fig e) . obviously, recombinant mouse il- β protein ( ng/ml) is able to significantly induce bgp expression in pems and raw . cells in vitro (fig f) , and in concurrence, il- β treated raw . cells appear to produce more virus than the pbs treated controls post-infection ( fig f) . in validation, we injected the virus-infected wt mice with il- r antagonist (il- ra, mg/kg/day), a naturally occurring cytokine that blocks il- β biologic response [ ] , and observed a significant limitation of il- β secretion (p = . , s a fig mhv- fails to induce fgl production and liver neutrophil infiltration in il- r -/mice fgl plays an essential role in inducing hepatocellular necrosis following mhv- infection [ ] . we firstly examined fgl expression in pems isolated from mhv- infected il- r -/mice and observed substantial lower levels of fgl as compared to the wt controls ( fig a) . the limited fgl expression in macrophages correlates with the low concentrations of fgl observed in the virus infected il- r -/liver and serum (fig b and c ). therefore, in response to mhv- viral infection, il- r -/mice responded with limited fibrinogen formation, leading to a down modulation of liver coagulation and necrosis ( fig d) . similarly, il- ra-treated wt mice displayed with reduction of fgl and fibrinogen deposition in liver tissues, which was followed with decrease in liver damages and enhance the survival time (s b, s d and s e fig) . neutrophils and cd + foxp + regulatory t cells (tregs) have been well recognized as important players in viral fh [ , ] . to determine the role of il- β in regulating these cells during viral fh, we firstly examined liver neutrophil infiltration status. flow cytometry showed that in the liver-tissue samples from and h post mhv- infection, the infiltration of cd + gr- high neutrophils was substantially higher in the wt livers than that in the il- r -/littermates (fig e and f ). the number of cd + foxp + treg in the virus-infected livers appeared to increase significantly after mhv- infection, nevertheless, little difference was observed between il- r -/mice and their wt controls (s fig). similarly, serum concentration of c a, a cytokine that deteriorates the pathogenesis of mhv- -mediated fh [ ] , was not changed dramatically between virus infected il- r -/mice and their wt controls (s a fig). these results suggest that attenuation of viral fh by il- r deficiency could be the consequence of both ineffective fgl production by macrophages and limited cd + gr- high neutrophil infiltration in the affected liver. a reduction of fgl expression was observed in il- r -/mice in response to mhv- infection, together with il- β and fgl were co-expression in pems (fig a) , implying that il- β/ il- r interactions may directly regulate fgl expression in macrophages. to address the issue, we treated raw . cells, a macrophage line capable of expressing fgl , with the recombinant mouse il- β protein ( ng/ml) in vitro. qpcr and western-blotting data showed that il- β alone is incapable of stimulating flg expression, nevertheless, it synergistically enhances tnf-α-induced fgl levels (fig b and c ). the expression of fgl has been proposed to be mediated through the activation of nf-κb and mitogen-activated protein kinase (mapk) signaling pathways under inflammatory conditions [ , ] . to further investigate the molecular mechanisms through which il- β promotes fgl production, we examined these signaling pathways in il- β-treated raw . cells. results showed that either il- β or tnf-α treatment alone, had a minimum stimulation on phosphorylation of the nf-κb chaperone iκbα (p-iκbα) and the nf-κb subunit p (p-p ), appearing only at extended incubation time point ( h). however, synergistic effects of il- β and tnf-α (il- β+tnf-α) seemed to be significant for which substantial increases in phosphorylation of iκbα and p can be detected as early as h post infection ( fig c) . furthermore, the inhibition of nf-κb activation by pyrrolidinedithiocarbamic acid (pdtc) successfully prevented fgl upregulation after il- β+tnf-α treatment ( fig d) . the combination of il- β and tnf-α is capable of potently stimulation the phosphorylation of mapks, including extracellular signal-related kinase (p-erk / ) and p (pp ) (fig c) . nevertheless, the erk inhibitor-pd and the p -mapk inhibitor-sb seemed to be incapable of blocking fgl upregulation. moreover, blocking all of these three pathways did not show additive effect on inhibition of fgl expression ( fig d) . these results suggest that nf-κb rather than the mapk pathways is responsible for il- β+tnf-α-mediated fgl upregulation in viral infected macrophages. it has been established that the caspase- -mediated bio-activation of proil- β is under the control of nlrp inflammasome [ ] . mhv- infected pems and raw . cells exhibited with a significantly enhanced nlrp , asc, pro-caspase- and its activated form (caspase- p ) within h of mhv- infection ( fig a) . in accordance, qpcr analyses illustrated that the mrnas for nlrp and procaspase- were significantly higher in the virus infected livers, this correlates with observation that these virus infected livers also manifest with higher expression of the respective protein ( fig b) . next, we infected nlrp -/mice and caspase- -/mice with mhv- to address the importance of nlrp inflammasome in the causing the virusinduced liver injuries. remarkably, a h viral infection largely failed to induce il- β expression in the livers, which was associated with significant reductions in liver fgl accumulation (fig c) , fibrinogen deposition and local tissue damages, along with significant decreases in serum alt/ast enzymes as compared with the infected wt mice (fig d) . in agreement with these results, we also observed that bgp expression was significantly lower in nlrp -/and caspase- -/livers during infection ( fig c) . meanwhile, nlrp -/mice and caspase- -/mice appeared to produce much less viruses at h of infection as compared to the wt controls ( fig e) . finally, nlrp -/and caspase- -/mice presented with considerably prolonged survival rates toward mhv- infection in comparing to the wt controls ( fig f) . the serum c a in the viral infected nlrp -/and caspase- -/animals was also significantly increased but no different from the wt control mice (s b fig), indicating that c a up-regulation during the viral infection, appears to either additively or synergistically work with other inflammatory factors to cause viral fh. together these observations further validate that the nlrp /caspase- -inflammasome regulates the bio-processing of proil- β for causing the mhv- mediated viral fh. assembly and activation nlrp inflammasome, being critical for bio-processing and activation of il- β, has been suggested to also involve in the bio-activation of il- , another member of the il- superfamily [ ] . the mhv- -infected mice showed a significant up-regulation of proil- mrna in pems and livers (fig a) , as well as enhanced il- protein in serum ( fig b) . however, the recombinant mouse il- protein ( ng/ml) alone, or in the combination with tnf-α and inf-γ, was unable to stimulate fgl mrna transcription in raw . cells or sve- endothelial cells in vitro (fig c) . moreover, mhv- induced liver fgl production remained high in il- -/mice (fig d) , showing with consequentially high levels of fibrinogen deposition, liver damages and hepatocyte necrosis ( fig e) . additionally, liver tissues isolated from il- -/mice appear to up-regulate bgp expression after mhv- infection. in accordance, these mice also manifested with high virus duplication ( fig f) . overall, il- -/mice are still sensitive to mhv- infection (fig g) , suggesting that il- is not essential in mhv- -mediated fulminant hepatitis. many factors contribute to activating the nlrp inflammasome and among which, ros is lately gaining particular attentions [ ] . in order to examine the role of ros in nlrp inflammasome hyperactivation, we first detected the release of nadph oxidase-derived ros by using a permeable dichlorohydrofluorescein (dcfh) upon mhv- infection. flow cytometry showed that the releasing of dcfh from mhv- infected pems and raw . cells significantly increased, especially at h and h post-infection (fig a) . this result correlates with the up-regulation of gp phox , p phox and nox , the subunits that are essential for acute ros secretion in raw . cells (fig b) . however, the dcfh level dropped dramatically at h in addition to nadph oxidase-derived ros, mitochondria may provide an alternate source of ros [ ] . we therefore assessed the functional mitochondrial pool in mhv- infected cells. the viral infection in pems and raw . cells caused an increase in mitochondrial damage, especially at h and h post-infection, as detected by mitotracker green fm, a dye that stains mitochondria with no influence on their membrane potentials (fig a) . similarly, electron microscopy showed with swollen mitochondria in the mhv- infected raw . cells at h and h (fig c) . this sign of mitochondrial damage seemed to strongly correlate with the increase in mitosox release within the same time frame (fig a) . to further elucidate the role of ros in nlrp inflammasome hyperactivation, we treated mhv- infected raw . cells with a ros inhibitor diphenyliodonium chloride (dpi), which is capable of preventing both nox-dependent ros and mitoxos secretion [ ] . noxoriginated dcfh was successfully inhibited by dpi in a dose dependent manner (fig d) . however, mitoxos release was not prevented by the dpi treatment, even at a very high dose ( μm) (fig d) . the efficiency of nox-originated ros inhibition by dpi appeared to correlate with the reduction in il- β activation in the infected raw . cells and pems in dose dependent manners (fig e) . together, these results suggest that the hyperactivation of nlrp inflammasome in macrophage is partially mediated by mhv- induced, nox-derived ros. p phox-/mice are resistance to mhv- induced fh by limiting nlrp inflammasome hyperactivation cells in deficiency of p phox exhibit a reduced capacity in generating ros [ ] . to further investigate the role of nox-originated ros in regulating nlrp inflammasome hyperactivation, we infected p phox-/mice with mhv- and examined the severity of liver pathology. as anticipated, pems isolated from mhv- infected p phox-/mice showed with limited dcfh (fig a) . interestingly, the p phox-/mice also displayed considerable resistance to mhv- infection, presenting with reduced disease severity within the prolonged survival time as compared with the wt controls (p = . , fig b) . the lack of virus-induced ros response, which leads to prohibition of nlrp /caspase- activation and thus reduction in il- β production, seems to be responsible for this effect (fig c and d) . as a result, the virus infection is unable to generate significant fgl accumulation in the liver and serum (fig c and d) . therefore, these mice manifested with less severe fibrinogen deposition, liver injury and hepatocyte necrosis, accompanying with low levels of ast/alt enzymes released by the liver (fig e) . however, the limitation of il- β secretion in these p phox-/mice only slightly affected liver bgp expression (fig c) , and therefore live virus titers were still high at h of infection ( fig f) . conversely, the administration of il- β ( ng/mouse/day) in mhv- infected p phox-/mice was able to reinstate all aspects of disease severity typical in viral fh (figs g and s ) . taken together, these results clearly indicate that the ros/nlrp /il- β axis plays a critical role in the pathogenesis of viral fh. in the present work, we report that mice infected with mhv- , an animal model for viral fh, have significantly elevated levels of il- β in the serum and liver. the accumulation of il- β accelerated liver pathology through synergistically acting with tnf-α, one of the key inflammatory cytokines that has been previously shown to be essential for causing viral fh [ , ] , il- r signaling is responsible for stimulation of fgl expression in macrophages and enhancing infiltration of the inflammatory cd + gr- high neutrophils in the livers. interestingly, mhv- infection in il- r -/mice, or in wt mice treated with il- β signaling inhibitors, such as using il- ra, rescue the otherwise susceptible animals from the viral fh status, presenting with limited virus replication, attenuated disease progression and reduced mortality. we have also shown that the bioprocess of il- β maturation is under the control of a key signaling pathway, involving a mhv- virus inducible, ros-dependent nlrp inflammasome activation. animals lacking of nlrp , caspase- or nadph oxidase subunit p phox that controls acute ros secretion, all exhibited with reduced il- β bio-processing that results in prevention of the mhv- mediated disease severity. to the best of our knowledge, these data provide evidence for the first time showing that the ros/nlrp /il- β axis is an essential contributor for the virus-induced fh. although macrophage-mediated inflammation has been speculated to be critical for gauging the pathological susceptibility of viral fh caused by mhv- infection [ ] , the mechanisms underlying the pathogenesis are not well understood. il- β and il- are two key inflammatory cytokines produced by macrophages which play a pivotal role in antimicrobial immunity [ , ] . previous studies have showed that il- r -/mice appear to have markedly reduced inflammatory pathology in the lung, presumably due to the impaired neutrophil recruitment upon influenza virus infection [ ] . conversely, ramos et al. reported that il- r -/mice exhibited with a higher accumulation of the west nile virus (wnv) in the central nervous system due to a restrained activation of the virus-specific effector cd + t cells [ ] . similarly, il- β -/mice are more susceptible to herpes simplex virus (hsv )-mediated encephalitis due to an increase in viral load [ ] . we here further explored the role of il- β in mhv- mediated fh. interestingly, il- r -/animals display a significant reduction in viral duplication, amelioration of liver damage and a prolonged survival rate against mhv- infection (fig a and b) . these effects are probably due to il- r deficiency lead to limit liver recruitment of cd + gr- high neutrophils and decrease in production of the macrophage-derived fgl , which mediates sinusoidal fibrin deposition and hepatocellular necrosis in response to mhv- infection [ ] . bgp (also called carcinoembryonic cell adhesion antigen a,ceacam a) is the specific receptor for the mouse hepatitis virus (mhv), and down-regulation of bgp by ifn-γ is related to the antiviral state and resistance to mouse hepatitis virus infection [ ] . however, bgp does not appear to be involved in il- and tnf-α secretion from mhv- infected macrophages [ ] . in contrast to ifn-γ treatment, we here showed that the expression of bgp drops significantly in the il- r -/liver during the viral infection, suggesting bgp expression in macrophages is induced by il- β/il- r signaling, and lacking the pathway may compromise virus entrance and amplification. these unpredicted data implies that il- β has double-edge effects on the immune system, in which proper balancing with its signaling extent becomes essential (e) liver fibrinogen deposition was analyzed by immunohistochemistry, the architecture was analyzed by h&estaining and cellular apoptosis was analyzed using tunel staining (left). scale bar μm; arrow indicates positive cells; blue color indicates nuclear staining with dapi, n = per group. serum alt and ast activities were determined with an au automatic biochemistry analyzer (right). *p< . and **p< . , n = per group. (f) liver virus titers at h of infection were analyzed by plaque assay (left), and their levels were compared by statistical analysis (right). *p< . . (g) mhv- infected p phox-/mice were treated with mouse recombinant il- β protein ( ng/day/mouse) and the survival rate was monitored. one of three experiments with similar results is shown. *p< . compared top phox-/-+pbs+mhv- group. for the host in protection against various invading viruses and meanwhile, in prevention of the potential collateral damage. the molecular mechanisms that are responsible for triggering the expression of fgl prothrombinase, which plays a critical role in the development of mhv- mediated fh, are still unclear. mcgilvray et al. found that both erk and p -mapk proteins are activated in mhv- infected pems, and only inhibition of p -mapk can abolish fgl induction and its functional activity [ ] . jia et al. have illustrated that tnf-α upregulates fgl expression via activation of nf-kb and p -mapk in cardiac microvascular endothelial cells [ ] . our recent work also have showed that the inhibition of erk / and p -mapk efficiently block c amediated fgl upregulation [ ] . ning et al., have demonstrated that the hepatocyte nuclear factor- (hnf ) cis-elements and its cognate transcription factor, hnf α, are necessary for mhv- -induced fgl gene transcription [ ] . based on these studies, we further examined the molecular mechanisms underlying il- β-mediated fgl expression. the results show that il- β and tnf-α synergistically induce nf-κb, erk and p -mapk tyrosine-phosphorylation ( fig c) . however, the inhibition nf-κb pathway, but not the erk, or p -mapk signals, markedly prevented fgl expression (fig d) , suggesting that the nf-κb pathways are responsible for il- β+tnf-α-mediated fgl augmentation. the nlrp , rig-i and the aim are three main types of inflammasome complexes that have been shown to control caspase- activity and il- β maturation. it seems that aim is responsible for detecting dna viruses, while both nlrp and rig-i associate with recognition of rna viruses by cells [ , ] . recent evidences suggest that the host protective immunity requires the nlrp inflammasome for fighting against various kinds of viruses, including influenza a virus, modified vaccinia virus ankara, sendai virus, respiratory syncytial virus, encephalomyocarditis viruses, as well as adenoviruses [ ] . our study shows that the mhv- triggered nlrp , asc and caspase- mrna as well as protein expression in pems and raw . cells in vitro (fig a) . nevertheless, loss of either nlrp or caspase- in macrophages reduces il- β secretion upon mhv- challenge (fig c) . additionally, nlrp -/and caspase- -/mice essentially pheno-copied the manifestations of il- r -/mice in response to mhv- infections, these mice evidenced with reduction in mhv- virus-induced il- β production and lessening of disease progression (fig c- f ). these combined data suggest that nlrp -inflammasome acts as a predominant pathway for triggering il- β maturation by mhv- , and probably also by other corona viruses. previous study showed that raw . cells do not release mature il- β because they do not express asc [ ] . conversely, we here show that mhv- promotes il- β secretion from virus infected raw . cells through inducing asc expression. together with the recent work demonstrated that nlrp /asc/caspase- axis participates in the regulation of the generation of il- β in raw . cells, indicating that asc is inducible in the macrophage line raw . cells under circumstances, especially during mhv- infection [ ] . ros plays an essential role in mediating nlrp inflammasome activation [ ] . many different viruses, such as influenza virus, respiratory syncytial virus, and hepatitis c virus, trigger nlrp inflammasome activation through ros-dependent mechanisms [ ] [ ] [ ] . nox is an enzymatic complex consisting mainly of five subunits (p phox , p phox , p phox , p phox and gp phox ) and two gtp-binding proteins (rac /rac ). we here show that mhv- triggers nox-derived ros secretion in macrophages by inducing nox-subunits, including gp phox , p phox and nox- expression in the very early stages of the viral infection (fig a and c) . additionally, preventing nox-derived ros through dpi appeared to successfully down modulate nlrp hyperactivation and il- β maturation in vitro (fig f) . furthermore, virus infected p phox-/macrophages manifested with significant reduction in ros secretion, leading to the control of nlrp hyperactivation, which results in attenuation in severity of the viral fh (fig ) . these results are inconsistent with previous reports that have shown that nadph oxidase-derived ros are not involved in activating nlrp inflammasome [ , ] . one of the discrepancies is the different cell models are used in studies. silica crystals, lps, and uric acid crystals act as the stimulators in these studies, while mhv- virus is the activator in our research. conversely, it is worth mentioning that not all p phox-/mice are completely resistant to mhv- , and these animals eventually still died from the infections (fig b) , together with some virus infected mice still produce high levels of il- β and virus titers, suggesting the presence of other mediators that in response to the virus challenge, are capable of activating nlrp inflammasome in vivo. one of the potential activators is mitosox [ , ] . we have also observed a very high level of the mitosox production in the mhv- infected raw . cells at h and h post-infection in vitro, along with high frequency damage and destruction of mitochondria might simultaneously occur. however, the release of mitosox was unable to be successfully blocked by ros inhibitor-dpi ( um) (fig ) . additionally, dpi is harmful to animals and unsuitable in vivo experiments [ ] . the incapable of completely blocking ros production by using high dose of dpi in vitro suggests the existence of other sources of ros for activating nlrp inflammasome. interestingly, reduced mortality and pathology were seen in mhv- infected p phox-/mice compared to wt littermates despite a lack of significant reduction in virus replication, suggesting that mhv- -mediated pathology is due to inflammation and not direct virus infection. recent studies by warner greene's group demonstrate that hiv can trigger caspase- activation and pyroptosis, a highly inflammatory form of programmed cell death in which dying cells release their cytoplasmic contents, including inflammatory cytokines into the extracellular space where the virus infected cd + t-cells recite [ ] . a similar environment might also explain for the mhv- induced fh status. il- is another member of the il- superfamily that has been indicated to be important in the pathogenesis of mouse models of influenza virus, hbv, rhinovirus and vaccinia virus infection [ ] . for example, il- r -/mice appeared to be protected from influenza viral initiated inflammatory lung damages [ ] . consistent with previous reports, we have detected significantly high levels of matured il- in the serum of mhv- infected wt mice. however, il- deficiency does not prevent bgp expression, virus amplification and fgl accumulation in the liver following mhv- infection, and as the consequence, these mice stay high with fibrinogen deposition, liver damage and hepatocyte necrosis (fig ) . these results suggest that il- is not essential for causing mhv- mediated acute hepatitis. in conclusion, our study elucidates that nlrp inflammasome-dependent il- β production, a primary inflammatory signaling pathway of the host for mounting conventional immunity against pathogen invasions, plays a double-edged role in the host immune system. hepatotropic virus, like mhv- infection in mice, can induce exaggerated inflammation in the liver and cause life-threatening viral fh. these results shed lights on a novel strategy, for which the properly modulation of the il- β signaling pathway, in combination with blocking other inflammatory factors, might benefit the treatment of viral fh and other severe inflammatory diseases in human. the mice approximately weeks of age were used for these experiments. all animals received humane care according to the criteria outlined in the "guide for the care and use of laboratory animals" prepared by the national academy of sciences and published by the national institutes of health (nih publication - revised ). cells raw . cells were provided by the cell institute of the chinese academy of sciences (shanghai, china). peritoneal exudative macrophages (pems) were harvested as described previously [ ] . cells were cultured in -well plates and propagated in dmem supplemented with % fbs, u/ml penicillin, and μg/ml streptomycin. mhv- viruses were expanded in murine cl cells to a concentration of × plaque forming unit (pfu)/ml. the virus-containing supernatants were stored at - °c until use. macrophages were infected with mhv- (multiplicity of infection, moi = ) in vitro and mice were injected with pfu of mhv- via i.p. in some experiments, the virus infected mice were treated with il- r antagonist (il- ra, mg/kg/day) or recombinant mouse il- β protein ( ng/day/mouse) every day. mice were euthanized on the indicated days and the virus titers in liver were determined by plaque assay as described previously [ ] . the sources of antibodies and other reagents are detailed in s text. paraffin-embedded liver tissue blocks were cut into μm slices and mounted onto poly-lysinecharged glass slides, and tissue injury was stained by hematoxylin and eosin (h&e). cellular apoptosis was measured by tunel staining according to the manufacturer's instructions (roche, berlin, germany). the expression of fibrinogen and fgl was detected by immunohistochemistry as described previously [ ] . sections were scored in a blinded fashion for histological diagnosis. total rna was extracted from cultured cells or liver tissues with trizol reagent according to the manufacturer's instructions (invitrogen, ny, usa). first-strand cdna was synthesized with the primescript rt-pcr kit (takara, dalian, china). the expression of mrna encoding for nlrp , caspase- , proil- β and proil- was quantified by real-time quantitative pcr with the sybr premix extaq kit (takara) and was normalized to the expression of β-actin. sequences of the primers are provided in s table. results were calculated and compared by the −ΔΔct method. serum c a, fgl , il- and il- β levels were measured by elisa. the expression of fgl , procaspase- , caspase- -p , nlrp- , p phox , p phox , p phox , nox- , bgp , proil- β and il- β-p in mhv- infected livers or macrophages was detected by western-blotting described previously [ ] . the release of il- β/ros from virus infected macrophages, liver infiltration of cd + gr- high neutrophil and cd + foxp + regulatory t cells (treg), all were detected by flow cytometry (facsaria cytometer, bd, franklin lakes, nj, usa). the death cells were excluded firstly by staining with live/death fixable near-ir ded cell stain kit (life technologies, eugene, oregon, usa). the secretion of nox-derived ros was detected by means of an oxidation-sensitive fluorescent probe-dcfh according to the manufacturer's instructions (beyotime, shanghai, china). moreover, the mitochondria-derived ros was measured in cells stained with mitosox ( μm, invitrogen) for min. to measure mitochondrial damage, cells were stained for min with mitotracker green fm ( nm) and mitotracker deep red fm ( nm), two kinds of dye that stain mitochondria with no influence on their membrane potentials (invitrogen). a total of , live cells were analyzed. all the facs data were analyzed using cell-quest pro software. electron microscopy raw . cells or primary pems isolated from mhv- infected mice were fixed with % (v/ v) glutaraldehyde. sample preparation was conducted as described previously [ ] . mitochondrial morphology and virion was observed with jeol jem hc transmission electron microscopy. all data were analyzed using graphpad prism . software. an unpaired student's t-test (two-tailed) was used to assess comparisons between two groups when the data met the assumptions of the t-test. survival curves were generated by log-rank test. p< . was considered a significant difference. all animal experiments were performed in strict accordance with the guide for the care and use of laboratory animals issued by the ministry of science and technology of the people's republic of china. the protocol was approved by the third military medical university institutional animal care and use committee. supporting information s text. reagents and antibodies. (docx) s table. the primer sequences for qpcr of the indicated genes. acute-on-chronic liver failure: consensus recommendations of the asian pacific association for the study of the liver (apasl) fulminant viral hepatitis: molecular and cellular basis, and clinical implications the fgl /fibroleukin prothrombinase contributes to immunologically mediated thrombosis in experimental and human viral hepatitis tnf-α and fgl contribute to coagulation and complement activation in virus induced fulminant hepatitis c a/c ar pathway is essential for the pathogenesis of murine viral fulminant hepatitis by way of potentiating fgl /fibroleukin expression intracellular nod-like receptors in host defense and disease interleukin- in the pathogenesis and treatment of inflammatory diseases il- receptor antagonist ameliorates inflammasome-dependent alcoholic steatohepatitis in mice toll-like receptor promotes steatohepatitis by induction of interleukin- beta in mice interleukin- participates in the progression from liver injury to fibrosis type i interferons protect from toll-like receptor -associated liver injury and regulate il- receptor antagonist in mice nlrp inflammasome activation: the convergence of multiple signalling pathways on ros production? signaling by ros drives inflammasome activation innate immune activation through nalp inflammasome sensing of asbestos and silica a role for mitochondria in nlrp inflammasome activation mitochondrial reactive oxygen species promote production of proinflammatory cytokines and are elevated in tnfr -associated periodic syndrome (traps) a mitochondrial love-hate triangle interleukin- beta and the autoinflammatory diseases tissue and cellular distribution of an adhesion molecule in the carcinoembryonic antigen family that serves as a receptor for mouse hepatitis virus tumor necrosis factor α (tnf-α) receptor-i is required for tnf-αmediated fulminant virus hepatitis caused by murine hepatitis virus strain- infection the novel cd + cd + regulatory t cell effector molecule fibrinogen-like protein contributes to the outcome of murine fulminant viral hepatitis tnf-α upregulates fgl expression in rat myocardial ischemia/reperfusion injury il- in inflammatory and autoimmune disease enhanced autoimmunity, arthritis, and encephalomyelitis in mice with a reduced oxidative burst due to a mutation in the ncf gene expression of b and t lymphocyte attenuator (btla) in macrophages contributes to the fulminant hepatitis caused by murine hepatitis virus strain- interleukin- is responsible for acute lung immunopathology but increases survival of respiratory influenza virus infection il- β signaling promotes cns-intrinsic immune control of west nile virus infection tumor necrosis factor-alpha and interleukin- beta play a critical role in the resistance against lethal herpes simplex virus encephalitis down-regulation of bgp (a) viral receptor by interferon-gamma is related to the antiviral state and resistance to mouse hepatitis virus infection macrophage interleukin- and tumour necrosis factor-alpha are induced by coronavirus fixation to toll-like receptor /heparan sulphate receptors but not carcinoembryonic cell adhesion antigen a murine hepatitis virus strain induces the macrophage prothrombinase fgl- through p mitogen-activated protein kinase activation induction of prothrombinase fgl by the nucleocapsid protein of virulent mouse hepatitis virus is dependent on host hepatic nuclear factor- alpha aim recognizes cytosolic dsdna and forms a caspase- -activating inflammasome with asc recognition of rna virus by rig-i results in activation of card and inflammasome signaling for interleukin beta production central roles of nlrs and inflammasomes in viral infection p x receptor differentially couples to distinct release pathways for il- beta in mouse macrophage lipopolysaccharide/adenosine triphosphate induces il- β and il- secretion through the nlrp inflammasome in raw . murine macrophage cells hcv and oxidative stress in the liver inhibition of nox oxidase activity ameliorates influenza a virus-induced lung inflammation suppressing production of reactive oxygen species (ros) for influenza a virus therapy superoxide dismutase regulates caspase- and endotoxic shock reactive oxygen species-independent activation of the il- beta inflammasome in cells from patients with chronic granulomatous disease human nlrp inflammasome activation is nox - independent silica crystals and aluminum salts activate the nalp inflammasome through phagosomal destabilization studies on the inhibitory mechanism of iodonium compounds with special reference to neutrophil nadph oxidase cell death by pyroptosis drives cd t-cell depletion in hiv- infection nlrs, inflammasomes, and viral infection interleukin- improves the early defence system against influenza virus infection by augmenting natural killer cell-mediated cytotoxicity gene deletion of gabarap enhances nlrp inflammasome-dependent inflammatory responses we wish to thank dr. dayan cao, huan xu and xi chen for their helpful comments and constructive suggestions. lxz is supported by the intramural research program of the us national institutes of health. key: cord- - y y authors: shoemaker, jason e.; fukuyama, satoshi; eisfeld, amie j.; zhao, dongming; kawakami, eiryo; sakabe, saori; maemura, tadashi; gorai, takeo; katsura, hiroaki; muramoto, yukiko; watanabe, shinji; watanabe, tokiko; fuji, ken; matsuoka, yukiko; kitano, hiroaki; kawaoka, yoshihiro title: an ultrasensitive mechanism regulates influenza virus-induced inflammation date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: y y influenza viruses present major challenges to public health, evident by the influenza pandemic. highly pathogenic influenza virus infections generally coincide with early, high levels of inflammatory cytokines that some studies have suggested may be regulated in a strain-dependent manner. however, a comprehensive characterization of the complex dynamics of the inflammatory response induced by virulent influenza strains is lacking. here, we applied gene co-expression and nonlinear regression analysis to time-course, microarray data developed from influenza-infected mouse lung to create mathematical models of the host inflammatory response. we found that the dynamics of inflammation-associated gene expression are regulated by an ultrasensitive-like mechanism in which low levels of virus induce minimal gene expression but expression is strongly induced once a threshold virus titer is exceeded. cytokine assays confirmed that the production of several key inflammatory cytokines, such as interleukin and monocyte chemotactic protein , exhibit ultrasensitive behavior. a systematic exploration of the pathways regulating the inflammatory-associated gene response suggests that the molecular origins of this ultrasensitive response mechanism lie within the branch of the toll-like receptor pathway that regulates stat phosphorylation. this study provides the first evidence of an ultrasensitive mechanism regulating influenza virus-induced inflammation in whole lungs and provides insight into how different virus strains can induce distinct temporal inflammation response profiles. the approach developed here should facilitate the construction of gene regulatory models of other infectious diseases. influenza viruses present major challenges to public health, evident by the influenza pandemic. highly pathogenic influenza virus infections generally coincide with early, high levels of inflammatory cytokines that some studies have suggested may be regulated in a strain-dependent manner. however, a comprehensive characterization of the complex dynamics of the inflammatory response induced by virulent influenza strains is lacking. here, we applied gene co-expression and nonlinear regression analysis to time-course, microarray data developed from influenza-infected mouse lung to create mathematical models of the host inflammatory response. we found that the dynamics of inflammation-associated gene expression are regulated by an ultrasensitive-like mechanism in which low levels of virus induce minimal gene expression but expression is strongly induced once a threshold virus titer is exceeded. cytokine assays confirmed that the production of several key inflammatory cytokines, such as interleukin and monocyte chemotactic protein , exhibit ultrasensitive behavior. a systematic exploration of the pathways regulating the inflammatoryassociated gene response suggests that the molecular origins of this ultrasensitive response mechanism lie within the branch of the toll-like receptor pathway that regulates stat phosphorylation. this study provides the first evidence of an ultrasensitive mechanism regulating influenza virus-induced inflammation in whole lungs and provides insight into how different virus strains can induce distinct temporal inflammation response profiles. the approach developed here should facilitate the construction of gene regulatory models of other infectious diseases. invading pathogens induce acute inflammation when molecular signatures are detected by pattern recognition receptors (prrs; e.g., rig-i like receptors [rlrs] and toll-like receptors [tlrs] ) expressed on tissue-resident immune cells and non-immune cell types. prr ligation triggers innate immune responses and leads to the induction of inflammatory and antiviral gene expression, which together function to limit pathogen growth, activate the adaptive immune response, and ultimately resolve the infection [ , ] . precise regulation of prr-mediated signaling is necessary to both avoid inadvertent tissue damage in response to non-pathogenic stimuli, and to prevent immunopathology resulting from excessive expression of inflammatory molecules. in essence, the ideal inflammatory response must exhibit a balance between appropriate activation against a genuine threat and self-limiting behavior once that threat has been controlled. despite its importance in maintaining normal tissue homeostasis and limiting pathogen-associated diseases, the mechanisms underlying the regulation of this balance are poorly understood. influenza a viruses are recognized by both tlrs and rig-i-like receptors (rlrs) [ ] [ ] [ ] [ ] [ ] , and some strains are potent inducers of inflammatory and antiviral gene expression. generally, lung tissues infected with pathogenic isolates exhibit high virus titers and robust inflammatory gene expression, as has been documented in in vivo studies with the spanish influenza virus [ , ] , highly pathogenic h n avian influenza viruses [ ] [ ] [ ] , and the h n pandemic influenza virus [ , ] . in contrast, seasonal influenza viruses typically replicate less efficiently, elicit more restrained inflammatory responses, and are usually not associated with lethal infections. recent evidence has implicated the level of virus replication in infected lung tissues as the primary phenotypic variable driving inflammation and lethal outcomes [ , ] . other data indicate that influenza viruses that exhibit significant differences in pathogenicity stimulate qualitatively similar host responses that differ primarily at the level of magnitude and kinetics [ ] . however, these studies have not revealed the mechanisms that account for the different profile dynamics observed in infections by high and low pathogenic viruses. such information would aid in clarifying not only how some influenza viruses induce lethal disease, but also the general mechanisms that regulate inflammatory balance. to characterize the dynamics of influenza virus-induced inflammation, we developed a novel approach to infer gene regulatory models from dynamic gene expression data. referred to as systems inference microarray analysis, our method builds on current approaches that use co-expression analysis to isolate modules of functional signatures in gene expression data and then extends these methods by fitting the gene expression modules to mathematical equations (models) by using segmented regression analysis. models can be created to look for strain-dependent responses and, unlike traditional differential expression analysis, to predict gene expression under new experimental conditions. by using this method, we set out to determine how influenza viruses that exhibit variable pathogenicity profiles influence the dynamics of the inflammatory response. to characterize the dynamics of the host immune response to specific virus isolates, we infected mice with pfu of three virus isolates with distinct pathogenicity profiles and harvested lung tissues at ). an initial inoculation of pfu was used as previous studies indicated that a high virus dose was needed to invoke different pathologies in h n and ph n -infected mice [ ] . as expected, lung virus titers (virus titers determined by plaque assay and reported in plaque forming units [pfu] per gram lung; fig ) indicated a clear hierarchy of mild, moderate, and severe virus-induced disease. specifically, the h n virus produced the highest lung titers and between days and post-infection, this virus also caused mortality in the animals whose lungs were to be collected on day post-infection (i.e., 'severe' disease). in contrast, all animals infected with the h n or ph n viruses survived the duration of the time course study; however, ph n -infected animals were visibly sicker and exhibited higher lung titers relative to those infected with h n at all time points observed , , , , , , , , , and h and , , and days), and virus titers in lung tissues were determined by plaque assays in mdck cells. error bars illustrate the standard deviation from the mean. the gray boxes at each time point identify significant pairwise differences between the means of the viruses indicated at the right of the figure panel (significance was determined by anova followed by tukey's honestly significantly different test, p < . ). *, three h n -infected mice intended for collection on day succumbed to their infections prior to sample collection. after the first h post-infection (i.e., 'moderate' and 'mild' disease, respectively). histopathological analysis of tissue samples collected on days , , and post-infection (fig ) also showed that h n -infected tissue exhibited the earliest, most severe signs of inflammation and inflammatory immune cell infiltrates followed by ph n -infected tissue, whereas h n -infected tissue showed mild evidence of inflammation and was most similar to tissue from the control mice (mock-infected mice). we next used co-expression analysis to integrate inflammation-associated gene expression differences between influenza-infected and control lungs into a systems level context. we first asked whether the expression of inflammation-associated genes clustered into modules of coexpressed genes. tissues from the same animals that were used to determine virus growth were used to evaluate changes in global lung transcriptional profiles. a total of microarrays were developed (three per time point for h n -infected, ph n -infected, h n -infected and control mice). one microarray was removed after reviewing replicate quality. after filtering transcripts for minimally confident variation (we required at least one time-matched, infected condition compared with mock-infected absolute fold change and a false discovery rate [fdr]-adjusted p-value < . ), the log of the normalized intensity of the retained transcripts ( , ) for all samples were then clustered by using the weighted gene co-expression network analysis (wgcna) algorithm [ ] . in all, distinct co-expression modules were identified (referred to as n , n , etc.; s table provides the module assignments for all transcripts). to identify the biological role of the host response modules, we performed functional enrichment analysis on each gene module by using david [ ] and toppcluster [ ] . because each module was comprised of positively and negatively correlated transcripts, we used the module eigengene (i.e., the first principle component of the gene expression matrix) to divide each module into two submodules containing transcripts that were positively or negatively correlated with the parent module's eigengene, denoted as kme+ and kme-(referred to as module membership), respectively. this procedure allowed us to look for biological processes with similar but opposing dynamic responses to the virus infections. functional enrichment analyses were then applied to each submodule by using two bioformatics platforms to ensure robust results. we identified two submodules (n kme+ and n kme-, referred to as simply n and n in the remainder of the text) that were enriched for inflammatory response and inflammationassociated pathway signatures by using both bioinformatics platforms, and these two modules became the focus of our study (table summarizes the functional enrichment results for the immune and inflammatory related annotations. the complete enrichment results from toppcluster and david are available in s table and s and s files). the n module was uniquely enriched for cytokine activity and type i interferon (ifn) regulating tlr and rlr pathways [ ] , as well as transcriptional signatures associated with ifn-regulated activity (i.e., the transcription factor binding sites [tfbs] of irf , irf , irf , isre, and nf-κb). additionally, n was the only module that exhibited significant enrichment with a compendium of established ifn-stimulated genes ('isgs'; table ; see methods. the list of isgs is available in s file). a more recent study identified ifn stimulated genes in immortalized, human airway epithelial (calu ) cells [ ] . of these, mouse homologs were annotated on the microarrays and of the homolog probes were assigned to the n module (fisher's exact test; pvalue < - ; odds ratio = . ), further associating n interferon-stimulated gene activity. in contrast, the n module was only weakly enriched for some cytokine activity related annotations and not enriched for any of the binding sequences of transcription factors that are members of canonical inflammatory pathways (such as interferons, interferon regulatory factor proteins, or nfκb). instead, it was primarily associated with several annotations related to leukocyte and lymphocyte activity (see summary of toppcluster enrichment results in table ; see s and s files). further analysis with cten [ ] , a platform for associating clustered gene expression data with specific cell types, found n to be highly enriched for genes expressed in macrophages in various cellular states (e.g., bone marrow-derived macrophages exposed to lipopolysaccharide [lps]) ( fig a; additional details available in s table) . the remaining immune-associated submodules (the kme+ n , n , n , and n submodules; described in table ) were enriched for several key immune processes such as antigen presentation, and t cell and natural killer (nk) cell activity, but their further assessment would be beyond our focus and the scope of this study. thus, bioinformatics analyses robustly associated the n module with inflammation, cytokine production, and type i ifn pathway activity-likely activated in resident lung cells-whereas the n module is associated with migration and activation of macrophages in the lung. each module was divided into submodules in which each member gene's expression was positively or negatively correlated with the module's eigengene (denoted kme+ and kme-, respectively). for each module, we provide the number of transcripts assigned to each submodule, the top david annotation clusters for each submodule (parenthesis shows the-log of the average enrichment p value for all annotations in the cluster), the top enriched biological processes determined using toppcluster (parenthesis shows the-log of the fdr-adjusted p value), the enrichment of established transcription factor binding sites in each submodule (tfbs; parenthesis shows the-log of the fdr-adjusted p value; performed using toppcluster), and the enrichment score of a set of ifn-stimulated genes (see methods; enrichment score is the-log of the fdr-adjusted p value). doi: . /journal.ppat. .t a closer examination of the expression dynamics of each of the inflammation response-associated modules revealed patterns of expression that were consistent with the biological roles predicted by our bioinformatics analyses. we used the scaled difference of the module eigengene to characterize the expression of all genes within each module. we subtracted the mean of the eigengene of the control samples from the eigengene of time-matched, virus-infected dynamics of inflammation-associated co-expression modules. panel (a) shows the enrichment scores (-log of the fdr-adjusted p value) of the genetic signatures of cell types in the n module as determined using cten [ ] . data corresponding to macrophage signatures in different cellular states are colored red (see s table for within the inflammation-associated modules (n and n ), the h n virus induced the earliest gene expression changes and the highest peak expression levels, corroborating previous observations that h n viruses are strong inducers of inflammatory and ifn response signaling in vivo [ , , ] . consistent with the prediction that n is involved in detecting virus in infected tissues, the n module eigengene was the most highly correlated with virus titer (pearson pairwise correlation, ρ = . ). module gene expression dynamics further suggested that the n module gene is associated with lymphocyte infiltration. exudate macrophages [ ] and neutrophil [ ] have been identified as factors of severe disease during influenza infection. to associate gene expression dynamics with changes of immune cell counts, a new population of mice were infected with the three influenza viruses, five mice per infection group were sacrificed on days , , and and the changes in the number of macrophages and neutrophils was assessed (see methods). unlike the previous study by brandes, et al. [ ] , strong neutrophil infiltration was not specific to fatal infections but, instead, infiltration of both cell types had a clear hierarchical relationship with the severity of the infection (fig d and e ). the n module eigengene exhibited a lesser correlation to virus titer (ρ = . ), but was tightly correlated to macrophage influx into the lung (ρ = . ; p-value < . student's t-test). of the module identified in the studied, n had the highest correlation to both macrophage and neutrophil influx (the correlation of macrophage and neutrophil influx and all module eigengenes are provided in s table; ρ = . ; pvalue < . ). the n module on the other hand was weakly but significantly correlated with immune cell infiltration (ρ = . [p-value = . ] for neutrophils; ρ = . [p-value = . ] for macrophages), but its eigengene was not the most highly correlated ( other module eigengenes had a greater absolute correlation). these results further associate n with immune cell-specifically macrophage-infiltration, while the sum of the bioinformatics, virus titer correlations and immune cell infiltration evidences associated n with inflammation and type i ifn pathway activity. a further advantage of a network approach is that the functional relevance of genes might be inferred from their positions within the co-expression network [ ] . we used the module membership (the correlation between the gene's expression and the module eigengene, kme) to isolate potential regulators of the n module. among the top intramodular hub genes (i.e., genes with the highest module memberships, see s table) , we found mnda, herc and cd and several interferon regulated, virus replication inhibitory genes such as oas and oas [ ] . herc is involved in ubiquitination [ ] . mnda is significantly up-regulated in monocytes exposed to interferon α [ ] while cd is a transmembrane protein expressed on antigen presenting cells and modulates activation of t cells, b cells and myeloid cells. we also observed that interferon stimulated genes tended to have higher intramodular hub rankings, suggesting a regulatory role for interferon (wilcoxon rank sum test, p-value < - ). we then considered the module membership rankings of transcription factors known to regulate interferon. of the established interferon regulatory factors that are members of the n module (e.g., irf , irf , irf , irf , stat , and stat . nfkb and nfkb were not assigned to n ), irf and irf had the highest module memberships (kme = . and . ; ranking = and , respectively). irf expression was also several orders of magnitude greater than irf (s table) . together, these findings corroborate our bioinformatics analyses by suggesting that n is regulated by interferon, and n expression likely results in enhanced cytopatchic effects and regulation of the lymphocyte immune response. network analysis further suggests that irf may play a regulatory role upstream of interferon transcription. previous studies have suggested that highly pathogenic influenza virus infections induce an irregular or disproportionate inflammatory response relative to seasonal influenza viruses, and that these differences occur early in the host response [ ] . for this reason, we sought to further explore the possibility of isolate-specific or isolate-independent response patterns of the cytokine-associated n module. we wanted to infer mathematical relationships that could describe when inflammatory-associated gene expression occurs and what magnitude of expression is expected. by using the eigengene as a representation of the scaled gene expression dynamics, we attempted to infer simple mathematical models that can be related to common signaling mechanisms. surprisingly, when we plotted the n sde for each isolate against the corresponding virus titer, we observed a consistent profile for all three viruses; regardless of intrinsic virulence, the fold change in n gene expression remained initially low and rapidly increased only after a virus titer of approximately~ pfu/g (of lung) was reached (fig a) . following activation, n gene expression increased as a function of virus concentration at the same apparent rate for all infection conditions, and more complicated dynamics were observed only during the later phase of the infection when virus clearance was observed (i.e., when the virus titers began to decrease). these observations suggest that ifn-regulated (n ) gene expression was induced by an ultrasensitive response mechanism controlled at the level of the virus titer rather than the virus's intrinsic virulence. ultrasensitive responses characterize the dynamics of several signaling pathways that regulate essential and often toxic biological processes such as the cell cycle [ ] and apoptosis [ ] . as shown in fig b, ultrasensitive responses are typified by an attenuated response to low levels of stimulation but a strong response occurs once a threshold level of stimulus is reached. cooperativity [ ] and positive feedback [ ] are two mechanisms that produce ultrasensitive responses. to formalize the hypothesis that the inflammatory gene response follows an ultrasensitive response profile, we selected a segmented linear model (slm, defined in fig c) to be a simplified representation of the ordinary differential equations normally used to model ultrasensitive responses, and we fit the n sde to an slm that was strictly a function of the virus titer. the optimal fit showed a threshold of . ± . pfu/g is required for n module activation to occur, after which the sde's rate of activation (a ) was . ± . log (pfu/g) - with an intercept (b ) of - . (unitless) (see the methods and s fig for additional details) . below this threshold, the model predicted minimal gene expression (a = . ; b = . ). the slm goodness of fit on the training data was an adjusted r = . while an adjusted r = . was observed when the data was fit to a linear model. a davie's test confirmed that a segmented model was a significantly better fit than a linear correlation model (p-value < . e- ). while the h n -infected lung tissue did not exceed an average peak virus titer of . pfu/g (peak titer occurs at hpi in fig a) , we observed increased transcriptional activity in h n -infected mouse lung tissues after this time point, suggesting either that the actual peak virus titer occurred between hpi and the subsequent time point ( hpi), or that the model-predicted threshold was slightly over-approximated. we next sought to validate the threshold model by attempting to predict cytokine-associated gene expression in influenza virus-infected lung when only the virus titers are known. for this, we selected the h n virus, which has previously been associated with an excessive illustration of an ultrasensitive response to increasing stimulus. in our model, the response is the change in inflammatory-associated gene expression and the stimulus is the lung virus titers. a segmented linear model (slm) can approximate key aspects of the ultrasensitive response. in this study, the response is gene expression and the stimulus is the concentration of virus in the lung (c) the mathematical definition of an slm. below some threshold virus titer (thr), the scaled eigengene (se) is approximated by a linear model that is a function of the log of the virus titer, the slope (a ), and the intercept (b ). after the threshold virus titer is reached, the slope and intercept parameters change (a and b ) to describe the se after activation. the parameters were fit to the data shown in panel (a) and used to predict the n se in newly infected mice. (d) shows a comparison of the h n n eigengene from an infection at a dose of pfu (as shown in fig ) and an infection at a dose of pfu. panel (e) compares the predicted scaled eigengene based on the slm versus the measured eigengene behavior in mice infected with pfu of the h n virus. panel (f) shows gene expression changes for selected constituent genes of the n module as a function of virus titer. cytokine response [ ] . we infected mice with pfu of the h n virus (a -fold reduced dose compared with that used in the experiments to fit the model), determined lung virus titers at the same time points used for the initial experiment (s fig), and then evaluated the segmented linear model's ability to predict cytokine-associated gene expression. first, we confirmed that the original transcripts assigned to the n module were again co-expressed, and thus we used the same transcripts originally assigned to the n module to determine the eigengene (see s fig for an analysis of the conservation of the n module between the two experiments). in this experiment, as expected, the peak average virus titer ( . ± . pfu/g) for the pfu dose was delayed compared with that for the pfu dose (s fig, compare to fig ) . moreover, the sde exhibited an -h delay in activation and a -h delay in peak expression compared with the pfu n eigengene (fig d) . importantly, based on the virus titers alone, the fitted segmented linear model accurately predicted n -like sde behavior (r = . for all time points and r = . for time points up to peak expression; fig e and s fig), and this could be further demonstrated at the individual gene level for specific n -associated transcripts (e.g., herc , stat and irf ; fig f) . these observations provide strong evidence that activation of inflammatory-associated gene expression is dictated by a specific virus concentration in infected tissue, and further suggest the novel possibility that the pulmonary innate inflammatory response has a nonlinear, ultrasensitive-like activation profile that promotes tolerance to low concentrations of virus. the dynamics of key inflammatory cytokines are consistent with an ultrasensitive response although transcriptional activation of ifn-stimulated and inflammatory gene expression is a reasonable measure of the effects of inflammation response stimulation, we reasoned that a bona fide ultrasensitive mechanism that regulates this response should be reflected in other aspects of the associated signaling pathway(s). indeed, of the cytokines associated with the n module, -including key inflammatory proteins, such as interleukin (il- ) and monocyte chemoattractant protein- (mcp- )-were significantly correlated with the n module eigengene (pearson's ρ . ; fdr-adjusted p-value < . ; see s table) . in addition, when the protein expression levels of these cytokines were plotted against the corresponding titer data, we observed dynamics similar to that of the inflammatory-associated n gene module. initially, protein expression was low but strongly increased only after the virus titers exceeded the threshold of~ pfu/gram determined in the gene regulation model (fig a) . in contrast, most of the protein levels of the other measured cytokines with transcripts that were not assigned to the n module did not show any obvious relationship to the proposed threshold response (s fig). the only major exceptions were lif, rantes, and il (s fig). lif's gene transcript was not annotated on the arrays whereas il 's transcript was not identified as differentially expressed and therefore was not included in the clustering study. the rantes transcript was included in the clustering study and assigned by the wgcna algorithm to n although the transcript's correlation to the n eigengene suggests it could also have been assigned to the n module (pearson correlation = . and . to the n and n eigengene, respectively). some proteins appeared to conform to the threshold model in ph n -infected, but not h n -or h n -infected, mice. these may be cytokines that have strain-dependent responses and do not conform to the model. overall, changes in n -associated cytokine protein levels in influenza virus-infected mouse lungs were consistent with the proposed virus-titer regulated threshold-mechanism underlying the ifn-mediated response. threshold-like behavior is observed on upstream and downstream components of the ifn signaling pathway. (a) lung tissues from the same infected mice used for the original gene expression analysis were subjected to cytokine array analysis. thirty-two cytokine protein concentrations were measured and log scaled (data points lower than the limit of detection for each protein were set to . to allow scaling), and the average fold change relative to uninfected, time-matched lung samples was determined. the heat map illustrates protein expression values for the cytokines that had transcripts assigned to the n module (a blue-to-yellow scale shows expression levels, as indicated by the color key at the top of the panel; time points in which the change in the cytokine levels were not significant [fdr-adjust p < . ] were set to zero); of these, exhibited expression profiles that were highly significantly correlated with the corresponding transcript and the n module eigengene (pearson's ρ . and fdr < . ; eotaxin and tnf-α were the only two that did not exhibit a significant, direct correlation). non-n cytokines are shown in s (fig b) . for the h n data, significant levels of pstat were observed at hpi which-as noted previously-corresponds to the time immediately after the virus titers in h n -infected mice reached their peak (see previous discussion). on the other hand, significant increases in phosphorylated irf (pirf ) were observed only in ph n and h n infections, whereas significant increases in ifn-β were observed only in the h n infection. changes in the levels of ifn-β and pirf occurred after significant increases in pstat and ifn-α occurred. as such, the change in the percentage of pstat was more closely correlated to the n eigengene than was that of pirf (correlation = . ± . and . ± . respectively), and significant increases in pstat corresponded to time points at which the mean virus titer exceeded the threshold level identified in the gene expression analysis (~ . pfu/g). the greater correlation of ifnα and stat activation to the inflammation-associated, n module's gene expression dynamics and the enrichment of the n module for the irf binding sequence (table ) suggest that the primary driver of the threshold-regulated, inflammatory gene response originates along the irf ! ifn-α ! stat axis (fig c) . our data reveal that the activation of the ifn-associated inflammatory and antiviral response program in influenza-infected mouse lung is characterized by an ultrasensitive response driven by the virus load. the power of the threshold model is illustrated by its ability to accurately predict gene expression in infected mice, and the data further suggest that the molecular basis of threshold behavior originates upstream of ifn-α production. threshold-like and ultrasensitive mechanisms are hypothesized to be necessary for effective management of critical cellular machinery in noisy environments, and are recognized players in the activation of the cell cycle [ ] , mitogen-activated protein kinase signaling [ ] , and apoptosis [ , ] . however, while a role for threshold behaviors have been postulated to be essential for filtering noise or errant signaling in complex biomolecular environments [ ] , our study is the first to directly link threshold-like behavior to the virus-induced innate immune response. the ultrasensitive response observed in this study provides additional insight into the mechanisms that drive severe pathologies during influenza infection. several works have suggested that viral load is a key determinant of pathology [ , ] while other works suggest that highly pathogenic influenza viruses induce early, strong inflammatory responses that are independent of the viral load [ , , ] . recently, it was observed that fatal influenza infections in mice map, with titers surpassing the threshold level identified in the slm indicated in red. (b) another set of mice was infected with pfu of h n , ph n , or h n and lung tissues were harvested ( mice per infection per time point) for immunoblot (stat , phosphorylated stat , irf , and pirf ) or elisa (ifnα, ifn-β) analysis; virus titers were also determined. the heat map indicates the mean percentage of phosphorylated protein (purple) or the mean interferon concentration (pg/ml; green), and virus titers for each time point are indicated below. only significant changes are shown in the heat map (fdr-adjusted pvalue < . relative to mock-infected samples). titers highlighted in red indicate that the threshold level (as determined in the initial analysis) was exceeded. panel (c) shows a schematic of known canonical pathways that regulate ifn production in response to virus infection. black arrows denote induction whereas red lines denote points at which influenza virus is known to impair these pathways. ifn-α is primarily produced by antigen-presenting cells (apcs) whereas ifn-β is produced in virus-infected, non-apc cells. both ifns can induce stat phosphorylation and expression of isgs in responding cells. doi: . /journal.ppat. .g ultrasensitive mechanism regulates influenza-induced inflammation coincide with a strong influx of neutrophils in what the author's describe as a viral load-independent, "feedforward" inflammatory circuit [ ] . the ultrasensitive response suggested by our study consolidates these hypotheses by suggesting that viral load drives cytokine production (and in turn immune cell infiltration) in a nonlinear manner which is capable of producing states of high and low innate immune responses. characterization of key aspects of the inflammatory response, such as the onset and peak inflammatory gene expression, require a high temporal resolution of the virus growth and host response dynamics; an experimental design that was unique to our study. the ultrasensitive response model does not negate the significance of neutrophil infiltration [ ] in determining fatal infections but suggests that viral load drives the high and low innate immune states. the observed threshold may represent the transition to immunopathology; as indicated by the histopathology results (fig ) . moreover, the influenza virus' ns protein is crucial for inhibiting the interferon-mediated antiviral response [ ] . the ns protein of three viruses used in this study have the sumo acceptor site that indicates interferon antagonism capability [ ] [ ] [ ] . additional studies with ns -mutated viruses and other pathogens may better reveal strain-dependencies for the observed thresholding behavior. the ultrasensitive response further suggests that the innate immune response has a limited capacity to respond to influenza virus infection and supports the development of immunomodulatory therapies. interestingly, after the threshold was exceeded, the rate of activation for inflammatory and interferon-associated gene expression (n ) was conserved for a moderately pathogenic and deadly viruses (fig a) . the conserved rate of activation implies that the immune response detects the virus concentration but not the virus growth rate; suggesting the innate immune response is naturally limited in its ability to respond to high growth influenza viruses. additionally, studies in knockout mice indicate that type i ifn-associated pathways are essential for protection during primary infection [ ] and that earlier initiation of these pathways coincides with increased survival in mice infected with highly pathogenic isolates [ ] . in combination with these studies, the findings here suggest a novel means of protecting high risk groups by treating them with compounds that target the molecular mechanisms responsible for the threshold behavior. lowering the threshold required to induce the cytokine response may be a means of providing protection from severe influenza infection. since these compounds would target host proteins, such treatments would be effective against various influenza virus strains. data from the viruses studied here suggest that post-threshold, inflammatory gene expression primarily reflects interferon-regulated tissue damage, but time-course data from additional highly pathogenic viruses are needed to assess the degree to which interferon activity is associated with virus growth suppression. the a/california/ / h n virus (ph n ) was received from the centers for disease control and prevention. the a/kawasaki/utk- / h n virus (h n ) served as a reference for a seasonal influenza, whereas a fatal human isolate, a/vietnam/ / h n virus (h n ), served a highly pathogenic virus. all mouse experience were performed in accordance to the university of tokyo's regulations for animal care and use. these regulations were approved by the animal experiment committee of the institute of medical science, the university of tokyo (approval number: pa - ). the committee acknowledged and deemed acceptable the legal and ethical responsibilities for the animals, as detailed in the fundamental guidelines for proper conduct of animal experiment and related activities in academic research institutions under the jurisdiction of the ministry of education, culture, sports, science and technology, . all experiments with h n viruses were performed in biosafety level containment laboratories at the university of tokyo, which are approved by the ministry of agriculture, forestry, and fisheries, japan. five-week old c bl/ j, female mice were obtained from japan slc. for all experiments, mice were anesthetized with isoflurane and intranasally inoculated with either or pfu of virus. initially, mice were inoculated with pfu of the h n , ph n , or h n virus or mock-infected with pbs (a total of mice). at time points, mice per group were humanely sacrificed and their lungs harvested. the lungs were sectioned and used to assess virus titers (right-upper lobe), cytokine levels (right lower), and the initial gene expression that was used to train the segmented linear model (left-lower). separately, mice were infected with pfu of the h n , sacrificed at the same time points, and lungs sections obtained as described previously to provide the model validation data. the same inoculation method was used for all mice in this study. the numbers of mice used for flow cytometry, western blot, and interferon protein assay experiments are specific in the corresponding sections. for cytokine and chemokine measurements, mouse lungs were treated with the bio-plex cell lysis kit (bio-rad laboratories, hercules, ca) according to the manufacturer's instructions. concentrations of other cytokines were determined with the bio-plex mouse cytokine -plex and -plex panels (bio-rad laboratories) array analysis was performed by using the bio-plex protein array system (bio-rad laboratories). virus titers were determined by plaque assay using mdck cells. mouse lung tissues were placed in rnalater (ambion, ca), an rna stabilization reagent and stored at - °c. all tissues were thawed together and homogenized for minutes at hz using a tissuelyser (qiagen, hilden, germany) as per the manufacturer's instructions. from the homogenized lung tissues, total rna was extracted with the rneasy mini kit (qiagen, hilden, germany) in accordance with the manufacturer's instructions. cy -labeled crna preparations were hybridized onto agilent- whole mouse genome x k microarrays for h at °c. feature extraction software version (agilent technologies) was used for image analysis and data extraction, and takara bio provided whole array quality control metrics. microarray normalization, replicate quality, annotation, and statistical analysis differential expression was assessed by using a linear regression model. by using the limma package [ ] version . . from bioconductor, probe intensities were background corrected by using the "norm-exponential" method, normalized between arrays (using the quantile method), and averaged over unique probes ids. replicate quality was assessed using hierarchical clustering, resulting in the removal of a single array (the array corresponded to a sample collected at hpi in h n -infected mice.) probe intensities were fit to a linear model that compared data from infected samples to time-matched data collected from uninfected mice. probes were annotated by matching to the probe names in the mgug a version . mouse annotation database available from bioconductor. all arrays in this study have been deposited on the geo expression omnibus (gse ). unsigned co-expression networks were constructed by using the blockwisemodules program from the wgcna package version . . [ ] in r. the analysis was performed with several different parameterizations to ensure robust clustering. for the results reported in this text, we removed all probes that did not have a confident fold-change greater than (fdr < . ) for at least one infected-tissue to control-tissue, time-matched comparison. we then clustered the log of the normalized intensities for all microarrays (corresponding the three samples for each time-point for each infected or control population with the exception of data from h n -infected mice at hpi which had two samples). based on the scale-free topology characteristics curve, a power of n = with no reassignment after clustering (reassignthreshold = ) and a maximum cluster size of probes was used. we generally observed that allowing gene reassignment between the modules led to poorer clustering based on the distribution the gene's module memberships (the correlation between a gene and the eigengene of the module to which it had been assigned. s fig illustrates the distribution of the gene kmes for modules n and n ). we then repeated the clustering using different powers (ranging from to ), allowing different cluster sizes, different subsets of the expression data (e.g., clustering data from each infection separately or together), or relaxing the differential expression condition. in all clusterings performed, the two modules discussed in the text were identifiable. fisher exact tests between each clustering run were used to determine whether the initial modules were significantly conserved under different parameterizations. we also considered if the n module would be isolated when using signed versus unsigned network construction. we constructed a signed co-expression network and found that % of the kme+ n genes are again clustered and confirmed that the gene expression dynamics were maintained (see s fig) . toppcluster [ ] and david [ ] were used for gene ontology and pathway enrichment, and toppcluster was also used for transcription factor binding site enrichment analyses. david uses clusters of related annotations constructed from several annotation databases (e.g., pathway and gene ontology annotations) to determine the function of a set of genes and scores the enrichment by averaging the unadjusted p-values (determined by fisher's exact test) of the annotations within the cluster. toppcluster uses hypergeometric tests to determine the enrichment between a set of genes and gene lists contained in categories (databases) detailed in the toppgene suite [ ] . the databases include cis-regulatory motif data [ , ] , referred to as transcription factor binding sites (tfbs) in the text. both tools were used with their default settings and the gene universe was considered to be all annotated mouse genes. for each module, we considered the enrichment of all genes assigned to the module and the kme+ and kmesubsets. generally, the enrichment analysis of the whole module gene set reiterated the enrichment results of the kme+ and kme-subsets albeit with slightly lower but still significant enrichment. since both tools returned similar go and pathway enrichment results, we summarized the functional and pathway enrichment results in table using the results from david. the enrichment of interferon stimulated genes was determined by using a list of interferon stimulated genes from the interferon stimulated gene database [ ] that was downloaded on may , (see s file). for each module, all module genes and the kme+ and kme-subsets were tested for enrichment using fisher's exact test in r. the p values were adjusted to control the false discovery rate. cten [ ] was used to determine enriched cell signatures in select co-expression modules. the enrichment score reported is the-log of the false discovery rate. model fitting and validation was performed in r using the 'segmented' package [ ] . five mice per time point per infection were infected with pfu of the described virus. five uninfected (naïve) mice served as negative controls. whole lungs were collected from mice, and incubated with collagenase d (roche diagnostics; final concentration: μg/ml) and dnase i (worthington; final concentration: u/ml) for minutes at °c. single-cell suspensions were obtained from lungs by grinding tissues through a nylon filter (bd biosciences). red blood cells (rbcs) in a sample were lysed with rbc lysis buffer (sigma). samples were resuspended with pbs containing mm edta and . % bovine serum albumin (bsa), and cell number was determined by using a disposable cell counter (onecell). to block nonspecific binding of antibodies mediated by fc receptors, cells were incubated with purified anti-mouse cd / (fc block, bd biosciences). cells were stained with appropriate combinations of fluorescent antibodies to analyze the population of each immune cell subset. the anti-f / (bm ; ebioscience) antibodies were used. all samples were also incubated with -aminoactinomycin d (via-probe, bd biosciences) for dead cell exclusion. data from labeled cells were acquired on a facsaria ii (bd biosciences) and analyzed with flowjo software version . . (tree star). three mice per time point per infection group were infected with pfu of the described virus. the primary antibodies of mouse anti-stat (phospho tyr ) mab (ab , abcam), rabbit anti-irf (phospho ser ) mab ( , cell signaling), and mouse anti-βactin (a ; sigma-aldrich) were used; the secondary antibodies were hrp-conjugated antimouse igg antibody (ge healthcare) and hrp-conjugated anti-rabbit igg antibody (ge healthcare). mouse lungs were collected and homogenized with ripa buffer (thermo scientific, rockford, il, usa) containing proteinase inhibitor (roche, mannheim, germany) and phosphatase inhibitor cocktails (sigma-aldrich, saint louis, missouri, usa). the lysates were then briefly sonicated and centrifuged. each sample was electrophoresed on sodium dodecylsulfate polyacrylamide gels (bio-rad laboratories, hercules, ca, usa) and transferred to a pvdf membrane (millipore, billerica, ma, usa). the membranes were blocked with blocking one (nacalai tesque, kyoto, japan) for min at room temperature, and then were incubated with the primary antibodies overnight at °c, followed by the secondary antibodies. they were then washed times with pbs plus tween (pbs-t) for min and incubated with secondary hrp-conjugated antibodies (as described above) for min at room temperature, followed by three washes with pbst. specific proteins were detected by using supersignal west femto maximum sensitivity substrate (thermo scientific, rockford, il, usa). photography and quantification of band intensity were conducted with the versadoc imaging system (bio-rad laboratories, hercules, ca, usa). the quantity of target bands from each sample was standardized by their respective β-actin. three mice per time point per infection group were infected with pfu of the described virus. half the lung of each mouse was dissolved in ml of ripa buffer. we measured the interferon-alpha and interferon-beta by using elisa kits (# , # , pbl assay science, nj, usa) according to the manufacturer's instructions. plates were read at an absorbance of nm using a versa max plate reader (moleculardevices, menlo park, ca). additional gene set overlap tests were performed in r with all of the genes annotated on the array as the reference (background) set. statistical tests to compare means within the western blot, flow cytometry, immune cell count and protein assay data sets were performed in r using the 'multcomp' package [ ] . supporting information s fig. the scaled difference of the module eigengene. the eigengene is the first principle component (equivalently the first eigenvector) of a matrix of gene expression data. in clustered data in which all genes are highly correlated (as in the case of wgcna), the eigengene is a scaled approximation of how gene expression changes for all genes in a module (i.e. cluster) across experimental conditions. the eigengene of module n is shown in (a) for the experimental conditions considered in this study ( infection conditions and time points). each point represents data from a single animal in each experimental condition ( animals per condition except for h n -infected animals at hpi which had ; total of samples). while the eigengene illustrates the changes in gene expression of module member genes, it is difficult to interpret the changes with respect to the control data. we therefore scaled the eigengene by subtracting from time-matched data the average of the eigengene from mock-infected animals and then dividing by the highest average eigengene for any experimental condition. the scaled difference of the eigengene (sde) is shown in (b). the sde now represents the fraction of greatest gene expression (i.e., the fraction of the largest log fold change in gene expression observed between all time-matched, infected-to-control samples). for example, the sde peaks in experimental condition (h n -infected animals at hpi), corresponding to the condition in which the log fold change was greatest for n member genes (see fig ) . the sde for ph n -infected animals at day pi (condition ) is~ . . we would expect the log fold change to be approximately half of that observed in h n -infected animals at hpi (see the heatmaps in fig ) . for completeness, we overlay the mean (lines) and the standard deviation to create the segmented linear model to describe the relationship between the n module eigengene and the virus titer data, we selected all titer data that occurred prior to and included the peak of the eigengene and then scaled the data such that the eigengene was bound between [ , ]. the model was then fit to the scaled data. panel (a) shows the time points selected as training data from each infection data set (indicated by the orange dots); and panel (b) shows the number of data points available for different ranges of the virus titer (top) and the segmented model's fit to the training data (bottom). in panel (c), we used the residuals (i.e., the difference between the predicted values and the actual values) to compare the slm's accuracy to that of a simple linear model. the line is the running average and the shaded region is the % confidence interval of the mean. the mean of the residuals of the segmented model was always near zero for the full range of virus titers and, thus, is a better fit than the linear model. two procedures were applied to determine whether the n genes that we identified as co-expressed in the original clustering analysis were also co-expressed in tissue from mice infected with pfu of h n virus. (a) compares the correlation between all , n gene transcripts and the eigengene (referred to as the kme) in the original gene expression data ( pfu infection conditions (referred to as ' pfu')) and the pfu infection condition (referred to as ' pfu'). more than % of the genes exhibited a kme > . . (b) the wgcna algorithm was repeated with all differentially expressed genes (fdr-adjusted p-value < . and fold change > for at least time point) from the pfu h n virus infection, and then the fisher's exact test was used to identify modules enriched for n genes. of the modules in the newly constructed co-expression network, only one module was enriched with the n transcripts. specifically, of the genes originally assigned to n , (benjimini-hochberg-adjusted p-value < . e- ) were differentially expressed and assigned to the same module in the pfu infection condition, as illustrated by the venn diagram. . the h n - pfu eigengene was constructed using the same set of probes assigned to the n module from the pfu infection condition and scaled to between [ , ]; and then the segmented linear model trained to the pfu h n , ph n , and h n data was used to predict h n - pfu scaled eigengene values. panel (a) shows a comparison of the predicted (black) and actual (pink arrows depicting the temporal evolution of the gene response) eigengenes, with error bars illustrating the standard deviation of the eigengene and of the log of the virus titer. panel (b) shows how the prediction residuals are distributed over time. for each time point, individual data points (black points) are shown, as well as the average (red points) and standard deviation (gray bars). the greatest deviations occurred at d and d , which was expected as the model was designed only to predict the onset of gene activation and the peak gene expression (peak of the eigengene). on days and post-infection, both the virus titers and gene expression has already peaked and are declining. panel (c) shows how the prediction residuals are distributed across the spectrum of observed virus titers, as compared to a linear model directly fit to the h n - pfu n eigengene. individual residuals are indicated by points, and the running average and the % confidence intervals are shown by the colored lines and the gray shading, respectively. as for the pfu infection condition, the segmented model performed well across the entire virus titer spectrum, and was significantly better than a linear model fitted directly to the data. (tif) s fig. protein concentrations of non-n module cytokines. as described in the fig leg end, cytokine concentrations were assayed in the lungs of mice infected with pfu of h n , ph n , and h n and compared with those in lung tissues from mock-infected animals. this heat map illustrates protein expression values for non-n -associated cytokines ( cytokine expression profiles are shown; il- (p )( ) was not detected at any time point and is not included), with a blue-to-yellow scale indicating expression levels (see the key to the right of the panel). the module to which the protein's mrna transcript was assigned during clustering is shown on the right hand side of the heat map (proteins whose gene transcripts were not de are labeled 'na'), and the average virus titers are shown below the heat map (red indicates that titers exceeded the threshold concentration predicted by the segmented linear model). while il- and leukemia inhibitory factor (lif) exhibited protein expression patterns consistent with n module behavior, the transcripts mapping to these proteins were not differentially expressed and were excluded from the co-expression network construction. (tif) s fig. virus titer and immunoblot data from an additional infection experiment with pfu of h n , ph n , or h n virus. as described in the fig legend (panel b) , an additional set of mice was infected with pfu of h n , ph n , or h n virus to determine total and phosphorylated levels of transcription factors by means of immunoblotting. here, virus titers (with standard deviation indicated by gray bars) for each infection condition are shown in panel (a), and immunoblot results are shown in panels (b) and (c). for immunoblot analyses, relative protein concentrations were determined by calculating the ratio of the gray intensity of the measured protein (iκbα, total irf , total irf ['irf '], phosphorylated irf [pirf '], total stat ['stat '], and phosphorylated stat ['pstat ']) relative to the gray intensity of actin (i.e., the loading control; referred to as the relative signal intensity, rsi) in each tissue sample. a linear model was used to compare the mean rsi of each protein at each time point to the mean rsi measured in uninfected animals (referred to as 'naïve'), and a significant difference was defined as having a false discovery rate (fdr) < . . the mean rsi of total irf , irf , and iκbα did not significantly deviate from the naïve data, but pstat , pirf and total stat significantly differed from the naïve data at several time points (signifi- the intramodular correlation (kme or correlation between each transcript and the eigengene) can be used to assess clustering quality. we show boxplots to illustrate the distribution of the kmes for all transcripts belonging to the n and n modules. (tif) s fig. applying signed co-expression network analysis also clusters the n kme+ transcripts. the n module presented in this work was identified by developing an unsigned co-expression network and then focusing on the kme-and kme+. therefore, the n kme+ set of genes should also cluster when developing a signed co-expression network. we confirmed this by repeating the wgcna procedure for signed network (power = ) . of the n kme+ transcripts, . % were assigned to the same cluster in the signed co-expression network (the new module is referred to as n signed). furthermore, . % if the n signed transcripts were n kme+ transcripts. we then confirmed that the same eigengene dynamics were observed. the scaled difference of the eigengene (sde) of the n signed module is shown versus time (a) and versus virus titers (b). (tif) s file. each of the kme+ submodules, labeled n , n ,. . .,n were analyzed using david bioinformatics tool to determine enriched biological functions. for each module, we report the top enriched annotation clusters as well as various measurements of enrichment (the bonferroni, benjamini, and the false discovery [fdr] adjusted p value. additional information on the enrichment statisticis can be found at david.abcc.ncifcrf.gov (xls) s file. each of the kme-submodules, labeled n , n ,. . .,n were analyzed using david bioinformatics tool to determine enriched biological functions. for each module, we report the top enriched annotation clusters as well as various measurements of enrichment (the bonferroni, benjamini, and the false discovery [fdr] adjusted p value. additional information on the enrichment statistics can be found at david.abcc.ncifcrf.gov (xls) s file. a list of interferon stimulated genes received from the interferon stimulated gene database. (xlsx) s table. the co-expression module to which each transcript was assigned. for each transcript we provide the entrez gene id, the gene symbol, the module it was assigned to and the correlation between the transcript's expression dynamics and the expression dynamics of its assigned eigengene (kme). module n is the set of transcripts which the algorithm did not identify as co-expressed. (xlsx) s table. enriched annotations identified using toppcluster. each kme+ or kme-submodule was analyzed in toppcluster for enriched domain, go biological process, go molecular function, go cellular component, mouse phenotype, pathway or transcription factor binding site annotations. columns a-d provide information on the annotation, including the category to which the annotation belongs, the database specific annotation id and the full title of the annotation. columns d-at contain the enrichment score for each annotation in each submodule. the enrichment score is the-log of the false discovery rate [fdr]-adjusted p value. a threshold enrichment score of (fdr-adjusted p < . ) was required to be considered as significantly enriched. (xlsx) s table. cten analysis of n module. the probes assigned to module n were analyzed in cten-a platform for identifying the genomic signatures of select cell type in microarray data. the enrichment score reported for each cell type is the-log of the false-discovery adjusted p value. (xlsx) s table. a heatmap of the gene expression of the probes assigned to the n and n modules. arrays were developed from the lungs of mice infected with h n , ph n , or hpai virus at time points. one array from the hpai infected data set was removed due to quality concerns. for each transcript, we provide annotation information (probe name, systematic name, entrez id, gene symbol, and gene name), identify to which module the transcript was assigned, the kme (the pearson correlation coefficient between the transcripts expression and the eigengene of the module it was assigned to), and the log fold change in the transcript's expression across all arrays used the study is illustrated by a heatmap. (xlsx) s table. correlation between the changes macrophage and neutrophil cell counts and each module eigengene. (xlsx) s table. correlation between changes in cytokine protein levels and cytokine gene transcript levels. (xlsx) regulation of toll-like receptor signaling pathways in innate immune responses immune signaling by rig-i-like receptors cutting edge: influenza a virus activates tlr -dependent inflammatory and rig-i-dependent antiviral responses in human lung epithelial cells recognition of single-stranded rna viruses by toll-like receptor differential roles of mda and rig-i helicases in the recognition of rna viruses rig-i-mediated antiviral responses to single-stranded rna bearing '-phosphates '-triphosphate rna is the ligand for rig-i aberrant innate immune response in lethal infection of macaques with the influenza virus genomic analysis of increased host immune and cell death responses induced by influenza virus lethal dissemination of h n influenza virus is associated with dysregulation of inflammation and lipoxin signaling in a mouse model of infection h n influenza viruses: outbreaks and biological properties fatal outcome of human influenza a (h n ) is associated with high viral load and hypercytokinemia in vitro and in vivo characterization of new swine-origin h n influenza viruses integrated network analysis reveals a novel role for the cell cycle in pandemic influenza virus-induced inflammation in macaque lungs h n influenza virus pathogenesis in genetically diverse mice is mediated at the level of viral load viral replication rate regulates clinical outcome and cd t cell responses during highly pathogenic h n influenza virus infection in mice specific mutations in h n mainly impact the magnitude and velocity of the host response in mice wgcna: an r package for weighted correlation network analysis resources: expanded annotation database and novel algorithms to better extract biology from large gene lists toppcluster: a multiple gene list feature analyzer for comparative enrichment clustering and network-based dissection of biological systems snapshot: pathways of antiviral innate immunity pathogenic influenza viruses and coronaviruses utilize similar and contrasting approaches to control interferon-stimulated gene responses cten: a web-based platform for identifying enriched cell types from heterogeneous microarray data lethal influenza virus infection in macaques is associated with early dysregulation of inflammatory related genes integrated clinical, pathologic, virologic, and transcriptomic analysis of h n influenza virus-induced viral pneumonia in the rhesus macaque ccr + monocyte-derived dendritic cells and exudate macrophages produce influenza-induced pulmonary immune pathology and mortality a systems analysis identifies a feedforward inflammatory circuit leading to lethal influenza infection transcriptomic analysis of autistic brain reveals convergent molecular pathology extracellular '- ' oligoadenylate synthetase stimulates rnase l-independent antiviral activity: a novel mechanism of virus-induced innate immunity systematic and quantitative assessment of the ubiquitin-modified proteome the transcriptional landscape of the mammalian genome ultrasensitivity in the regulation of cdc c by cdk bistability in apoptosis: roles of bax, bcl- , and mitochondrial permeability transition pores ultrasensitivity and positive feedback to promote sharp mitotic entry ultrasensitivity in the mitogen-activated protein kinase cascade identifying fragilities in biochemical networks: robust performance analysis of fas signaling-induced apoptosis the ability of pandemic influenza virus hemagglutinins to induce lower respiratory pathology is associated with decreased surfactant protein d binding influenza a virus lacking the ns gene replicates in interferon-deficient systems identification of the non-structural influenza a viral protein ns a as a bona fide target of the small ubiquitin-like modifier by the use of dicistronic expression constructs modification of nonstructural protein of influenza a virus by sumo sumoylation affects the interferon blocking activity of the influenza a nonstructural protein ns without affecting its stability or cellular localization myd signaling is indispensable for primary influenza a virus infection but dispensable for secondary infection toll-like receptor pre-stimulation protects mice against lethal infection with highly pathogenic influenza viruses linear models and empirical bayes methods for assessing differential expression in microarray experiments toppgene suite for gene list enrichment analysis and candidate gene prioritization systematic discovery of regulatory motifs in human promoters and utrs by comparison of several mammals molecular signatures database (msigdb) . functional classification of interferon-stimulated genes identified using microarrays segmented: an r package to fit regression models with broken-line relationships simultaneous inference in general parametric models we would like to thank susan watson for editing the manuscript. key: cord- -vxcj sn authors: chen, yuxin; tong, xin; li, yang; gu, bin; yan, jiawei; liu, yong; shen, han; huang, rui; wu, chao title: a comprehensive, longitudinal analysis of humoral responses specific to four recombinant antigens of sars-cov- in severe and non-severe covid- patients date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: vxcj sn there is an urgent need for effective treatment and preventive vaccine to contain this devastating global pandemic, which requires a comprehensive understanding of humoral responses specific to sars-cov- during the disease progression and convalescent phase of covid- patients. we continuously monitored the serum igm and igg responses specific to four sars-cov- related antigens, including the nucleoprotein (np), receptor binding domain (rbd), s protein, and ectodomain (ecd) of the spike protein among non-severe and severe covid- patients for seven weeks since disease onset. most patients generated humoral responses against np and spike protein-related antigens but with their distinct kinetics profiles. combined detection of np and ecd antigens as detecting antigen synergistically improved the sensitivity of the serological assay, compared to that of using np or rbd as detection antigen. . % of convalescent sera from covid- patients revealed that the varying extents of neutralization activities against sars-cov- . s -specific and ecd-specific iga responses were strongly correlated with the neutralization activities in non-severe patients, but not in severe patients. moreover, the neutralizing activities of the convalescent sera were shown to significantly decline during the period between days to days after hospital discharge, accompanied by a substantial drop in rbd-specific iga response. our data provide evidence that are crucial for serological testing, antibody-based intervention, and vaccine design of covid- . a a a a a the ongoing pandemic of severe acute respiratory syndrome coronavirus (sars-cov- ) that first emerged in china, has rapidly spread worldwide. as of june , , the coronavirus disease (covid- ) had been confirmed in more than million cases worldwide [ ] , carrying a mortality rate of . % [ ] . there is an urgent need for effective treatment and preventive vaccine to contain this devastating global pandemic, which requires a comprehensive understanding of humoral responses specific to sars-cov- during the disease progression and convalescent phase of covid- patients. we recently reported that differential longitudinal patterns of nucleic acid and serology testing among severe patients, non-severe patients, and asymptomatic carriers of sars-cov- [ ] . we demonstrated that early, arising antibody responses were detected concurrent with positive viral rna among severe patients, while antibody responses which often facilitate the viral clearance were observed from non-severe patients. this prompted us to further explore the antigen specificity and humoral responses among severe and non-severe patients. the kinetics of antibody response to nucleoprotein (np) and spike protein receptor binding domain (rbd) from sars-cov- have been reported [ , ] . however, in addition to rbd, the antibody response targeting the full-length s domain or ectodomain (ecd) of the spike protein is still not known. the rbd of sars-cov- is relatively small containing amino acids, compared to the full-length s protein or ectodomain (ecd) of the spike glycoprotein. therefore, the sensitivity of antibody seroconversion might be compromised by exclusively including the rbd instead of alternative recombinant antigens such as s and ecd protein for covid- serological testing. additionally, recent studies demonstrated that transfusion of convalescent plasma containing the neutralizing antibodies resulted in clinical improvement [ , ] , which necessitates a comprehensive understanding of the specificity, isotypes, potency and persistence of the neutralizing antibody components present in the convalescent sera of clinically recovered covid- patients. in this retrospective study, we successively monitored the serum igm and igg responses specific to four sars-cov- related antigens, including the np protein, rbd protein, s protein, and ecd protein in non-severe and severe covid- patients during the disease progression. moreover, the neutralization activities of the serum collected at hospital discharge were determined, and their correlations with the specific antibody of different isotypes targeting the above four recombinant antigens were identified. furthermore, in order to determine the duration of antibody responses, the neutralization activities and anti-sars-cov- antibody responses after hospital discharge were also analyzed. our data provide important insights for serological testing, antibody-based intervention, and vaccine design. four sars-cov- related proteins, recombinant spike protein receptor binding domain (rbd) protein, s protein, the ectodomain of the spike protein (ecd), and nucleocapsid protein (np) were used as detected antigens, respectively (fig a) . the genes encoding spike rbd (residues arg to phe of spike protein), s protein (residue val to arg of spike protein), the ectodomain of spike protein (ecd) (residue met to tyr of spike protein), and the full-length np protein were codon-optimized and synthesized (genewiz, china). genes encoding for the spike rbd, s , and ecd were cloned into the mammalian expression vector pcdna . , while the genes encoding the np protein were inserted into the humoral responses in severe and non-severe covid- patients expression vector pet- (b), in frame respectively and upstream of the series of six histidine residues. the rbd, s , and ecd proteins were expressed in f cells, while the np protein was from escherichia coli, followed by affinity purification. the purity of the np protein and rbd proteins was determined by % sodium dodecyl sulfate (sds) polyacrylamide gel electrophoresis (fig b) . this study was approved by ethics committee of affiliated hospital of xuzhou medical university and nanjing drum tower hospital in jiangsu province (protocol number: xyfy -kl - and - - ). written informed consent was waived by the ethics commission due to public health outbreak investigation. we retrospectively recruited patients with covid- from jan to feb , , at the affiliated hospital of xuzhou medical university and nanjing drum tower hospital in jiangsu province. patients with suspected sars-cov- were confirmed after two sequential positive respiratory tract sample results. throat swab samples were collected every - days. the serum samples retrieved from routine biochemical or immunological testing were inactivated at ˚c for min. these samples were later stored at - ˚c for later serological detection. the severity of covid- was judged based on the sixth revised trial version of novel coronavirus pneumonia diagnosis and treatment guidance [ ] . those who met the criterion as follows were defined as severe cases: ( ) respiratory distress with respiratory rate over per minute; ( ) hypoxia (oxygen saturation less than � % in the resting state); ( ) arterial blood oxygen partial pressure (pao )/oxygen concentration (fio ) less than mm hg; or ( ) severe disease complications including respiratory failure which requires mechanical ventilation, septic shock, or non-respiratory organ failure. an in-house enzyme-linked immunosorbent assay (elisa) was performed as previously described ( ) . briefly, -well plates were coated with ng/ml of individual recombinant viral antigen overnight, respectively. the plates were incubated with serum samples in a dilution of : , followed by incubation with either anti-human igm conjugated with hrp (abcam, ab , : dilution) and igg conjugated with hrp (abcam, ab , : dilution). optical density (od) value at nm was measured. for antibody isotyping of the sera collected during convalescent covid- patients, the plates were firstly coated with the desired recombinant antigen, respectively. later, the plates were added with serial diluted serum samples starting from : to : . after washing, the plates were coated with anti-human iga conjugated with hrp (abcam, ab , : dilution), anti-human igg conjugated with hrp (abcam, ab , : dilution), anti-human igg conjugated with hrp (abcam, ab , : dilution), anti-human igg conjugated with hrp (abcam, ab , : dilution), and anti-human igg conjugated with hrp (abcam, ab , : dilution) for antibody isotyping analyses. subsequently, the plates were incubated with tmb substrate for hour and the reaction stopped with m h so . the preliminary cut-off value for each elisa assay was calculated as the mean of the negative control serum od values plus standard deviation (sd) from archived healthy individuals from the year of . antibody endpoint titer was determined by the highest dilution which gives an od value higher than cut off value of the healthy control group at the same dilution. humoral responses in severe and non-severe covid- patients pseudoviruses expressing the sars-cov- spike protein were obtained as a general gift from the institute of biological product control from national institute for food and drug control of china. sars-cov- pseudoviruses were prepared using vsv g pseudotyped viruses (g � Δg-vsv) that package the expression cassette for firefly luciferase instead of vsv-g in the vsv genome, and the serum neutralization capability was performed as described recently [ ] . briefly, the sars-cov- pseudoviruses were preincubated with heat-inactivated serum samples at a -fold serial dilution starting at : at ˚c for one hour, together with the pseudovirus control and cell control wells. the sera from healthy controls were served as the negative control in hexaplicate. next, the -well plates were seeded with μl of freshly trypsinized huh cells ( x cells/well). after hours of incubation in a % co environment at ˚c, the luminescence was measured using luciferase substrate (promega, e ). the titer of nabs was calculated as % inhibitory dose (id ), expressed as the reciprocal serum dilution which resulted in a % reduction in relative light units (rlus) compared to the virus control wells after subtraction of background rlus. data including demographic information, medical history, symptoms, laboratory findings, treatment regimen were retrieved from the patients' medical record. laboratory results included blood routine, lymphocyte subsets, and crp. the total number of lymphocytes in peripheral blood was counted by hemocytometer. the percentage of lymphocyte subsets were analyzed with facs flow cytometry for those covid- patients on admission. binding antibody titers or neutralizing antibody titers were expressed as geometric mean titers (gmts). the mean (standard deviation (sd)) or median (interquartile range (iqr)) was used to present the continuous variables. categorical variables were described as the count and percentage. the independent group t test (normal distribution) and mann-whitney u (non-normal distribution) were used to compare continuous variables between groups. spss software program version . (chicago, il, usa) were used for data analysis. p< . was considered to be statistically significant. when multiple testing was performed in the correlation analysis, the bonferroni correction adjusted significance level was . / = . . a total of covid- patients were enrolled in our study (table and s table) . the median age was years (iqr, . - . ), and patients were male. the median age for severe covid- patients was significantly higher compared to the non-severe group (p = . ). type diabetes ( . %) was the most common comorbidities. fever ( . %), cough ( %), and myalgia ( . %) were the most common symptoms. blood test on admission revealed that the lymphocyte counts were significantly lower in the severe cases ( . x /l) than in the non-severe cases ( . x /l) (p< . ). lymphocyte subset analyses further indicated that the numbers of t lymphocytes, cd + t cells and b cells were markedly reduced in severe patients, in comparison to non-severe patients (p< . , p = . , p = . , respectively) (s table) . all the severe and non-severe patients were administered with empirical antimicrobial treatments including interferon α inhalation, arbidol, lopinavir- humoral responses in severe and non-severe covid- patients ritonavir combination and darunavir. as of march th, , the patients were clinically recovered from covid- and subsequently discharged. serum specimens were obtained from covid- patients (mean . serum samples per patient). we serially monitored the antibody profile specific to rbd, s , ecd and np during the disease progression phase of covid- patients (fig and s fig) . overall, ( . %) out of patients were seroconverted, whereas patients (p and p ) showed a barely detectable level of both igm and igg response against four sars-cov- antigens during our observation period. we cannot rule out the possibility that these two patients might have been seroconverted later after hospital discharge. an increase in serum igm and igg antibody levels against four proteins for most patients at days or later after symptom onset was noticed, as illustrated by the od values in fig . throughout our observation period, . % ( / ) of patients exhibited a strong response of igm or igg against four antigens, while three patients (p , p and p ) had a relatively low level of antibody response against spike protein derived antigens. furthermore, distinct kinetics of antibodies with different specificities were identified. the patients initially reached the np-specific igm peak and s -specific igm peak on day (iqr: - and iqr: - , respectively), then anti-rbd igm, anti-ecd igm, and anti-np igg peaked at day (iqr: - , iqr: - , iqr: - , respectively), followed by the rbd-specific igg peaked on day (iqr: - ), and the peak igg responses specific of s and ecd antigens on day (iqr: - , iqr: - , respectively). humoral responses in severe and non-severe covid- patients interestingly, antibody responses were sharply dropped in % ( / ) of patients after reaching their peak, whereas the antibody responses in the remaining patients were sustained. our data suggested that sars-cov- infection induced a distinct temporal profile of humoral responses against viral np, rbd, s and ecd antigens. correlation analyses of od values derived from four elisa assays were performed (fig ) . the anti-rbd igm response was correlated with anti-np igm (r = . , corrected p< . ), anti-s igm (r = . , corrected p< . ), and anti-ecd igm (r = . , corrected p< . ), while the anti-rbd igg response was associated with the levels of anti-np igg (r = . , corrected p< . ), anti-s igg (r = . , corrected p< . ), and anti-ecd igg responses (r = . , corrected p< . ). besides, the anti-s igm response was associated with the anti-n igg response (r = . , corrected p< . ) and anti-ecd igm responses (r = . , corrected p< . ). the anti-s igg response was linked with the anti-np igg (r = . , corrected p< . ) and anti-ecd igg response (r = . , corrected p< . ), anti-ecd igm response was correlated with the anti-np igm response (r = . , corrected p< . ), whereas anti-ecd igg response was correlated with anti-n igg response (r = . , corrected p< . ). overall, the level of rbd-specific igm or igg was well correlated with either the anti-s or anti-ecd antibody responses, while the np-specific igm or igg was weakly related with the level of igm or igg response targeting various subunits of the spike protein. our data indicated a distinct recognition pattern of np protein and various domains of spike protein in the serum of covid- patients. humoral responses in severe and non-severe covid- patients since the temporal profiles of np-specific and spike protein-specific antibody responses were distinct, we determined the sensitivity of serological testing using different sars-cov- humoral responses in severe and non-severe covid- patients derived antigens from week to week after disease onset. since the number of sera collected after week of disease onset was relatively limited in our cohort, the sensitivity of our elisa assay with the clinical samples within the first four weeks was analyzed. as shown in table , among the eight anti-sars-cov- specific igm or igg parameters, serological testing of np protein-specific igg was able of achieving optimal sensitivities of . %, . %, . %, and . % from week to week after disease onset, respectively. however, the combined testing of ecd-specific igm and np-specific igg responses were able to further synergistically boost the assay sensitivity to . %, . %, %, and . % from week to week , respectively, yielding the highest sensitivity if compared to that obtained using either combined anti-rbd igm and anti-rbd igg or combined anti-np igm and anti-np igg. to dissect the antibody component and specificity from the sera of covid- clinically recovered patients, the magnitude of igm, iga, igg, and igg isotypes including igg to igg targeting rbd, s , ecd, and np were further analyzed by elisa (fig ) . of note, barely detectable levels of anti-sars-cov- igg and igg were observed from all the patients in our cohort. compared to non-severe patients, severe patients tended to generate a higher level of antibody responses. specifically, remarkably higher level of rbd-specific igg, iga, igg , and igg titers were found in severe patients compared to non-severe patients (p = . , p = . , p = . , p = . , respectively). likewise, considerably higher levels of s -specific iga, np-specific iga and igg were also present in severe patients when compared to non-severe patients (p = . , p = . , and p = . , respectively). the neutralization activities were subsequently determined by the pseudovirus microneutralization assay as previously described. . % ( / ) of covid- patients, including . % ( / ) of the severe patients and . % ( / ) of the non-severe patients, generated neutralization activities at the time point of discharge with a geometric mean titer of (fig a and b) . meanwhile, / ( . %) of patients elicited low levels of neutralization activities (id titer between to ), while / ( . %) of patients produced a moderate level of neutralization activities (id titer between to ), and / ( . %) patients exhibited a potent level of neutralization activity (id titer over ). there was no statistically significant difference in terms of neutralizing titers between the severe group (geometric titer, ; iqr: - ) and non-severe group (geometric titer: ; iqr, . - . ) (p = . ), despite the fact that the severe patients had considerably higher level of anti-sars-cov- specific binding antibody titers. using the antibody binding titers and neutralizing activities form the non-severe patients, we discovered that the neutralizing antibody titers moderately correlated with the ecd-specific iga responses (r = . , p< . ), s -specific iga response (r = . , p< . ), rbd-specific igg responses (r = . , p< . ), rbd specific iga responses (r = . , p = . ), and np-specific igm responses (r = . , p< . ) (fig d) . furthermore, we also noticed that the ecd-specific igg titer (r = . , p = . ), ecd-specific igg titer (r = . , p = . ), and rbd-specific igg titer (r = . , p = . ) were weakly correlated with the serum neutralization activities. additionally, in addition to igg antibodies, iga also greatly contributed to the anti-sars-cov- neutralizing activities. it is worth mentioning that in contrast to the correlation between the neutralizing activities and the binding antibody titers established in non-severe patients, we did not find any strong relationship between the neutralization activities and binding titers in the severe patients, suggesting a complex involvement of non-neutralizing antibodies during disease progression. whether clinical recovered covid- patients might be susceptible to sars-cov- re-infection remains questioned. serum samples from sixteen covid- patients (eleven non-severe and five severe patients) were collected at the time point of discharge and during follow-up visit between and days. then, their neutralizing activities and binding antibody titers were further compared (fig a) . the neutralizing activities of serum obtained at the followup visit between days and days after discharge were significantly lower than those obtained at the time point of hospital discharge (p = . , paired-t test), concurrent with significantly reduced anti-rbd iga responses (p = . , paired-t test), which might be responsible for the declining trend of neutralizing activities. nevertheless, we did not observe a profoundly reduced igm or igg response targeting the np or spike protein-related antigens. additionally, the reduced fold of igm, igg and iga binding titer and neutralization activities at the time point of hospital discharge and follow-up visit between days and days after discharge were also analyzed between severe patients and non-severe patients (fig b) . the severe patients and non-severe patients had comparable reduced fold of igm, igg, and iga binding titer specific to rbd, ecd, s , and np protein and neutralization activities. using four recombinant sars-cov- related antigens, we were able to monitor the dynamic antibody responses with various specificities. we demonstrated that the kinetics profile of igm or igg responses specific for four antigens might be distinct in time and magnitude aspects. the elisa assay was developed using rbd [ , ] , s [ ] , stabilized full-length spike protein [ ] , np protein [ ] , combined rbd and np protein ( ), respectively, as serological assay, humoral responses in severe and non-severe covid- patients humoral responses in severe and non-severe covid- patients which is a feasible approach to analyze the sars-cov- specific humoral responses in covid- patients and perform large-scale sero-epidemiology studies. our current study suggested that combined detection of np-specific igm and ecd-specific igg could greatly improve the sensitivity of serological assay especially during the first weeks after symptom onset, compared to the mere inclusion of either the np-specific antibody or rbd-specific antibody. despite of improved sensitivity of our elisa assay, a small portion of non-severe covid- patients was still identified as seronegative. this was consistent with recent findings. first, it is well-established that the non-severe covid- patients generated significantly lower level of viral-specific humoral response compared to that in severe patients [ , ] , consistent with our results. besides, asymptomatic sars-cov- carrier patients had a remarkably reduced level of virus specific igg levels, compared to symptomatic group, accompanied by prolonged the reduced folds of igm, igg and iga titer specific to rbd, ecd, s , and np protein and neutralization activities at the time point of hospital discharge and follow-up visit between days and days after discharge were analyzed between severe patients and non-severe patients. https://doi.org/ . /journal.ppat. .g humoral responses in severe and non-severe covid- patients presence of viral rna and remarkable reduction of igg and neutralizing activities [ ] . the differential pattern of specific humoral responses elicited by sars-cov- might be contributed by host factors such as age and host inflammatory responses [ , ] . it is unclear whether clinically recovered patients acquire the protective immunity from reinfection. limited information regarding the neutralization activities of the clinically recovered patients was available. in this study, we reported that . % of convalescent sera had varying degrees of neutralization activities, and only a small portion of patients elicited a potent level of neutralization activity. preliminary studies indicated that the major factor associated with the efficacy of convalescent plasma therapy is the neutralizing antibody titer of the convalescent plasma obtained from the donor [ , ] . our data also demonstrated the importance of prior selection of convalescent serum using neutralization assays. additionally, the rapidly declined neutralizing activities in covid- clinically recovered patients within days after discharge, suggesting that the circulating anti-sars-cov- neutralizing antibodies might have a relatively short half-life. this finding was consistent with previous studies demonstrating that the humoral immunity rapidly waned over time in patients that recovered from sars-cov [ ] and mers-cov [ ] infections. this study also revealed a pivotal role of mucosal immunity in humoral protection against sars-cov- . first, we discovered that the neutralization activities were strongly associated with s -specific iga and ecd-specific iga responses, and moderately correlated with rbdspecific iga and igg responses, suggesting that mucosal immunity might contribute greatly to viral neutralization. even though our current temporal antibody profile did not include the iga responses, previous studies demonstrated the presence of np or rbd-specific iga responses in the early phase during sars-cov- infection, whereas the systemic igm and igg responses occurred later [ , ] . of note, the rbd-specific iga response declined sharply after hospital discharge, accompanied by the rapidly waned neutralization activities. due to the relatively short half-life of iga [ ] , our finding of rapidly declined rbd-specific iga response is not surprising, which might be responsible for the clinical observation of prolonged viral rna shedding in fecal samples [ ] or re-occurrence of positive viral rna in rectal swabs [ ] . furthermore, significantly higher level of iga response was detected from the clinically recovered severe patients in comparison to non-severe patients. consistent with our findings, vaccine-induced potent mucosal iga was associated with lower level of viral load and reduced pulmonary pathological damages upon challenge with sars-cov [ ] . the beneficial role of iga during the covid- disease course still requires thorough investigation. it is important to further analyze the magnitude of iga responses between the survivor and nonsurvivor groups in a large cohort. the antibody-dependent enhancement (ade) phenomenon has been a major concern in viral infections [ ] [ ] [ ] . antibody specificity, concentration, neutralization ability, and isotype might define the ability of antibody to neutralize virions and protect the host or promote ade and acute inflammation [ ] . previous animal studies of sars-cov indicated that the spike protein rbd region or hr domain-specific antibodies might exert a beneficial role in viral clearance, whereas the antibodies specific for np and other s protein epitopes other than the rbd and hr domain might induce ade and escalate acute inflammation [ , ] . besides, igg isotype also controls the effector function, in which igg and igg engage fcrriia and fcrriib with high affinity leading to the possible occurrence of ade. our data showed that severe patients generated significantly higher level of antibody titers, especially for np-specific igg and rbd-specific igg and igg compared to the non-severe patients. consistently, we did not identify any correlation between the binding antibody titers and the neutralizing activities from the convalescent sera of severe covid- patients, suggesting that a large amount of non-neutralizing antibodies might have been present in the severe group. whether such humoral responses in severe and non-severe covid- patients higher magnitude of np-specific igg or rbd-specific igg and igg contributed to the severity of covid- disease progression remains to be determined. our data also have important implications for the current development of the covid- vaccine. the majority of patients generated potent humoral responses recognizing spike protein-related antigens, including rbd, s and ecd proteins, implying their high immunogenicity makes them vaccine candidates. additionally, our correlation analyses showed that the neutralizing activities were strongly correlated with ecd-specific iga and s -specific iga responses, compared to rbd-specific iga responses. several human neutralizing monoclonal antibodies (mabs) isolated from convalescent covid- patients were not exclusively targeted at rbd region. for example, the epitope of a potent neutralizing mab, a , is within n terminal of spike protein [ ] , while other mabs targeted at cryptic epitopes on spike trimeric interface [ ] . collectively, our study and others suggested that the antibodies targeting at diverse domains of the spike protein might greatly contribute to higher neutralization activities. consequently, the inclusion of the full-length spike protein might be ideal to elicit humoral responses targeting to the major neutralizing targets. moreover, the igg subclass responses in covid- patients were skewed toward igg and igg , and induction of optimal antibody isotypes such as iga and certain igg subclasses such as igg might be desired in vaccine studies of sars-cov- . our study also has some limitations. first, we only included a small number of recovered covid- patients and did not have the deceased covid- patients. second, the cytokines and antigen-specific cellular responses were not serially monitored, which could facilitate our understanding of innate and adaptive immune responses during covid- disease. thirdly, the mucosal iga responses such as the iga responses in saliva warrant further investigation. in conclusion, our study demonstrated that the majority of covid- patients generated a distinct profile of immune response against np and spike protein-related antigens in both time and magnitude aspects. therefore, combining np and ecd as detecting antigens could further enhance the sensitivity of the serological assay. furthermore, . % of the convalescent sea from covid- patients displayed varying levels of neutralization activities against sars-cov- , which correlated with s -specific and ecd-specific iga responses in non-severe patients. a rapid decline in these neutralizing activities was observed, accompanied by a sharply reduced rbd-specific iga response. our comprehensive, longitudinal analysis provides clues for the optimization of future serological testing, antibody-based intervention, and vaccine design. supporting information s fig. dynamic igm or igg responses specific to np, rbd, s , and ecd antigens for four severe covid- patients (p to p ) and sixteen non-severe covid- patients (p to p ) over time since symptom onset, determined by elisa assay. (tif) s table. laboratory findings and drug treatment of severe and non-severe covid- patients. (docx) conceptualization: yuxin chen, rui huang, chao wu. humoral responses in severe and non-severe covid- patients formal analysis: yuxin chen, rui huang. investigation: yuxin chen, rui huang. clinical characteristics of coronavirus disease in china different longitudinal patterns of nucleic acid and serology testing results based on disease severity of covid- patients temporal profiles of viral load in posterior oropharyngeal saliva samples and serum antibody responses during infection by sars-cov- : an observational cohort study serological assays for severe acute respiratory syndrome coronavirus (sars-cov- ) treatment of critically ill patients with covid- with convalescent plasma effectiveness of convalescent plasma therapy in severe covid- patients chinese management guideline for covid- (version . ) establishment and validation of a pseudovirus neutralization assay for sars-cov- antibody responses to sars-cov- in patients of novel coronavirus disease severe acute respiratory syndrome coronavirus -specific antibody responses in coronavirus disease a serological assay to detect sars-cov- seroconversion in humans profiling early humoral response to diagnose novel coronavirus disease (covid- ) bin he, tianying zhang, shengxiang ge, lei liu, jun zhang, ningshao xia, zheng zhang. antibody responses to sars-cov- antibody profiles in mild and severe cases of covid- . clin chem. . plos pathogens humoral responses in severe and non-severe covid- patients viral and host factors related to the clinical outcome of covid- neutralizing antibody responses to sars-cov- in a covid- recovered patient cohort and their implications. medrxiv treatment with convalescent plasma for critically ill patients with sars-cov- infection disappearance of antibodies to sars-associated coronavirus after recovery feasibility of using convalescent plasma immunotherapy for mers-cov infection, saudi arabia iga interaction with the asialoglycoprotein receptor prolonged presence of sars-cov- viral rna in faecal samples positive rectal swabs in young patients recovered from coronavirus disease (covid- ) intranasal vaccination of recombinant adeno-associated virus encoding receptor-binding domain of severe acute respiratory syndrome coronavirus (sars-cov) spike protein induces strong mucosal immune responses and provides long-term protection against sars-cov infection the potential danger of suboptimal antibody responses in covid- anti-spike igg causes severe acute lung injury by skewing macrophage responses during acute sars-cov infection antibody-dependent enhancement of severe dengue disease in humans immunodominant sars coronavirus epitopes in humans elicited both enhancing and neutralizing effects on infection in non-human primates prior immunization with severe acute respiratory syndrome (sars)-associated coronavirus (sars-cov) nucleocapsid protein causes severe pneumonia in mice infected with sars-cov a neutralizing human antibody binds to the nterminal domain of the spike protein of sars-cov- . science identification of human single-domain antibodies against sars-cov- humoral responses in severe and non-severe covid- patients key: cord- - a qde authors: johnson, christopher j; phillips, kristen e; schramm, peter t; mckenzie, debbie; aiken, judd m; pedersen, joel a title: prions adhere to soil minerals and remain infectious date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: a qde an unidentified environmental reservoir of infectivity contributes to the natural transmission of prion diseases (transmissible spongiform encephalopathies [tses]) in sheep, deer, and elk. prion infectivity may enter soil environments via shedding from diseased animals and decomposition of infected carcasses. burial of tse-infected cattle, sheep, and deer as a means of disposal has resulted in unintentional introduction of prions into subsurface environments. we examined the potential for soil to serve as a tse reservoir by studying the interaction of the disease-associated prion protein (prp(sc)) with common soil minerals. in this study, we demonstrated substantial prp(sc) adsorption to two clay minerals, quartz, and four whole soil samples. we quantified the prp(sc)-binding capacities of each mineral. furthermore, we observed that prp(sc) desorbed from montmorillonite clay was cleaved at an n-terminal site and the interaction between prp(sc) and mte was strong, making desorption of the protein difficult. despite cleavage and avid binding, prp(sc) bound to mte remained infectious. results from our study suggest that prp(sc) released into soil environments may be preserved in a bioavailable form, perpetuating prion disease epizootics and exposing other species to the infectious agent. transmissible spongiform encephalopathies (tses, prion diseases) are a group of fatal neurodegenerative diseases that affect a variety of mammalian species and include bovine spongiform encephalopathy (bse, ''mad cow'' disease), chronic wasting disease (cwd) of deer and elk, sheep scrapie, and creutzfeldt-jakob disease in humans [ ] . the agricultural, economic, and social impacts of prion diseases have been intensified by evidence suggesting transmissibility of bse to humans [ ] . the putative infectious agent in these diseases, designated prp sc , is a misfolded isoform of the normal cellular prion protein (prp c ). the amino acid sequences of prp sc and prp c are identical [ ] ; normal and abnormal forms of the protein differ only in conformation. no differences in posttranslational covalent modification have been demonstrated [ ] . circular dichroism and infrared spectroscopy indicate that the disease-specific isoform has a higher b-sheet and lower a-helix content than prp c [ ] . the normal isoform is soluble and primarily monomeric in solution, whereas prp sc forms insoluble aggregates. sheep scrapie and cervid cwd are unique among tses, because epizootics can be sustained by horizontal (animal-toanimal) transmission [ , ] . routes of natural transmission remain to be clarified, but available evidence indicates that an environmental reservoir of infectivity contributes to the maintenance of these diseases in affected populations [ ] [ ] [ ] . the expanding range of cwd (several regions of north america and korea) increasingly brings domestic livestock, companion animals, and wildlife species into contact with infected animals and carcasses, and shedded tse agent, raising the possibility of cross-species transmission. this was demonstrated by the recent detection in colorado, usa, of a free-ranging, cwd-infected moose, a species not previously known to be affected by the disease in the wild [ ] . although other modes of environmental transmission of scrapie and cwd have been proposed (e.g., flesh flies [ ] , hay mites [ ] ), several lines of evidence point to soil as a reservoir for tse infectivity. tse infectivity exhibits remarkable resistance to inactivation by most chemical agents, radiation, and heat [ ] and has been shown to persist after burial in soil for at least y [ ] . anecdotal observations of healthy sheep contracting scrapie after occupying fields previously containing diseased animals have been reported [ , ] . although these older studies did not account for the genetic susceptibility of the sheep under study, they suggest that scrapie agent can persist in the environment for years. recent controlled field experiments provide more compelling evidence of the environmental persistence of prions. miller et al. [ ] demonstrated that naïve mule deer could contract cwd when housed in paddocks previously inhabited by infected animals or containing decomposed infected carcasses. tse agents directly enter the environment when carcasses of infected animals decompose [ ] , through alimentary shedding of the agent from gut-associated lymphoid tissue [ , ] , or from urinary excretion from infected, nephritic animals [ ] . furthermore, bovine, sheep, and deer tse agents have been introduced to soil environments through the burial of diseased carcasses and other infected material [ ] . animals ingest soil both deliberately and incidentally [ ] . cattle, deer, sheep, and other animals can consume hundreds of grams of soil daily [ , ] . taken together, these data support the notion that prp sc -contaminated soil may allow intraspecies tse transmission and enhance the likelihood of spread to other species. as a first step toward understanding the role of soil as a reservoir of tse infectivity, we investigated the binding of prp sc to common soil minerals and whole soils and examined the infectivity of mineral-bound prions. we examined the sorption of purified prp sc to three common soil minerals (table s ) : quartz, montmorillonite (mte, an expandable layered silicate clay mineral), and kaolinite (kte, a nonexpandable phyllosilicate mineral). quartz of two particle sizes was employed in sorption experiments: fine sand (hydrodynamic diameter [d h ] ¼ - lm), representing quartz concentrated in the sand and silt fractions of soils, and microparticles (d h ¼ - lm), representing quartz present in the coarse clay fraction [ ] . purified prp sc (; . lg) was introduced into aqueous suspensions (ph . ) of each soil mineral and subjected to -h mixing. unbound prp sc was separated from bound protein by centrifugation through a -mm sucrose cushion. bound and unbound fractions were analyzed by sds-page and immunoblotting. the extent of prp sc sorption differed among the mineral particles examined. all detectable prp sc adsorbed to the expandable clay mineral mte ( figure a ). x-ray diffraction analysis provided no evidence that prp sc entered mte interlayer spaces (mte d spacings were . nm and . nm before and after prp sc adsorption, respectively); prion protein appeared to adsorb to only external clay surfaces. prp sc did not associate with an equal mass of fine quartz sand at levels detectable by immunoblotting ( figure a) . a large degree of prp sc binding to the nonexpandable clay mineral kte was observed when the surface area was matched to that of external mte surfaces ( figure a ). the limited association of prp sc with fine quartz sand was at least in part attributable to the much smaller specific surface area of these particles as compared to kaolinite and external mte surfaces (table s ). when quartz surface area was matched to that of external mte surfaces, all detectable prp sc adsorbed to quartz ( figure a ). the amount of prp sc adsorbed to mte was semiquantitatively assessed by serial dilution of samples to the limit of immunoblotting detection. the dilution at which no detectable immunoreactivity remained provided a basis for comparison with samples lacking immunoreactivity before dilution. prp sc desorbed from mte still exhibited immunoreactivity after -fold dilution, indicating that the amount of prion protein adsorbed to mte exceeded that in samples without immunoreactivity (e.g., unbound prp sc in experiments with mte) by at least two orders of magnitude ( figure b) . furthermore, this result suggests that fine quartz sand was saturated by at least -fold less prp sc ( . lg) than used for sorption experiments ( figure a) . to assess the prp sc -binding capacity of the other soil minerals, increasing quantities of prp sc were added to each mineral. protein desorbed from mineral particles was serially transmissible spongiform encephalopathies (tses) are a group of incurable diseases likely caused by a misfolded form of the prion protein (prp sc ). tses include scrapie in sheep, bovine spongiform encephalopathy (''mad cow'' disease) in cattle, chronic wasting disease (cwd) in deer and elk, and creutzfeldt-jakob disease in humans. scrapie and cwd are unique among tses because they can be transmitted between animals, and the disease agents appear to persist in environments previously inhabited by infected animals. soil has been hypothesized to act as a reservoir of infectivity, because prp sc likely enters soil environments through urinary or alimentary shedding and decomposition of infected animals. in this manuscript, the authors test the potential for soil to serve as a reservoir for prp sc and tse infectivity. they demonstrate that prp sc binds to a variety of soil minerals and to whole soils. they also quantitate the levels of protein binding to three common soil minerals and show that the interaction of prp sc with montmorillonite, a common clay mineral, is remarkably strong. prp sc bound to mte remained infectious to laboratory animals, suggesting that soil can serve as a reservoir of tse infectivity. diluted and subjected to sds-page and immunoblotting to semiquantitate the amount of sorbed protein. the binding capacity of a mineral was attained when subsequent prp sc additions did not further increase the dilution factor required to reach the limit of immunoblotting detection (table ). of the minerals examined, mte exhibited the highest prp sc adsorption capacity (; lg protein mg mte À ). the adsorption capacity of the quartz microparticles was nearly -fold less (; . lg protein mg microparticle À ), and that of kte was nearly -fold less than mte (; lg protein mg kte À ). when expressed on a surface-area basis (table ) , the adsorption capacities of mte and quartz microparticles were indistinguishable by our measurement method; that of kte was times less. these data demonstrate that mineral surface properties contribute to differences in the amount of prp sc bound. unexpectedly, prp sc desorbed from mte surfaces exhibited a lower molecular mass (; - kda) than the starting material (; - kda) ( figure a ). neither contaminant proteases nor metal oxide coatings on mte particles appeared responsible for prp sc cleavage, as treatments to counteract each did not prevent cleavage (unpublished data). prior to sorption experiments, mte was boiled in a solution of mm nacl for min to denature contaminant proteases, or binding experiments were performed in the presence of a cocktail of protease inhibitors to inactivate them. neither treatment prevented prp sc cleavage. amorphous metal oxide coatings on clay mineral particles can alter their surface reactivities and could potentially be responsible for prp sc cleavage. the size-fractionated mte used in this study has been reported to not contain such impurities at levels detectable by x-ray diffraction analysis [ ] , and precautionary pretreatment of the clay with a buffered neutral citrate-bicarbonate-dithionate solution to remove metal oxide coatings [ ] failed to prevent cleavage. prion protein desorbed from kte and quartz did not exhibit a change in molecular mass ( figure a ), suggesting that surface properties specific to mte were responsible for the cleavage. previous studies on protein interaction with mte have not noted reductions in molecular mass upon desorption [ , ] . we incubated prp sc with mte for short time periods ( - min) to qualitatively investigate initial adsorption and cleavage kinetics. adsorption of prp sc to mte was apparent within min, and reduction in protein molecular mass was discernable (figure a ). prion protein cleavage consistently occurred early within the first min of contact with mte and appeared maximal by min. cleavage of prp sc caused by sorption to or desorption from mte seemed to be a phenomenon specific to this protein. we examined sorption and desorption of scrapie-infected hamster brain homogenate (bh) to mte. desorption of brain proteins from mte produced no changes in the overall molecular mass distribution as visualized by coomassie blue staining (unpublished data). subunit c of the s proteasome (; kda), an unrelated protein similar in size to prp likewise did not appear cleaved upon desorption from mte ( figure b ). in contrast, prp sc in bh was cleaved ( figure c ). cleavage of prp sc involved loss of the n-terminal portion of the protein, which is not necessary for infectivity [ ] . prion protein desorbed from mte lost immunoreactivity with an antibody directed against amino acids - on the protein n terminus, indicating that all or part of the epitope of this antibody was missing from the desorbed protein ( figure d ). in contrast, probing identical samples with a polyclonal antibody against full-length prp demonstrated that prp sc was desorbed from the mte. although the precise cleavage site was not determined, these data suggest that the n terminus of prp sc was removed; the fate of the cleaved amino acid residues is not known, as they may have remained bound to the clay or may have been extracted but not detected. when the n-terminal ; amino acids were removed from prp sc by pretreatment with proteinase k (pk) prior to adsorption to mte, we observed sorption to the mte, but no further reduction in molecular mass upon desorption, evidence that other regions of the protein remain intact when associated with mte ( figure e ). these results also indicate that the n terminus of prp sc is not necessary for adsorption to mte. prp sc attachment to mte was avid, and sorbed prp sc was stable. washing mte-prp sc with the background solution used in sorption experiments did not induce detachment of detectable amounts of prp sc from mte (unpublished data). contact of prp sc with mte for up to wk did not result in additional degradation, indicating that the protein was not rendered more susceptible to cleavage by further structural rearrangements on the clay surface ( figure a ). the strength of prp sc attachment to mte was surprising, even in light of reports of protein sorption-desorption hysteresis on mineral surfaces [ ] . conditions previously employed to desorb other proteins from soil minerals were largely ineffective in detaching prp sc from mte surfaces [ , ] . in our experiments, described above, a solution containing % sds at c was used to remove the prp sc from mineral surfaces. changes in ph often alter interactions between clay surfaces and sorbed proteins [ , ] . incubation of mte-bound prp sc in mm phosphate buffer at ph . or . , proton activities substantially higher and lower than the reported isoelectric points for prp sc [ ] , failed to release the protein ( figure b ). likewise, increases in ionic strength ( . m or m nacl) failed to remove detectable prp sc from mte ( figure c ). strong chaotropic agents can be effective in desorbing proteins from soil minerals by disrupting hydrogen bonds [ ] ; however, neither m urea nor m guanidine released detectable amounts of prp sc from mte ( figure d ). our data indicate the interaction between prp sc and mte is strong and of high affinity. sorption of proteins to soil particles often results in structural rearrangements that cause loss or diminution of function [ , , ] . if binding to mte surfaces results in (partial) unfolding of prp sc , a reduction or loss of infectivity would be expected, as denaturation renders the protein noninfectious [ ] . we therefore tested whether prp sc adsorbed to mte remained infectious by intracerebrally inoculating hamsters with mte-prp sc complexes ( table ) . the time to onset of clinical symptoms after inoculation provides a measure of infectivity [ ] . hamsters inoculated with mte-prp sc exhibited clinical symptoms of scrapie dpi. to control for any unbound prion protein that may have cosedimented with mte particles, mineral-free prp sc suspensions were processed in the same manner as in sorption experiments. the sedimented fraction of these control samples (mock pellets) showed substantially less infectivity than mte-prp sc pellets with a mean incubation period of d, d longer than mte-prp sc pellets. hamsters inoculated with supernatants from these control samples (mock supernatants) showed clinical symptoms dpi. animals intracerebrally inoculated with mte alone and uninoculated animals did not exhibit tse symptoms during the course of the experiment ( d). to examine the extent of prion protein binding by whole soils, we conducted prp sc sorption experiments with four soils differing in texture and mineralogy (table s ) . when equal masses of soil ( . lg) were used, all soils bound prp sc to a similar extent ( figure ) ; no detectable prp sc remained in the supernatant at the level of protein used in this experiment. prion protein desorbed from the soils did not appear cleaved. several nonmutually exclusive factors may have contributed to this finding, including ( ) relatively small amounts of mte in some samples, ( ) occlusion of mte cleavage sites by metal oxide and/or natural organic matter coatings, and ( ) competition among the various sorption domains (both inorganic and organic) for prp sc , limiting interaction with mte. the amount of immunoreactive prp sc recovered from each soil differed slightly; for example, the immunoreactive protein desorbed from the elliot soil was less than that from the boardman soil. this may have been due to stronger interaction of prp sc with the elliot soil than with the boardman soil, leading to incomplete extraction, consistent with the larger fraction of clay-sized particles in the elliot soil (table s ). environmental transmission of prion diseases has been noted for decades [ , , ] . in this study, we provide evidence indicating that soil and soil minerals serve as a reservoir of tse infectivity. while extrapolation of in vitro studies to the environment must be made with caution, our findings suggest that prp sc released from diseased animals may be sequestered near the soil surface, maintaining the tse agent in an environmental medium with which livestock and wildlife come in contact. our experiments demonstrate that mtebound prp sc remains infectious and suggest that soil may harbor more tse agent than previously assumed on the basis of water extraction of prions from garden soil [ ] . our results demonstrate that all soil mineral surfaces examined bound prp sc and that mte and quartz have larger specific binding capacities for prp sc than does kte (figure ). although not relevant to tse transmission, nonglycosylated, recombinant prp c has been shown to bind to mte [ ] . interestingly, the n terminus of prp sc desorbed from mte was truncated ( figures a and ) . while mte is known to catalyze several reactions, including the deamination of free glutamine and aspartic acid [ ] and the polymerization of rna into oligomers [ ] , protease activity has not been noted previously. the interaction between mte and prp sc is remarkably avid, as the only extractant used in this study that effected desorption was a solution containing % sds at c ( figure b- d) . prion protein appears unlikely to readily desorb from mte in the environment. the propensity for prp sc to tenaciously bind to mte could be exploited in landfills to isolate prion-infected materials and prevent migration of the infectious agent. the observation that prions remained infectious when bound to mte is intriguing in light of the results of the desorption experiments; prp sc adsorbed to mte was extremely difficult to remove. current mechanistic models for conversion of prp c to the pathological form require direct prp c -prp sc interaction [ ] . the brain is unlikely to possess microenvironments capable of extracting significant amounts of prp sc from clay surfaces. the -d increase in incubation period for mte-adsorbed prp sc relative to clay-free controls (mock supernatant) was statistically significant (p , . ) and would correspond to approximately a -log increase in infectivity [ ] . this result suggests that prp sc -mte complexes are inherently more infectious than the unbound protein and/or adsorption to mte reduces clearance from the brain. we consider it likely that prp sc adsorbed to mte surfaces was available to convert prp c in the brain to the pathological isoform. our findings are reminiscent of reports in which metal wires exposed to scrapie agent harbored significant infectious agent despite attempts to remove attached prp sc [ , ] . the infectivity of soil-and soil mineral-sorbed prp sc following oral exposure warrants investigation. the binding of prp sc to soil particles could reduce oral bioavailability such that soil serves as a sink rather than a reservoir for infectivity. conversely, association with mineral particles may protect the agent from degradation in the gastrointestinal tract, possibly enhancing transmission [ ] . for example, bovine rotaviruses and coronaviruses retain infectivity via the oral route when bound to clay minerals [ ] . while desorption of the protein from soil particles is more likely to occur in the gut than in the brain, removal of prp sc from mineral particles may not be necessary to initiate infection. in conclusion, soil and soil minerals have the potential to bind prp sc and maintain infectivity. these findings will serve as the basis for further study on the interaction of prp sc with other soil components (humic substances, quartz, and other minerals), the stability of soil-bound prp sc under typical environmental conditions (uv light, freeze-thaw cycles) and the effect of soil microorganisms and extracellular enzymes on protein integrity. our current results suggest that sorption of prp sc to clay minerals may limit its migration through the soil column. maintenance of prion infectivity at the soil surface may contribute to the propagation of cwd and scrapie epizootics and enhance the likelihood of interspecies transmission of these diseases. preparation of soil minerals and soils. montmorillonite (swy- ) and kaolinite (kga- b) clays, obtained from the clay minerals society source clays repository (west lafayette, indiana, united states), were size-fractionated by wet sedimentation to obtain particles with d h ¼ . - lm and saturated with sodium. these reference clay samples were extensively characterized previously [ , ] . fine quartz sand (d h ¼ - lm) and sio microparticles (d h ¼ - lm; % purity) were obtained from sigma (st. louis, missouri, united states). the fine quartz sand was soaked for h in n hcl to remove impurities. x-ray diffraction analysis and infrared photoacoustic spectroscopy indicated that the sio microparticles were composed of quartz. we examined prp sc sorption to four soils (table s ). the elliot soil was a silty clay loam purchased from the international humic substances society (st. paul, minnesota, united states). organically amended dodge soil (sandy clay loam) was obtained from a glaciated upland area in madison, wisconsin. the bluestem soil was a sandy clay loam collected from a fluvial deposit in cedar rapids, iowa. the boardman soil was a silt loam taken from an eolian deposit in boardman, oregon. characteristics of these soils are presented in table s . source of prp sc . syrian hamsters (cared for according to all institutional animal care and handling protocols of the university of wisconsin, madison) were experimentally infected with the hyper strain of hamster-adapted transmissible mink encephalopathy agent. prp sc was purified to a p pellet from brains of infected hamsters by a modification of the procedure described by bolton et al. [ , ] . the p pellet prepared from four brains was resuspended in ml of mm tris (ph . ) with mm nacl. for experiments employing pktreated prp sc , % brain homogenate was treated with lg ml À of proteinase k for min at c. after blocking pk activity with mm phenylmethylsulfonyl fluoride, purification was performed as above. batch sorption experiments. larger prion aggregates were removed from purified prp sc by collecting supernatants from two sequential -min centrifugations at g (clarification step). clarified prp sc (; . lg) was added to lg of mte or fine quartz sand, , lg of kte, or . mg of quartz microparticles in mm nacl buffered to ph . with mm -n-morpholinopropanesulfonic acid (mops) ( ll final volume). in some cases, mte experiments were conducted in unbuffered mm nacl. sorption experiments with mte performed in buffered and unbuffered mm nacl yielded comparable results. experiments with mte, kte, and quartz microparticles each employed equivalent (external) mineral surface areas. in sorption experiments with whole soil samples, ; lg of clarified prp sc was added to -ml suspensions of each soil ( mg) in mm cacl . samples were rotated at ambient temperature for h or an indicated time period. sorption appeared complete within h, as longer incubation times did not result in changes in levels of bound protein. each prp sc -mineral suspension and a -ll aliquot of each prp scsoil suspension was placed over a mm sucrose cushion prepared in a solution of the same composition as the background solution in the sorption experiment, and centrifuged at g for min to sediment mineral or soil particles and adsorbed prp sc . a sucrose cushion was found necessary to prevent a fraction of unbound prp sc from sedimenting during centrifugation. clarified prp sc did not sediment through the sucrose cushion ( figure s ). unbound prp sc remaining in the supernatant was precipitated with four volumes of cold methanol and resuspended in sds-page sample buffer ( mm tris [ph . ], % sds, . mm edta, mm dithiothreitol, and % glycerol). prp sc was extracted from pelleted mineral particles with sds-page sample buffer at c for min. the same procedure was followed for prp sc -soil suspensions. to determine mineral adsorption capacities for prion protein, varying volumes of clarified prp sc preparation were added to a : dilution of each mineral suspension. all adsorption experiments were repeated at least three times. for bh sorption experiments, % bh was clarified by collecting supernatants from two sequential -min centrifugations at g. aliquots ( or ll) of clarified bh were rotated with mte in mm nacl at ambient temperature for h; complexes of mte and bh constituents were then sedimented through a sucrose cushion and processed as described in the preceding paragraphs. all samples prepared for sds-page were separated on %À % precast gels (biorad, hercules, california, united states) under reducing conditions. proteins were transferred to polyvinyl difluoride membranes and immunoblotted with mab f ( : , dilution), r n-terminal pab ( : , dilution), rab pool full-length prp pab ( : , dilution), or anti- s proteosome subunit c pab ( lg ml À ; a.g. scientific, san diego, california, united states). detection was achieved with an hrp-conjugated goat anti-mouse immunoglobulin g (igg) (biorad) for mab f and an hrp-conjugated goat anti-rabbit igg (biorad) for all pabs. x-ray diffraction analysis. prp sc preparation ( lg) was added to lg of mte in mm nacl (final volume of . ml). samples were rotated at ambient temperature for h and centrifuged at , g for min. after centrifugation, the bulk of the supernatant was removed, leaving a small amount of solution above the clay pellet. the clay was resuspended in the remaining supernatant, and the slurry was placed on silica wafer slides and stored in a desiccator for over h. the basal d spacings of near homoionic na þ -swy- before and after adsorption of prp sc were determined by x-ray diffraction on a scintag pad v diffractometer (cupertino, california, united states) using cuka radiation and continuous scanning from to h with a step size of . and a dwell time of s. extraction experiments. prp sc adsorbed to mte was incubated for min at room temperature in m urea or m guanidine hcl ( ll per pellet), . or m nacl ( ll per pellet), or mm sodium phosphate (ph . or . ; ll per pellet). primary extractions with these solutions were followed by secondary extractions with sds-page sample buffer at c to assess the efficacy of the primary extraction. urea and guanidine primary extracts were dialyzed against double distilled water for h (nominal molecular weight cutoff, - kda; fisher scientific, pittsburgh, pennsylvania, united states) prior to sds-page analysis. infectivity bioassay. prp sc -mte pellets prepared as above were resuspended in ph . pbs ( ll per pellet) and intracerebrally inoculated into male, weanling syrian hamsters (harlan, indianapolis, indiana, united states). equivalent amounts of prp sc starting material or mte without prp sc were inoculated into control animals. hamsters were monitored every d for the onset of clinical symptoms [ , ] . brains from clinically positive hamsters and uninfected controls were analyzed for protease-resistant prp by immunoblotting. figure s . sucrose cushion prevented sedimentation of unbound prp sc under conditions necessary to pellet soil minerals a substantial amount of unbound prp sc pelleted when centrifuged under conditions required to remove na þ -mte from suspension, but was prevented from sedimenting by a sucrose cushion. sucrose cushions were therefore employed in batch sorption experiments to prevent sedimentation of unbound prp sc . results from representative mock adsorption experiments are shown. prp sc was rotated in a solution of mm nacl in the absence of soil minerals for h and was either placed above a mm sucrose cushion and centrifuged (two right lanes), or centrifuged without a sucrose cushion (two left lanes). supernatants (sup) and pellets (pel) were analyzed by immunoblotting with mab f . found at doi: . /journal.ppat. .sg ( kb pdf). the genbank (http://www.ncbi.nlm.nih.gov/) accession number for prp sc is m . the prion diseases the public health impact of prion diseases secondary structure analysis of the scrapie-associated protein prp - in water by infrared spectroscopy a review of the epidemiology of scrapie in sheep prion disease: horizontal prion transmission in mule deer scrapie: observations on the transmission of the disease by mediate contact rida (scrapie) in iceland and its epidemiology hunter harvested moose tests positive for cwd fly larvae and pupae as vectors for scrapie characteristics of scrapie isolates derived from hay mites inactivation of transmissible degenerative encephalopathy agents: a review survival of scrapie virus after years' interment environmental sources of prion transmission in mule deer natural infection of suffolk sheep with scrapie virus oral transmission and early lymphoid tropism of chronic wasting disease prp res in mule deer fawns (odocoileus hemionus) coincident scrapie infection and nephritis lead to urinary prion excretion bse: the final resting place geophagy and potential contaminant exposure for terrestrial vertebrates adaptations of white-tailed deer to naturally occurring sodium deficiencies ingestion of sludge applied organic chemicals by animals silica in soils: quartz and disordered silica polymorphs baseline studies of the clay minerals society source clays: powder x-ray diffraction analyses soil chemical analysis. revised nd ed adsorption and binding of amplitaq dna polymerase on the clay minerals, montmorillonite and kaolinite kinetics and interaction constants of protein adsorption onto mineral microparticles-measurement of the constants at the onset of hysteresis adsorption, desorption, and activity of glucose oxidase on selected clay species interpretation of the ph dependence of protein adsorption on clay mineral surfaces and its relavance to the understanding of extracellular enzyme activity in soil scrapie prp - is a sialoglycoprotein structural changes of cytochrome c( ) from thermus thermophilus adsorbed on anionic and hydrophobic surfaces probed by ftir and d-ftir spectroscopy scrapie infectivity correlates with converting activity, protease resistance, and aggregation of scrapie-associated prion protein in guanidine denaturation studies molecular properties, partial purification, and assay by incubation period measurements of the hamster scrapie agent fate of prions in soil: trapped conformation of full-length ovine prion protein induced by adsorption on clays glutamic acid deamination in the presence of montmorillonite synthesis of long prebiotic oligomers on mineral surfaces interactions between prion protein isoforms: the kiss of death? infectivity of scrapie prions bound to a stainless steel surface transmission of scrapie by steel-surface-bound prions gastric acidity protects mice against prion infection? in vitro studies on the use of clay, clay minerals and charcoal to adsorb bovine rotavirus and bovine coronavirus data handbook for clay minerals and other non-metallic minerals isolation and structural studies of the intact scrapie agent protein reversibility of scrapie inactivation is enhanced by copper identification of two biologically distinct strains of transmissible mink encephalopathy in hamsters the most infectious prion protein particles we thank richard rubenstein (suny downstate medical center) for the gift of mab f and byron caughey (national institute of allergy and infectious diseases, national institutes of health, rocky mountain laboratories) for pab r . we thank allen herbst, chad johnson, mine ekenler, juan gao, and laura sullivan for technical assistance. we thank harry read and beatriz quinchia-rios for their critical reading of this manuscript. we gratefully acknowledge the constructive comments of three anonymous reviewers.author contributions. cjj, dm, jma, and jap conceived and designed the experiments. cjj, kep, and pts performed the experiments. cjj, kep, pts, dm, jma, and jap analyzed the data. jma and jap contributed reagents/materials/analysis tools. cjj, dm, jma, and jap wrote the paper.funding. this work was supported by usepa grant c-r -naex (jap) and dod grant damd - - - (jma).competing interests. the authors have declared that no competing interests exist. key: cord- -m k i g authors: ziegler, christopher m.; eisenhauer, philip; bruce, emily a.; weir, marion e.; king, benjamin r.; klaus, joseph p.; krementsov, dimitry n.; shirley, david j.; ballif, bryan a.; botten, jason title: the lymphocytic choriomeningitis virus matrix protein ppxy late domain drives the production of defective interfering particles date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: m k i g arenaviruses cause severe diseases in humans but establish asymptomatic, lifelong infections in rodent reservoirs. persistently-infected rodents harbor high levels of defective interfering (di) particles, which are thought to be important for establishing persistence and mitigating virus-induced cytopathic effect. little is known about what drives the production of di particles. we show that neither the ppxy late domain encoded within the lymphocytic choriomeningitis virus (lcmv) matrix protein nor a functional endosomal sorting complex transport (escrt) pathway is absolutely required for the generation of standard infectious virus particles. in contrast, di particle release critically requires the ppxy late domain and is escrt-dependent. additionally, the terminal tyrosine in the ppxy motif is reversibly phosphorylated and our findings indicate that this posttranslational modification may regulate di particle formation. thus we have uncovered a new role for the ppxy late domain and a possible mechanism for its regulation. observed, but it is not known whether these rnas have interfering properties or are selectively incorporated in di particles [ ] . the interfering activity of arenavirus di particles can be blocked by neutralizing antibodies but is maintained even after treatment with ultra-violet (uv) radiation, unlike standard particles, which are highly susceptible to both treatments [ ] . arenaviruses generate high levels of di particles both in cell culture [ ] and in host rodents [ ] . it has long been postulated that arenavirus dis are an important factor in the establishment of persistent infection [ , , ] but a causal link between arenavirus di particles and persistence has yet to be firmly established. there are several outstanding questions regarding the arenavirus matrix protein, including how its functionality is regulated and how, in the context of a fully replicating virus, encoded late domains contribute to the production of standard and di particles. herein we demonstrate that lcmv's sole late domain, ppxy, is not required for standard virus budding but instead is the driving force of di particle release. further, standard virus appears to bud independently of escrt machinery while di particle release is escrt-dependent. finally, we show that the lcmv ppxy motif is tyrosine phosphorylated and that this post-translational modification appears to regulate di particle formation. the lcmv matrix protein is reversibly phosphorylated the matrix protein plays a multifactorial role in the arenavirus life cycle yet little is known regarding how its various functions are regulated. given the importance of phosphorylation for regulating the functionality of matrix proteins of other virus families [ ] [ ] [ ] [ ] [ ] , we were interested in whether lcmv's matrix protein might also be phosphorylated. lcmv strain armstrong b particles grown in vero e cells were purified via sucrose-banding ( fig a) and subjected to mass spectrometry. this analysis revealed a tyrosine phosphorylation site near the c-terminus of the lcmv z protein at position (y ) (fig b and s fig) , which lies within lcmv z's pppy late domain (fig c) . both phosphorylated and unphosphorylated peptides containing this residue were observed at a ratio of to , respectively, which suggests that~ % of the total z protein in this virion preparation is phosphorylated ( fig b and s c fig) . because the virion preparation contained a mixture of both standard infectious virus and di particles, we were not able to determine whether the phosphorylated z was derived from standard particles, di particles, and/or both types of particles. to confirm the phosphorylation site, plasmids encoding either wt z or a phenylalanine mutant (y f) that cannot be phosphorylated were transfected into hek t cells and days later the cells were treated with either water or the tyrosine phosphatase inhibitor, hydrogen peroxide. wt z and y f z were affinity purified and probed with a phosphotyrosine-specific antibody. the phosphotyrosine signal detected from wt z was greatly enhanced following inhibition of tyrosine phosphatases (fig d) . substitution of tyrosine with phenylalanine, to prevent phosphorylation, resulted in a complete loss of detectable phosphotyrosine signal in both settings indicating that y may be the only tyrosine of the encoded in lcmv z that is phosphorylated (fig d) by endogenous kinases in these cells. to determine whether lcmv z is tyrosine phosphorylated in the context of a relevant rodent cell line, we infected murine l cells with a rlcmv that encodes z with a c-terminal streptavidin binding peptide (sbp) tag. two days later, cells were either treated with hydrogen peroxide or not and z was affinity purified from cell lysates for western blot analysis. as shown in fig e, a phosphotyrosine signal was clearly detectable from z and was enhanced following treatment with hydrogen peroxide. the finding that lcmv z is phosphorylated at y was intriguing as this residue is part of lcmv's only late domain, pppy. this motif is well conserved among most old world arenavirus z proteins (fig a) and its importance for the budding activity of lcmv and lassa virus z in the context of vlp-budding assays has been well described [ , ] . to investigate the role of this late domain in the context of authentic virus and to determine whether tyrosine phosphorylation may regulate its function, we generated recombinant (r)lcmv encoding either phenylalanine or alanine at position to prevent phosphorylation at this site or glutamic acid to mimic constitutive phosphorylation. the alanine mutant was included as a reference to previous studies on the function of this late domain for lcmv and lassa virus z, which used alanine substitutions at y to assess the contribution of this late domain to z's budding hek t cells were transfected with an empty vector or a plasmid encoding lcmv z (either wt or y f) with a c-terminal streptavidin binding peptide (sbp) tag. following a minute exposure to either water or the tyrosine phosphatase inhibitor, h o , z was affinity purified from cell lysates using magnetic streptavidin beads and screened via western blot using antibodies specific for phosphotyrosine or the sbp tag. results are representative of independent experiments. (e) lcmv is phosphorylated in rodent cells. l cells were infected or not with a rlcmv that encodes a streptavidin binding peptide (sbp) fusion tag at the c terminus of z. two days later, cells were exposed to either water or the tyrosine phosphatase inhibitor, h o , for minutes. sbp-tagged z was then affinity purified from cell lysates using magnetic streptavidin beads and screened via western blot using antibodies specific for phosphotyrosine or the sbp tag. results are representative of independent experiments. doi: . /journal.ppat. .g the lcmv z ppxy late domain is dispensable for the production of infectious lcmv particles. (a) sequence alignment of arenavirus z proteins reveals conservation of y among most old world, but not new world, arenaviruses. (b) recombinant (r)lcmv containing substitutions at z y that either mimic constitutive phosphorylation (y e) or cannot be phosphorylated (y f and y a) were generated using reverse genetics as described in the materials and methods. vero e cells were infected at an moi of . and the quantity of infectious virus released at each of the indicated time points was efficiency in vlp assays [ , ] . viruses containing all three mutations were recoverable despite the well-described defect in z's budding efficiency caused by mutation of this residue ( fig b) [ , ] . the growth kinetics of rlcmv z-y f and z-y a during the first hours (hr) post-infection (pi) were nearly identical, but impaired~ -fold compared to rlcmv wt (p . ; fig b and c) . the growth rate of the rlcmv z-y e phosphomimetic was also attenuated compared to wt virus over this same time frame (~ -fold less pfu at hr pi, p . , fig b and c ). however, the phosphomimetic virus grew to~ -fold higher titers than the alanine or phenylalanine mutants (p . ; fig b and c) . additionally, the mean plaque size for rlcmv z-y e was significantly increased compared to the z-y f and z-y a viruses ( . vs . or . mm ; p . ; fig d) , indicating that virus spread was partially restored in the phosphomimetic virus. notably, each mutant virus eventually reached peak wt titers. given the delayed kinetics observed in the mutant viruses, we tested for reversion mutations at hr pi and confirmed that each virus retained its respective mutated residue at position and its small plaque phenotype ( fig d) . collectively, these results demonstrate that the ppxy late domain is not absolutely required for the formation and release of standard infectious particles. further, phosphorylation of y may have a positive regulatory impact on viral propagation. point mutations made at y suggested that dynamic phosphorylation of this residue was important for the function of the matrix protein. given the important role of the lcmv matrix protein and its late domain motif in regulating viral budding [ , ] , we next investigated the specific effect these point mutations had on z's budding efficiency in a vlp release assay. because the lcmv z protein is sufficient for the production of vlps in the absence of any other viral proteins [ , ] , we were able to assess the budding activity of plasmid-derived wt or y -mutant z proteins. as a control, we also included the lcmv z g a mutant, which exhibits a pronounced defect in vlp formation due to its inability to be myristoylated at this glycine residue [ ] . hek t cells were transfected with plasmids encoding wt or y mutants and day later the vlp-containing supernatant and cells were collected and analyzed by quantitative western blotting. the budding activity of all three z y mutants was significantly reduced compared to wt z, indicating that mutations in this region reduce the efficiency of vlp release (fig e) . in particular, the impaired vlp release exhibited by the z y a mutant confirmed earlier findings by perez et al. [ ] . we did not observe a significant difference between the budding of the z-y e phosphomimetic compared to y f and y a ( fig e) . this suggests that the partial gain of fitness observed with the phosphomimetic rlcmv-z-y e virus in fig b is not due to an increase in budding activity and as such y phosphorylation does not appear to directly regulate the budding function of this late domain. however, because the vlp budding assay measures only the release of matrix protein, in the determined via plaque assay. data are presented as mean pfu ± standard error of the mean (sem) of independent experiments. (c) summary of two way analysis of variance (anova) with holm-sidak's test for multiple comparisons of log-transformed data for virus growth curves in (b). (d) the area of plaques from rlcmv wt or z y mutants was measured using image j. data represent the mean ± sem of plaques analyzed from wells from -well plates. mean values were compared using the kruskal-wallis non-parametric test with dunn's multiple comparisons test. (e) mutation of the ppxy domain reduces z budding function in a vlp assay while phosphorylation of this domain at y does not further impact budding. a plasmid encoding lcmv z wt, z g a, or the indicated z y mutants was transfected into hek t cells and day later cells and vlp-containing supernatants were collected and screened via quantitative western blot for z. the percent vlp release was calculated as the amount of z protein found in the cell culture media relative to the amount in cells. data are presented as mean release ± sem relative to wt z from independent experiments. a one way anova with holm-sidak's test for multiple comparisons was used to compare the mean values. (c-e), n.s. (not significant), *p < . ; **p < . ; ***p < . ; ****p < . , as determined by the indicated statistical tests. absence of other viral proteins, it is possible that this assay does not recapitulate all the facets of infectious virion production. to investigate the protein and genome composition of virions containing mutated late domains, an equivalent quantity of cell-free infectious virus particles from each rlcmv strain was concentrated for screening. quantitative western blotting revealed substantial reductions in the total amount of np, gp, and z in the y mutant particles relative to wt virus (fig a- d ). however, no difference was observed in the levels of these proteins among the three y mutant viruses (fig a- d ). the quantity of z protein detected in the y mutant virus preparations was < % of wt virus (fig d) whereas np and gp quantities were~ % of wt virus (fig b and c ). viral genome content in particles was assessed by qrt-pcr. on a per pfu basis, the quantity of either l or s segment genomic rna in the non-phosphorylatable mutants, y f and y a, was significantly reduced versus wt (p . , fig e and f ). however, genome levels in the phosphomimetic virus, y e, were not significantly different than wt (fig e and f) , which may explain a component of its partially restored growth kinetics ( fig b) . the observation of reduced viral proteins and/or genomes released from cells infected with the y mutant viruses combined with the fact that wt lcmv is known to produce relatively large quantities of di particles [ ] led us to hypothesize that the ppxy mutants may have defects in their ability to generate di particles, which could explain their greatly reduced levels of viral protein and genome relative to pfu. the ppxy late domain drives the production of di particles a substantial fraction of virus particles produced by lcmv are di particles [ ] . accordingly, inoculation of lcmv at low multiplicities of infection (moi) results in efficient production of standard virus and spread, while high mois do not. this seemingly contradictory phenomenon is caused by di particles, which inhibit the propagation of standard virus and its ability to cause cytopathic effect with one hit kinetics [ , ] . monolayers inoculated with high concentrations of standard infectious lcmv exhibit no cytopathic effect due to di particle inhibition, but as the inoculum is diluted, standard virus particles that infect cells in the absence of a coinfecting di particle will subsequently form plaques. we exploited this phenomenon to initially evaluate the relative amounts of di particles generated by the ppxy mutant viruses. equal infectious doses of wt virus and each y mutant, spanning a range of to , pfu, were applied to vero e cell monolayers in a standard plaque assay (fig a and b ). evidence of possible di particle interference is clearly seen in wt virus, where the most concentrated viral sample ( , pfu) resulted in no cell death while in successive -fold dilutions ( , and pfu) the number of di particles per cell is lowered allowing standard virus to enter cells in the absence of di particles and form plaques (fig a) . in contrast, the ppxy-mutant viruses exhibited a considerable increase in cytopathology (fig a, , and , pfu). quantification of the observed cytopathology confirmed the striking phenotype and revealed significant differences between the mutant and wt viruses ( fig b) . intriguingly, the cytopathology of the rlcmv z-y e phosphomimetic at , pfu was significantly less than both y f or y a viruses and therefore more closely resembled wt virus ( fig b) . to confirm that the interfering activity observed in fig was indeed due to lcmv di particles, we next established an assay to directly and quantitatively measure lcmv di particle activity. at present, no consistent biochemical or genetic signature exists to distinguish lcmv di particles from standard infectious particles [ , ] . in an attempt to uncover such a signature, we separated preparations of rlcmv wt or y mutants via density ultracentrifugation. similar to previous studies [ ] [ ] [ ] [ ] [ ] , we were unable to isolate fractions containing pure di particles as abundant levels of standard virus were detectable across all fractions (s fig) . therefore, it was not possible to identify a di particle-specific signature for screening purposes. despite this limitation, several assays, including a yield reduction assay [ ] , a plaque reduction assay [ ] , and a focus interfering assay [ ] have historically been used for accurate measurement of lcmv di particle abundance and activity levels. indeed, these assays were originally used to define lcmv di particles. we utilized the plaque interference assay (also known as the plaque reduction assay) analogous to that used in [ ] but also capitalized on the strong uvresistance exhibited by lcmv di particles, but not standard virus particles [ ] . briefly, cellfree virus preparations containing both standard and di particles were treated with uv to neutralize standard virus particles while leaving the interfering properties of di particles intact ( fig a) . it should be noted that standard virus particles treated with uv do not acquire detectable interfering properties (fig a) [ ] . limiting dilutions of this uv-treated sample were applied to vero e cells, followed by the addition of a fixed quantity of lcmv pfus. as shown in fig a, this allows for the determination of lcmv di particle activity and is expressed as plaque interfering units (piu ) per ml of a given sample. importantly, we recapitulated a plaque interference assay for the measurement of lcmv di particle activity. in (a), a stock of rlcmv wt containing both standard infectious virus particles and di particles was subjected to uv irradiation for min to inactivate standard lcmv particles but spare di particles. serial -fold dilutions of this uv-treated virus preparation (uv-lcmv) were added to vero e cells followed by a fixed amount of pfu of the indicated challenge virus (rlcmv wt; junín virus candid , (junv c# ), or vesicular stomatitis virus (vsv)). additional controls were wells that received i) media only, ii) serial -fold dilutions of the stock lcmv virus preparation before uv treatment (no uv lcmv), iii) serial -fold dilutions of the stock lcmv virus preparation following uv treatment (uv-lcmv), or iv) pfu per well of standard lcmv, junv c# , or vsv, as indicated. following a hr incubation at °c to permit viral particle absorption, cells were overlaid with agarose and subsequently fixed and stained with crystal violet to visualize whether the uv-lcmv preparation impacted the ability of each virus to form plaques. the lcmv di titer is expressed as plaque interfering units (piu ) per ml of a given sample and was calculated as described in the materials and methods. in (b), rlcmv wt containing both standard and di particles was subjected to the indicated filtration or not and then subjected to uv irradiation as described in (a). serial -fold dilutions of each uv-lcmv preparation (filtered or not) were added to vero e cells followed by a fixed amount of pfu of rlcmv wt. as described in (a), the ability of these uv-lcmv preparations to interfere with the ability of standard lcmv to from plaques was measured via plaque assay and lcmv di titers are reported several key controls from previous studies to demonstrate the specificity of this assay for lcmv di particles. in particular, uv-treated lcmv di particle preparations only interfered, in a dose-dependent manner, with the growth of homologous lcmv, but not heterologous viruses such as vesicular stomatitis virus (vsv) or the new world arenavirus junín virus candid (junv c# ), which rules out a nonspecific antiviral factor as a mediator of interference (e.g. interferon) ( fig a) . further, passing lcmv di particle-containing supernatant through a series of filters ( . μm, . μm, kda, kda) showed that interference is not due to soluble factors that are smaller than kda (e.g. cytokines) or larger (> . μm) membrane bound entities such as bacteria ( fig b) . when this assay was applied to the rlcmv wt and y mutant samples examined in fig , it confirmed that the rlcmv wt samples exhibited substantial di particle interfering activity (mean piu /ml ± sem), but that the mutant y viruses had much less ( fig c) . there was no detectable di activity for either the y f or y a viruses while the y e virus contained intermediate levels of interfering activity (mean piu /ml ± sem). collectively, the findings in figs and support the hypothesis that the lcmv ppxy late domain is required for the efficient formation of di particles and that phosphorylation of y may play a regulatory role in di particle production and the inhibition of cytopathic effect. viral late domains can drive virus budding by recruiting components of the cellular escrt pathway to complete the final membrane scission step. given the important role that the lcmv ppxy late domain played in the production of di particles (figs a, b and c), we hypothesized that this late domain might be recruiting the escrt pathway machinery to drive di particle formation. to test this hypothesis, we utilized cell lines that lack a functional escrt pathway due to inducible expression of a dominant negative (dn), e q point mutant, of vps , an atpase involved in the final stages of escrt pathway function [ ] [ ] [ ] . because the escrt pathway can also affect lcmv entry [ ] , we first infected cells with lcmv for hr to allow the entire monolayer to become infected before inducing expression of wt or dn vps . the cells were washed and fresh media containing the induction agent was added to the cells hr after initial induction ( hr pi) and the virus-containing media was collected hr later ( hr pi) to determine levels of standard infectious particles and di particles (fig ) . western blot analysis of protein lysates at hr pi confirmed the strong induction of wt and dn vps b expression and examination of fixed coverslips showed that all cells were expressing both the induced vsp b as well as lcmv np (fig b) . this infection protocol was chosen to ensure that we were examining virus that was produced in cells expressing the induced vps b proteins, while minimizing the effect that these proteins could exert on viral entry. expression of dn vps b had no impact on the release of standard infectious lcmv (p = . ; fig b) . in contrast, expression of dn vps b led to a marked reduction in the release of infectious vsv particles (fig a) , which is consistent with previous studies [ ] and as piu /ml. in (a-b), the graphical results represent the mean lcmv di titer ± sem for independent experiments and representative wells for each condition are shown directly below each graph. (c) lcmv di particle production requires a functional ppxy domain. the rlcmv wt or y mutants examined in fig were subjected to the assay described in (a-b) to directly measure the di particle titer present in each virus preparation. briefly, each indicated rlcmv preparation was subjected to uv-irradiation to inactivate standard infectious lcmv particles while preserving di particle activity. serial -fold dilutions of each uv-treated sample were inoculated onto vero e cells, followed by the addition of pfu of standard lcmv. following a hr incubation at °c to permit viral particle absorption, cells were overlaid with agarose and subsequently fixed and stained with crystal violet to visualize whether the various uv-treated rlcmv preparations impacted the ability of standard lcmv particles to form plaques. for each rlcmv, di titer is reported as mean piu /ml ± sem for independent experiments. (a-c) n.s. (not significant), *p < . ; ***p < . ; ****p < . , determined by first substituting values of piu /ml (just below the limit of detection value of piu /ml) for samples that were below the limit of detection and then performing a one way anova. doi: . /journal.ppat. .g t-rex hek cells stably transduced with vectors for tetracycline-based induction of wt vacuolar protein sorting b (vps b) or the dn vps b mutant, eq, were treated with tetracycline to induce the expression of wt or dn vps b. both the wt and dn vps b proteins have gfp fusion tags. one hr following vps b induction, the media was removed and cells were infected with vsv in media containing tetracycline. one hr later, the viral inoculum was removed and replaced with fresh media containing tetracycline. six hr later ( hr post-vps b induction; hr post-infection), supernatants were collected to determine vsv pfu titers via plaque assay. cell protein lysates were also generated at this time to verify the induction of vps b wt or eq expression using an anti-gfp antibody. protein lysates were also screened for actin as a loading control. viral titers represent the mean vsv pfu ± sem from independent experiments and were tested for statistical significance with an unpaired t test with welch's correction. (b-e) lcmv requires a functional escrt pathway for the release of di particles, but not standard infectious particles. t-rex hek cells stably transduced with vectors for tetracycline-based induction of wt vps b or the dn vps b eq were infected with rlcmv wt and d later treated with tetracycline to induce the expression of wt or dn vps b. six hr after vps b induction ( hr pi), the cells were washed and given fresh media containing tetracycline. supernatants were collected hr later ( hr pi) and titered via plaque assay. similar to (a), protein lysates were collected at hr pi and screened for vps b wt or dn using an anti-gfp antibody or for actin as a loading control. extra wells containing cells grown on cover slips were also fixed at hr pi to examine, via immunofluorescent confocal microscopy, the expression and localization of wt or dn vps b (green) or lcmv np (red). a μm square is shown for each panel. the results shown in (b) represent the mean lcmv pfu ± sem from independent experiments and were tested for statistical significance with an unpaired t test with welch's correction. (c) equivalent pfus of virus (range x to x ) produced from wt or dn vps b cells were inoculated onto monolayers of vero e cells and a standard plaque assay was performed. representative images of crystal violet-stained wells are shown in (c). inhibition of standard infectious virus-induced cytopathic effect by di particles at each dose was determined in (d) by measurement of the mean pixel intensity of each well using image j software. the confirms the specificity of our findings for lcmv. measuring lcmv di particle activity as described in fig revealed that wt lcmv produced considerably fewer di particles per standard infectious virus particle in the dn vps b background when compared to cells expressing wt vps b (fig c and d) . a similar trend for both lcmv infectious virus and di particle activity was seen in cells expressing wt or dn vps a (s fig). we next used the assay described in fig to directly quantitate the lcmv di particle activity in these samples. consistent with the findings in fig c and d , this demonstrated that significantly fewer di particles are made in the context of the dn vps b background when compared to wt vps b (mean ± sem vs , ± piu /ml, respectively; p = . ). thus it appears that lcmv di particle formation requires a functional escrt pathway (fig c- e ) in addition to a canonical late domain (figs and c) while standard particles do not (fig b) . the ability of most arenavirus matrix proteins to drive viral budding is thought to be highly dependent upon one or more encoded late domains [ , ] . the arenavirus lcmv encodes a single late domain, pppy. the ppxy motif is found in the matrix proteins of several families of enveloped rna viruses and for many of these viruses is required for the release of infectious virions in an escrt-dependent fashion (for review see [ ] ). we demonstrate here that the ppxy late domain encoded by lcmv is not absolutely required for infectious virus release. further, our data suggest that infectious particle release can occur in the absence of a functional escrt pathway. strikingly, we show that the formation of lcmv di particles critically requires a functional ppxy late domain and that this process is escrt-dependent (see fig for our proposed model). last, our data demonstrate that the terminal tyrosine in the lcmv ppxy motif is phosphorylated and that this posttranslational modification may exert a regulatory effect on z's ability to drive di particle release. therefore, we have uncovered an unexpected role for the ppxy late domain and a possible mechanism for its regulation of di particle production. our findings raise the intriguing possibility that lcmv utilizes divergent pathways for the production of infectious and di particles. neither a functional ppxy motif nor escrt pathway were absolutely required for the release of standard infectious particles. these findings, combined with the fact that lcmv z does not encode additional canonical late domains, strongly suggest that infectious lcmv release occurs through a novel, unknown mechanism. while the rlcmv ppxy mutants studied here initially displayed a slight lag in infectious virus release, each ultimately matched wt levels. consistent with an earlier study by perez et al. [ ] , we observed that mutation of the ppxy domain impairs the ability of lcmv z to form vlps (fig e) . this finding in the vlp system may accurately reflect the initial lag in infectious virus release seen for rlcmvs bearing the same ppxy mutations (fig b and c ) and/or their decreased ability to form di particles (figs and ) . with regard to the importance of the escrt pathway for infectious virus production, expression of dn vps b had no impact on data in (d) are representative of the mean ± sem relative to wt vsp b (at , pfu per well) from independent experiments and were tested for statistical significance with a two way anova and holm-sidak's test for multiple comparisons. (e) the assay described in fig was used to directly measure the lcmv di titer present in the supernatants collected from the vsp b wt or dn cells at hr pi. briefly, each virus-containing preparation was subjected to uv-irradiation to inactivate standard infectious lcmv particles while preserving di particle activity. serial -fold dilutions of each uv-treated sample were inoculated onto vero e cells, followed by the addition of pfu of standard lcmv. following a hr incubation at °c to permit viral particle absorption, cells were overlaid with agarose and subsequently fixed and stained with crystal violet to visualize whether the various uv-treated rlcmv preparations impacted the ability of standard lcmv particles to form plaques. the lcmv di titers are reported as mean piu /ml ± sem for independent experiments and were tested for statistical significance with an unpaired t test with welch's correction. (a-b, d-e) n.s. (not significant), *p < . , **p < . , as determined by the indicated statistical tests. the ability of wt rlcmv to form standard infectious virus (fig b) . similarly, expression of dn vps b did not impair the ability of lcmv z to form vlps when compared to cells expressing wt vps b (see s fig) . this vlp-based result does not agree with an earlier finding by perez et al. whereby silencing expression of the escrt component tsg impaired the release of infectious lcmv vlps [ ] . this discrepancy may reflect differences in the particular vlp assays employed (vlp release versus infectious vlp release and transduction) or perhaps the format of the experiments (sirna silencing of tsg versus inducible expression of wt or dn vps b). the different vlp systems could also recapitulate different aspects of virus particle release, with one vlp assay perhaps mimicking infectious virus production while the other more closely resembles di formation. in similar experiments featuring rhabdoviruses [ , ] , ebola virus [ ] , retroviruses [ ] [ ] [ ] [ ] , and hepatitis b virus [ ] , loss of the ppxy motif and/or escrt resulted in standard virus growth that remained attenuated compared to wt. interestingly, the new world arenavirus pichinde remains attenuated following disruption of its encoded late domain (psap) [ ] , while the matrix protein encoded by the new world arenavirus tacaribe can form vlps in the absence of its late domain (yxxl), but requires vps [ ] . these findings suggest that arenaviruses have evolved diverse strategies to drive infectious virus release. one possible explanation for the observed late domain-independent generation of infectious lcmv particles is that the lcmv z protein, either by itself or in combination with other viral structural proteins, may be sufficient to drive particle release in the absence of recruited host proteins. alternatively, lcmv may contain additional sequence motifs, either in z or the other structural proteins, that recruit novel host protein machinery to facilitate budding. in contrast to standard virus particles, both the ppxy late domain and the escrt pathway appear critical for the release of di particles. to our knowledge, this is the first example of a virus utilizing a late domain to selectively drive the production of di particles independently of standard virus. that lcmv has evolved such a mechanism likely reflects the presumed importance of di particles for the successful establishment of an asymptomatic, persistent infection in reservoir rodents, which ultimately ensures the long term maintenance of lcmv in nature [ , ] . while the existence of arenavirus di particles has long been realized, surprisingly little is known about their exact composition and properties. our data demonstrates that cells infected with the rlcmv ppxy mutants release much less np, gp, and z per pfu of cell-free virus when compared to wt rlcmv. this is presumably due to reduced levels of di particles being released by the ppxy mutant viruses. interestingly, the degree of reduction was not equivalent among the viral proteins. in particular, z was reduced to the greatest extent (~ % of wt) when compared to np or gp (~ % of wt), which could indicate that z itself is enriched in di particles and is critical for the ability of di particles to interfere with the propagation of standard virus particles. consistent with this idea, z is able to render cells refractory to superinfection with homologous virus and treatment of di particles with rna-damaging levels of uv does not reduce their interfering ability [ , ] . therefore, it is possible that particles containing high quantities of z, or simply vlps consisting primarily of z, may represent a class of arenavirus di particles. arenavirus matrix proteins exhibit significant diversity in the type and number of late domains they encode. the ppxy domain is found in several old world arenavirus matrix proteins but not in new world arenaviruses (fig a and [ , ] ). we have observed that the new world arenavirus junv c# , which encodes both a ptap and yxxl late domain, generates considerably fewer di particles per standard infectious particle when compared to the ppxycontaining lcmv (s fig). while it is not known whether the ptap and/or yxxl motifs contribute to di particle formation in the case of junv c# , this observation may indicate that the ppxy domain is particularly strong in driving di particle assembly and release. it is possible that individual arenaviruses require different rates of di particle formation for optimal fitness and have evolved to encode particular late domain combinations to best meet those needs. we show that the lcmv z protein is phosphorylated, which suggests that phosphorylation may be important for the regulation of one or more of z's functions. the fact that this modification occurs at the terminal tyrosine of the ppxy late domain and can be detected in virionderived z led us to hypothesize that it may influence z's budding function. to study the impact of this modification we generated rlcmv with mutations at tyrosine that either prevented phosphorylation (y f or y a) or mimicked it (y e). relative to the mutants that cannot be phosphorylated, the y e phosphomimetic virus generated significantly more di particles per infectious particle (figs and c ). this suggests that reversible phosphorylation of the ppxy motif may act as a rheostat to regulate the rate of di particle production independent of standard virus, possibly through the recruitment of escrt machinery. interestingly, the terminal tyrosine of the ppxy late domains encoded by ebola virus and marburg virus vp is also phosphorylated. inhibiting phosphorylation of the ebola virus vp ppxy motif reduced the release of infectious virus [ ] whereas mutation of the marburg virus vp ppxy motif to prevent phosphorylation did not impact infectious vlp release, but instead impaired the recruitment and incorporation of nucleocapsids into vlps [ ] . the role of the ppxy domain and/or its phosphorylation with regard to di particle production in the filovirus model and other ppxy-containing virus families remains an open question. in the current model, it will be important to identify the host kinase responsible for phosphorylating the lcmv ppxy domain, although this may require a comprehensive tyrosine kinase screen as the flanking residues surrounding y were not recognized by kinase motif prediction tools [ , ] . how does the ppxy late domain of lcmv promote the release of di particles? while it is well known that the ppxy motif can drive viral budding, the exact mechanism by which it recruits escrt proteins to promote this process is not fully understood. several ppxy-dependent viruses also require nedd family e ubiquitin ligases (e.g., nedd , itch, or wwp ), which can directly bind to the ppxy motif via their ww domains (for review see [ ] ). recruitment of escrt machinery could occur due to ubiquitination of z by a nedd e ubiquitin ligase, which in turn could recruit escrt components (e.g. tsg ) that can bind ubiquitinated proteins [ ] . alternatively, arrestin-related trafficking adaptors (arts) may provide an escrt linkage as these proteins interact with both nedd e ubiquitin ligases as well as escrt proteins and have been shown to influence both multivesicular body formation and viral budding [ ] . under this scenario, phosphorylation of the ppxy motif could regulate di particle release by modifying the accessibility of the ppxy domain to nedd e ubiquitin ligases. all viruses must strike a balance between pathogenesis and persistence, with the ultimate goal of ensuring their own maintenance in nature. it has long been postulated that di particles may be one way in which lcmv is able to tip the scales towards persistence, thus lowering its immunological profile and fitness cost to its host. by identifying the specific cellular pathway required to form these di particles, and the apparent importance of viral phosphorylation in accessing the pathway, our findings raise the possibility that arenaviruses can dynamically adjust di particle production in response to external environmental factors. further, the ppxy mutant viruses we have developed represent a new tool that will allow the field to formally test the importance of di particles for the establishment of persistent lcmv infection in reservoir rodents. the ability of the ppxy late domain to drive the production of di particles is a novel finding with important implications for understanding host-pathogen relationships and the design of vaccines and antivirals. in particular, ppxy-containing viruses such as ebola virus and lassa virus are known to persist for long periods following the resolution of acute human disease [ ] [ ] [ ] . it is possible that di particles play a significant role in allowing these viruses to persist in humans, similar to their presumed importance for infection in reservoir species. therefore, targeting di particle formation could be a promising approach to clear persistent infection in humans. finally, the possibility that the ppxy late domain and escrt machinery could broadly drive di particle production in other virus families represents an exciting area of future research. l. whitton (the scripps research institute, la jolla) and grown in dmem supplemented with % fbs, % penicillin-streptomycin, and % hepes buffer solution. t-rex hek cells stably transduced with a tetracycline-inducible plasmid encoding wt or dominant negative eq mutant vacuolar protein sorting a (vps a) or vps b as described in [ ] [ ] [ ] were generously provided by m. kielian (albert einstein college of medicine, bronx) and were maintained in dmem supplemented with % fbs, % penicillin-streptomycin, % mem non-essential amino acids solution, % hepes buffer solution, % glutamax, and μg/ml zeocin (r - , invitrogen). vps expression was induced by incubating cells in the above growth medium containing μg/ml tetracycline as described [ ] . all cell lines were grown at °c in a humidified incubator containing % co . lymphocytic choriomeningitis virus (lcmv) strain armstrong b was kindly provided by j. l. whitton. wild-type vesicular stomatitis virus expressing green fluorescent protein (vsv-gfp) as described in [ ] was kindly provided by j. hiscott (vaccine and gene therapy institute of florida, port st. lucie) and m. shaw (icahn school of medicine at mount sinai, new york). junín virus (junv) c# , which is an attenuated vaccine strain derived from the virulent wt junv strain xj as described in [ , ] , was kindly provided by m. buchmeier (university of california, irvine) and r. tesh (the university of texas medical branch at galveston). working stocks of these viruses were amplified and titered (via plaque assay) on vero e cells. see below under "generation of recombinant lcmv" for a description of the recombinant (r)lcmv strain armstrong b that were generated for this study. the lcmv armstrong b z protein (wt, y a, y e, or y f) was subcloned into a modified pcaggs expression vector [ ] and different combinations of these plasmids were used to screen for the phosphorylation of z (fig d) or the budding efficiency of z (fig e) . the wt and y mutant z genes were fused to the streptavidin binding peptide (sbp) (mdekttgwrgghvveglageleqlrarlehhpqgqrep) through an base pair linker at the c-terminus of z to permit affinity purification and western blot detection of z. the nucleotide sequence of the wt z gene matches ncbi gene identifier number ay while the translated amino acid sequence for the wt z gene matches protein locus number aax . wt z was amplified by pcr from the pt -l(+) plasmid generously provided by j. c. de la torre (the scripps research institute, la jolla) [ ] using the forward primer lcmvzf ( '-acaagtttgtacaaaaaagcaggctgatatcgccaccatgggtcaaggcaag tccaga- '), which has a ' overhang containing gateway attb and kozak sequences and the reverse primer lcmvzr ( '-acctccacctccagctgcctcttcgtagggaggtgg aga- '), which has an overhang containing the linker sequence. the sbp tag was amplified from the pt -flag-sbp- plasmid (p , sigma-aldrich, st. louis, mo) via pcr using the forward primer sbpf ( '-gcagctggaggtggaggtatggacgaaaaaaccaccgg t- '), which has a ' overhang containing the linker sequence, and the reverse primer sbpr ( '-accactttgtacaagaaagctgggtcttacggttcacgctgaccctgcgg- '), which contains a ' overhang with a stop codon preceding an attb sequence. the two products were fused by pcr using the z forward primer and the sbpr primer. the cassette was subcloned into the pcaggs vector using the gateway cloning system (invitrogen) following the manufacturer's instructions as has been previously described [ , ] . the plasmids pc-np and pc-gp, which express the lcmv armstrong b nucleoprotein (np) and glycoprotein (gp), respectively, and plasmids pol-i s and pol-i l, which express the lcmv l and s genome segments, respectively, were used to generate rlcmv. these reagents were generously provided by j. c. de la torre and are described in [ ] . each of the y mutant z genes used in these studies were synthesized and subcloned into the pcaggs or pol-i l vectors, respectively, by biobasic, inc. (markham, on, canada) . a pol-i l vector containing an sbp-tag directly fused to the c-terminus of z was also generated by biobasic, inc. all plasmid sequences were verified by dna sequencing. to identify phosphorylation sites on lcmv z via mass spectrometry, vero e cells were infected with lcmv strain armstrong b and hr later cell-free virions were purified by sucrose-banding as described previously [ ] . purified virions were then lysed in triton buffer ( . % np , % triton x- , mm nacl, and mm tris-hcl containing a protease inhibitor cocktail ( , roche applied science, indianapolis, in)) and mixed with laemmli sample buffer ( . mm tris-hcl, % glycerol, % sodium dodecyl sulfate and . % bromophenol blue (b , fisher scientific, pittsburgh, pa)) containing % -mercaptoethanol. virion protein lysates were separated on a - % tris-glycine polyacrylamide gel (ec , invitrogen). the gel was stained with coomassie ( % methanol, % acetic acid, and . % brilliant blue r (b , sigma-aldrich)), destained with a solution of % methanol and % acetic acid, and then imaged using a canon canoscan f scanner. for mass spectrometry, the protein band corresponding to the z protein was excised and cut into mm cubes and processed with chemicals from fisher scientific as follows. the gel pieces were rinsed with hplc grade water and then incubated with destain solution ( mm ammonium bicarbonate and % acetonitrile) for minutes at °c. the destain solution was removed and the gel pieces were dehydrated by incubating twice with % acetonitrile for minutes. the gel pieces were reduced with mm dithiothreitol in mm ammonium bicarbonate for minutes at °c. after cooling for minutes at room temperature, the gel pieces were dehydrated by incubating with % acetonitrile for minutes and then alkylated in the dark with mm iodoacetamide in mm ammonium biocarbonate for minutes at room temperature. the gel pieces were washed two times in destain solution for minutes, dehydrated with % acetonitrile, then rehydrated with water for minutes. the gel pieces were further dehydrated with two minute incubations in % acetonitrile before removing all liquid and drying the gel pieces at room temperature for minutes. the gel pieces were rehydrated with a solution of . ng/μl sequencing grade chymotrypsin (v , promega, madison, wi) or . ng/μl sequencing grade modified trypsin (v , promega) in mm ammonium bicarbonate on ice for minutes, before digesting overnight at °c. peptides were extracted with a solution of . % formic acid in % acetonitrile while spinning in a microcentrifuge at , rpm for minutes. the supernatant was removed and saved while the gel pieces were subjected to further extraction and rinsing with % acetonitrile. the second extraction was combined with the initial extraction. all solvent was removed from the extracts using a vacuum centrifuge at °c. the peptides were resuspended in . % formic acid, . % acetonitrile prior to mass spectrometry analysis. peptides were separated over cm of magic c , μm, Å reversed phase material (pm / / , michrom bioresources, auburn, ca) in a microcapillary column using a microas autosampler (thermo scientific, pittsburgh, pa). following minutes of isocratic loading in . % acetonitrile, . % formic acid, the peptides were eluted from the column with a - % gradient of acetonitrile with . % formic acid over minutes using a surveyor pump plus hplc (thermo scientific). mass spectra were acquired either in an ltq-xl linear ion trap, or in a linear ion trap-orbitrap mass spectrometer (thermo scientific) as described previously [ ] . briefly, for most analyses data-dependent ms/ms spectra followed each survey scan. however, in several cases after obtaining the initial spectra for phosphopeptides we followed up with targeted ms/ms spectra in order to increase fragment ion coverage. the ipi human forward and reverse concatenated database was used to search the raw data using sequest software requiring tryptic peptides and either a da precursor mass tolerance (for precursor data acquired in the ltq) or ppm (for precursor data acquired in the orbitrap). in the searches the following precursor mass differences were allowed: serine, threonine, and tyrosine residues (+ . da); methionine (+ . da) and cysteines (+ . da or . ). to confirm that z was phosphorylated in human cells as well as cells from rodent cells, in fig d and e , plasmid-derived z expressed in hek t cells and z from rlcmv z-sbp-infected cells were both probed for phosphotyrosine signal via western blot. for plasmid-derived z, x hek t cells were seeded in a -well plate and transfected the next day with . μg per well of plcmv-z wt, plcmv-z y f, or an empty vector using . μl of a mg/ml solution of polyethylenimine ( , polysciences, inc., warrington, pa) per well. for z derived from rlcmv z-sbp-infected cells, . x l cells were seeded in -well plates and infected the next day at an moi of . . two days following the transfection or infection, h at a final concentration of . mm or an equivalent volume of h was spiked into the appropriate wells containing hek t or l growth media. after a minute incubation, the cells were lysed in triton buffer containing a protease inhibitor cocktail and phosstop phosphatase inhibitor cocktail ( , roche applied science) and the sbp-tagged z proteins were affinity purified using magnetic streptavidin beads as previous described [ ] . the purified proteins were separated via sds-page and screened for z or tyrosine phosphorylated-z via standard chemiluminescent western blotting and detected with film ( fig d) or with a li-cor c-digit digital imager (li-cor, lincoln, ne) (fig e) . rlcmv wt, rlcmv z-sbp and rlcmv containing z-y mutations (y f, y e, y a) were generated using the previously described reverse genetics system [ ] . briefly, μl of lipofectamine ( , invitrogen) was mixed with μl of optimem ( , invitrogen) and then added to a plasmid mixture consisting of . μg pc-np, . μg pc-l, . μg pol-i s, and . μg pol-i l (wt, z-sbp or containing the described y point mutations) in μl optimem and incubated at room temperature for minutes. μl of this transfection mixture and μl of optimem was then added to well of a -well plate which had been seeded the previous day with . x bhk- cells and washed prior to transfection with ml of optimem. the cells were incubated with the transfection mixture for hr after which the media was replaced with bhk- growth media diluted -fold in dmem. three days later the supernatant was collected, clarified by centrifugation at , rpm for minutes at °c, and used to infect a fresh monolayer of . x bhk- cells in a t- flask. following a hr absorption, the inoculum was removed and fresh bhk- growth media diluted -fold in dmem was added to the cells. three days later the supernatant of this flask was collected, clarified by centrifugation, and titered by plaque assay. to generate an expanded virus stock, vero e cells were infected with this material at an moi of . and or hr later, supernatants were collected, clarified, and titered by plaque assay. a portion of the l segment (most of the z gene, the intergenic region, and part of the l gene) of each rlcmv y mutant was sequenced to ensure that these viruses had not reverted. the material used for this sequencing was derived from the hr pi time point shown in fig b. viral rna from clarified supernatants was isolated using the qiagen viral rna mini kit ( , qiagen, valencia, ca) according to the manufacturer's protocol. viral rna was converted to cdna using primer l -( '-gcagg acttgagggctatga- '), superscript iii ( - , invitrogen), rnase out ( - , invitrogen), and μl of rna following the manufacturer's protocol for first strand cdna synthesis. a portion of the l-segment containing z was amplified with - cycles of pcr using platinum pfx dna polymerase ( - , invitrogen) and primers l + ( '-atag tacaaacagggccgaaatcc- ') and l -( '-tttgttgggttcagagataagtg t- ') following the manufacturer's protocol. the pcr product was prepared for sequencing using exosap-it ( , affymetrix, santa clara, ca) following the manufacturer's protocol and sequenced by the university of vermont cancer center dna analysis facility. protein lysates were diluted in laemmli sample buffer containing % -mercaptoethanol and separated on nupage - % bis-tris gels with mes buffer. protein was transferred to nitrocellulose membranes using iblot gel transfer stacks (ib or ib , invitrogen) and the invitrogen iblot device as directed by the manufacturer. efficient protein transfer was confirmed by staining membranes with a solution containing . % ponceau s (p , sigma-aldrich) and % acetic acid which was subsequently removed by washing with water. two methods were used for protein detection: quantitative li-cor-based detection or standard chemiluminescent-based detection. for quantitative li-cor analysis, membranes were blocked with a solution of % milk in pbs for hr and incubated overnight at room temperature with the indicated primary antibodies diluted in pbs containing % milk and . % tween (bp , fisher scientific). following washes in pbs with . % igepal ca- ( , mp biomedicals, solon, oh), the membranes were incubated for hr at room temperature with secondary antibodies diluted in pbs containing % milk, . % tween and . % sodium dodecyl sulfate, washed times in pbs with . % igepal ca- and time with pbs, then imaged using the li-cor odyssey clx imaging system. for quantitative li-cor analysis of vps b in figs a, b and s , membranes were probed using the ibind flex western device (slf , thermo scientific) with the ibind flex fluorescent detection solution kit (slf , thermo scientific) following the manufacturer's instructions. for chemiluminescent-based detection of phosphorylated proteins, the same general procedure was used with the following exceptions: i) membranes were blocked with either pbs containing % milk and . % igepal ca- or protein-free blocking buffer ( , thermo scientific), ii) primary and secondary antibodies were diluted in pbs containing % milk, . % igepal ca- , and % fetal bovine serum or protein-free blocking buffer, and iii) the secondary antibodies were incubated with the membrane for hr. the following primary antibodies were used for western blotting (at the indicated concentrations): mouse anti-streptavidin binding peptide (mab , millipore, billerica, ma) ( : , ), rabbit anti-actin (a , sigma-aldrich) ( : , ), mouse anti-actin (a , sigma-aldrich) ( : , ), rabbit anti-actin (a , sigma-aldrich) ( : , ), mouse antiphosphotyrosine (clone g , millipore) ( . μg/ml), mouse anti-green fluorescent protein ( , clontech, mountain view, ca) ( : , ), rabbit anti-lcmv z ( ) ( : ), mouse anti-lcmv gp ( . ) ( : , ), and rabbit anti-lcmv nucleoprotein ( ) ( : , ). antibodies , , and . were generously provided by m. j. buchmeier (university of california, irvine). for quantitative western blotting, the following secondary antibodies from li-cor were used: irdye cw goat anti-mouse ( - ) for the z release assay in fig e at : , and in figs a, b and s at : , (for probing by ibind) and irdye lt goat anti-mouse ( - ) and irdye cw goat anti-rabbit ( - ) were used at : , to detect proteins in fig a- d . irdye lt goat anti-mouse was used at : , (for probing by ibind) in s fig to detect actin. a horseradish peroxidase-conjugated antimouse secondary antibody ( , emd millipore, billerica, ma) diluted : , was used for chemiluminescent-based detection in fig d and e . to determine the growth kinetics of rlcmv in fig b, -well plates were seeded with . x vero e cells per well. the following day the cells were infected with each respective rlcmv at an moi of . . supernatants were collected at , , , , and hr pi, clarified by centrifugation at , rpm for minutes at °c, then titered by plaque assay. to determine the release efficiency of the y mutant z proteins in figs e and s , x hek t cells or t-rex hek cells stably transduced with a tetracycline-inducible plasmid encoding wt or dominant negative eq mutant vps b were seeded in a -well plate. the next day cells were transfected with . μg per well of plcmv-z wt, z-g a, -z y f, -z y e, or -z y a using . μl of a mg/ml solution of polyethylenimine per well. for the experiments shown in s fig, vps b expression was induced with μg/ml tetracycline at the time of transfection. the following day ( hr post-transfection) cells and vlp-containing media (which had been clarified) were collected, lysed with triton lysis buffer, and subjected to quantitative western blotting. for detection of z from vlps produced in vps b wt or dn cell lines, sbp-tagged z was affinity purified from lysed vlp-containing media using magnetic streptavidin beads prior to quantitative western blot analysis. to calculate the percent vlp release we first normalized each z protein value (from supernatants or cells) by the sum of all z protein bands on a particular gel as described in [ ] . the percent vlp release was then calculated as the quotient of the z protein quantity in vlps divided by the quantity of z in cells [(z mut vlp / z mut cells) / (z wt vlp/ z wt cells)]. to measure infectious virus titers, a standard plaque assay was employed as follows. six-well plates were seeded with x (lcmv and junv) or x (vsv) vero e cells per well and the following day inoculated with -fold serial dilutions of virus in a total volume of . ml of vero e growth medium. following a minute absorption at °c, the cells were overlaid with a solution of . % agarose ( - , apex industrial chemicals, aberdeen, united kingdom) in vero e growth media. the plates were fixed (vsv) or (lcmv and junv) days later with a solution of . % formaldehyde ( - l, sigma) in x pbs. following removal of the agarose plugs, the fixed monolayers were stained with . % crystal violet (c - , fisher scientific) and . % ethanol in water. to determine the plaque size of rlcmv in fig d or the overall level of cytopathic effect induced by these viruses in figs a, b, c and d, the wells were imaged with an alpha innotech digital camera paired to a computar h z m motorized zoom lens. the area of each plaque as well as the mean pixel intensity of each well was calculated using imagej software. to determine the titer of lcmv di particles, samples were transferred to clear snap cap tubes ( - - , thermo scientific) and irradiated for minutes with uv light in a uvp cl- ultraviolet crosslinker in to kill standard infectious virus. the samples were serially diluted in -fold increments and added to -well plates which had been seeded the previous day with , (lcmv and junv c# ) or , (vsv) vero e cells per well. subsequently, pfu per well of rlcmv wt (or pfu per well of junv c# or vsv in fig a) was added to each well containing uv-irradiated samples. uv-irradiated samples were also added to a second set of wells to which no standard virus was added to ensure that all infectious virus had been eliminated from the samples. after a minute absorption period at °c, the cells were overlaid with a solution of . % agarose in vero growth media and left at °c. the plates were fixed and stained (vsv) or (lcmv and junv c# ) days later as above for the plaque assay. the plaques were counted in each well and the plaque interfering unit (piu ) was calculated using the plaque reduction statistical web tool (https://exon.niaid.nih.gov/ plaquereduction). because a unique biochemical or genetic signature to differentiate standard infectious virus particles from di particles has not been defined, the assay we employed relied on measurement of the interfering activity of di particles as opposed to a direct physical measure of the particles themselves. for fig b, rlcmv wt was filtered with either . μm ( - , vwr, radnor, pa) or . μm ( - c, fisher scientific) syringe filters or amicon k (ufc , millipore) or k (ufc , millipore) centrifugal filters prior to treatment with uv light and di titering as above. to determine the role of the escrt pathway in lcmv release, . x t-rex hek cells stably transduced with a tetracycline-inducible vps a or vps b (wt or dominant negative eq in each case) were seeded in -well plates that were first coated with poly d-lysine (p , sigma-aldrich) for minutes then washed x with pbs. cells were infected hr later with rlcmv wt at an moi of . . forty-eight hr later (when all cells were productively infected) the cells were induced with growth medium containing μg/ml tetracycline or a medium only control. six hr after induction cells were washed x with pbs and fresh growth medium containing μg/ml tetracycline or medium alone were added. eighteen hr later the cells and supernatants were collected. in fig a and b , the cell lysates were probed for vps b dn or wt protein (via the gfp fusion tag on these proteins) or actin expression by quantitative western blotting. supernatants were titered by plaque assay for infectious virus and di particle levels by measuring the cytopathic effect in a plaque assay with equal pfus of virus in each well (as described under plaque assay) and/or by plaque interference assay. the role of vps b in vsv release was also tested. for the vsv challenge studies, x vps b wt or eq cells were seeded in poly d-lysine treated wells and hr later treated with either growth medium containing μg/ml tetracycline or medium alone. one hr later, the cells were infected with vsv at an moi of . one hr following infection, the cells were washed x with pbs and fresh growth medium containing μg/ml tetracycline or medium alone was added. six hr later the cells and supernatants were collected and assessed by quantitative western blotting and plaque assays, respectively. in order to verify uniform vps b expression as well as rlcmv wt infection by microscopy, in parallel to the experiment described above, x cells were seeded on poly d-lysinetreated mm glass coverslips in -well plates. at the time of harvest ( hr post-infection) the coverslips were rinsed with pbs, fixed with % paraformaldehyde ( , electron microscopy sciences, hatfield, pa) in pbs for minutes, then washed x with pbs for minutes. the cells were permeabilized with . % triton x- in % bovine serum albumin (bsa) in pbs, blocked with % normal goat serum ( - - , jackson, west grove, pa) in % bsa in pbs, and immunostained with anti-lcmv nucleoprotein antibody ( . - ) ( : ) kindly provided by m. buchmeier (university of california, irvine) and secondary anti-mouse alexafluor (a , thermo scientific) ( : , ) each for hr in % bsa in pbs. dna was detected with ', -diamidino- -phenylindole hydrochloride (dapi) (d , sigma aldrich) in % bsa in pbs. cells were washed with % bsa in pbs in between each step. images were acquired on a zeiss lsm laser scanning confocal microscope using a x objective lens. post-capture image processing was carried out in fiji and photoshop; the gfp fluorescence, np staining, and dapi signal are shown at equal exposures in all conditions. to determine the np, gp, and z protein content of rlcmv virions in fig a- d , x vero e cells were seeded in a t- culture flask and infected the next day at an moi of . , . , or . . at or hr following inoculation, the supernatant was collected, clarified by centrifugation, and screened for infectious virus by plaque assay. an equal number of plaque forming units of each virus (range to x pfu per experiment) were layered onto a solution of % sucrose in tne buffer, ph . ( mm tris base, mm edta, . m nacl) and centrifuged for hr at , rpm at °c in a thermo-scientific sorval wx ultra ultra centrifuge equipped with a sorval surespin rotor. the resulting virus pellet was resuspended in xconcentrated laemmli buffer containing % -mercaptoethanol, then analyzed by sds-page and quantitative western blotting. to separate rlcmv by gradient centrifugation in s fig, x vero e cells were seeded in a t- culture flask and infected the next day at an moi of . . at hr following inoculation, the supernatant was collected and clarified by centrifugation. the clarified supernatants were added to ml tubes ( , corning) containing polyethylene glycol (peg) ( , sigma-aldrich) and sodium chloride such that the final concentrations were % and %, respectively. the solutions were incubated at °c on a rotating platform for hr then were centrifuged for minutes at , rpm at °c in a thermo-scientific sorval legend rt+ centrifuge equipped with a sorval fiberlite f - x cy rotor. the supernatant was removed and the virus-peg pellet was resuspended in tne buffer and screened for infectious virus by plaque assay. density gradients were prepared by layering solutions of %, %, %, %, and % optiprep (d , sigma-aldrich) diluted in pbs in ml tubes ( , thermo scientific) then leaving overnight at °c to allow a continuous gradient to form. an equal number of plaque forming units of each virus (range x to x pfu per experiment) was layered onto the continuous gradient and centrifuged for hr at , rpm at °c in a thermo-scientific sorval wx ultra ultracentrifuge equipped with a sorval surespin rotor. the resulting separated virus was collected in ml fractions using a new era ne- g programmable peristaltic pump and titered via plaque assay. to enumerate copies of lcmv s and l segment genomic rna contained in virions for fig e and f , viral rna was extracted from cell-free virions using the qiagen viral rna mini kit according to the manufacturer's instructions and subjected to quantitative rt-pcr as previous described [ ] . briefly, cdna was generated in a μl rt reaction containing μl of viral rna, . μm of the gene specific primer s -( '-cagggtgcaagtggtgtggtaaga- ') or l -( '-tgggactgagtttcgagcattacg- '), which are complementary to the s or l segment genomic rna, μl of x pcr buffer ii (#e , applied biosystems, carlsbad, ca), μl of mm dntp mix ( , applied biosystems), μl rnase inhibitor (n - , applied biosystems), and . μl of multiscribe reverse transcriptase ( , applied biosystems). rt reaction conditions were °c for minutes, °c for minutes, and °c for minutes. quantitative pcr was then performed in a μl reaction volume consisting of μl of cdna, . μm each of the forward primer s + ( '-cgctggcctggg tgaat- ') or l + ( '-ggccttgtatggagtagcacctt- ') and reverse primer s -( '-atgggaaaacacaacaattgatctc- ') or l -( '-ggtctgtgag atatcaagtggtagaatg- '), . μm of the taqman probe s + ( '- fam-ctg caggtttctcgc-mgbnfq- ') or l -( '- fam-ctgaagaataccacctattat acca-mgbnfq- '), and . μl of the taqman universal pcr master mix ( , life technologies, grand island, ny). reaction conditions were °c for minutes followed by cycles of °c for seconds and °c for minute. copy numbers of lcmv s or l segment genomic rnas were calculated by comparison with a series of standard dilutions of the pt -s or pt -l plasmids as described [ ] . data was generated on an applied biosystems ste-poneplus real-time pcr system and analyzed with the provided software. statistical analysis was performed using graphpad prism software. for the virus growth curves in fig b, the data was first log transformed, then a two-way analysis of variance (anova) was performed with a holm-sidak's test for multiple comparisons to compare viruses at each time point. a one-way anova with holm-sidak's test for multiple comparisons was used to analyze the vlp release assay in fig e, the viral protein levels in concentrated virions in fig b- d , and the s and l segment to pfu ratios in fig e and f . to compare plaque area in fig d , the data were first tested for normality using the d'agostino and pearson omnibus normality test, then the kruskal-wallis non-parametric test was used and multiple comparisons were made with dunn's multiple comparisons test. to analyze the cytopathic effect induced by rlcmv wt or z-y mutants (fig b) or by rlcmv wt generated in vps b wt or dominant negative cells (fig d) , a two-way anova was performed with the holm-sidak's test for multiple comparisons. to compare vsv or lcmv virus titers, lcmv di particle titers, or z vlp levels produced in vps b wt or eq cells (fig a, b and e and s fig) a two-tailed unpaired t test with welch's correction was performed. to compare di particle titers in fig a- c , a value of piu /ml (just below the limit of detection value of piu /ml) was substituted for samples that were below the limit of detection and then a one way anova was performed. for all statistical analyses, the data utilized was generated from at least independent experiments as indicated in each respective figure legend. infectious rlcmv wt or y particles following separation via density ultracentrifugation. vero e cells were infected with rlcmv wt, y f, y e, or y a at an moi of . and hr later supernatants were clarified, precipitated with peg- , resuspended in tne, and titered for pfu via plaque assay. an equal number of pfu for each rlcmv was layered onto an optiprep gradient ( %, %, %, %, and %) and centrifuged for hr at , rpm at °c. the entire gradient was collected in fractions of ml each. each fraction was titered for pfu via plaque assay. shown are results from independent experiments. (tif) s fig. efficient di particle formation requires a functional escrt pathway. (a-c) t-rex hek cells stably transduced with vectors for tetracycline-based induction of wt vacuolar protein sorting a (vps a) or the dn vps a mutant, eq, were infected with rlcmv wt and d later treated with tetracycline to induce the expression of wt or dn vps a. hr after vps a induction ( hr pi), the cells were washed and given fresh media containing tetracycline. supernatants were collected hr later ( hr pi) and titered via plaque assay. the results shown in (a) represent the mean pfu ± sem from independent experiments that contained technical replicates and were tested for statistical significance with an unpaired t test with welch's correction. equivalent pfus of virus (range x to x ) produced from wt or dn vps a cells were inoculated onto monolayers of vero e cells and a standard plaque assay was performed. representative images of crystal violet-stained wells are shown in (b). inhibition of standard infectious virus-induced cytopathic effect by di particles at each dose was determined in (c) by measurement of the mean pixel intensity of each well using image j software. the data in (c) are representative of the mean ± sem relative to wt vsp a (at pfu per well) from independent experiments that contained technical replicates and were tested for statistical significance with a two way anova and holm-sidak's test for multiple comparisons. (c) à p < . , as determined by the indicated statistical tests. ) t-rex hek cells stably transduced with a tetracycline-inducible plasmid encoding wt or dominant negative eq mutant vacuolar protein sorting b (vps b) were simultaneously transfected with a plasmid encoding lcmv z wt and exposed to tetracycline to drive the expression of wt or dn vps b. one day later both the cells and vlp-containing supernatants were collected. z from vlp-containing supernatants was affinity purified with magnetic streptavidin beads. the quantity of z affinity purified from vlps or present in the corresponding whole cell lysates was determined via quantitative western blotting. the percent vlp release shown in (a) was calculated as the amount of z protein found in the cell culture media relative to the amount in cells. data are presented as mean release ± sem relative to wt z from independent experiments. a one way anova with holm-sidak's test for multiple comparisons was used to compare the mean values. n.s., not significant). in panel (b), cell lysates were also screened by western blotting to verify the induction of vps b wt or eq expression using an anti-gfp antibody and for actin as a loading control. mammalian reservoirs of arenaviruses arenaviridae: the viruses and their replication transmission of lymphocytic choriomeningitis virus by organ transplantation treatment of argentine hemorrhagic fever arenaviruses: genomic rnas, transcription, and replication cell entry by human pathogenic arenaviruses molecular mechanism of arenavirus assembly and budding multifunctional nature of the arenavirus ring finger protein z lassa virus z protein is a matrix protein sufficient for the release of virus-like particles the small ring finger protein z drives arenavirus budding: implications for antiviral strategies cellular factors required for lassa virus budding parallels between cytokinesis and retroviral budding: a role for the escrt machinery human escrt and alix proteins interact with proteins of the midbody and function in cytokinesis escrt complexes and the biogenesis of multivesicular bodies. current opinion in cell biology virus budding and the escrt pathway defective viral particles and viral disease processes defective interfering viruses a novel type of defective viral genome suggests a unique strategy to establish and maintain persistent lymphocytic choriomeningitis virus infections viral pathogenesis and resistance to defective interfering particles determinants of lymphocytic choriomeningitis interference defective interfering particles in mice infected with lymphocytic choriomeningitis virus glycoproteins of the arenaviruses viral persistence: mechanisms and consequences productive replication of ebola virus is regulated by the c-abl tyrosine kinase parainfluenza virus m protein interaction with host protein - - negatively affects virus particle formation the host cell map kinase erk- regulates viral assembly and release by phosphorylating the p (gag) protein of hiv- the thr phosphorylation site within respiratory syncytial virus matrix (m) protein modulates m oligomerization and virus production phosphorylation of marburg virus matrix protein vp triggers assembly of nucleocapsids with the viral envelope at the plasma membrane. cellular microbiology myristoylation of the ring finger z protein is essential for arenavirus budding arenaviruses: cellular response to long-term in vitro infection with parana and lymphocytic choriomeningitis viruses homologous interference of lymphocytic choriomeningitis virus: detection and measurement of interference focus-forming units characterization of a virus variant produced by l cells persistently infected with lymphocytic choriomeningitis virus protein analysis of defective interfering lymphocytic choriomeningitis virus and persistently infected cells biochemical composition of lymphocytic choriomeningitis virus interfering particles equilibrium sedimentation of virus in density-gradients of iodinated compounds structural components and replication of arenaviruses properties of defective lymphocytic choriomeningitis virus arenavirus defective interfering particles mask the cell-killing potential of standard virus interaction of the mammalian endosomal sorting complex required for transport (escrt) iii protein hsnf - with itself, membranes, and the aaa+ atpase skd distinct roles for the aaa atpases nsf and p in the secretory pathway ubiquitin depletion and dominant-negative vps inhibit rhabdovirus budding without affecting alphavirus budding old world arenaviruses enter the host cell via the multivesicular body and depend on the endosomal sorting complex required for transport ppey motif within the rabies virus (rv) matrix protein is essential for efficient virion release and rv pathogenicity mutations in the pppy motif of vesicular stomatitis virus matrix protein reduce virus budding by inhibiting a late step in virion release ebola virus vp late domains are not essential for viral replication in cell culture infectivity of moloney murine leukemia virus defective in late assembly events is restored by late assembly domains of other retroviruses a proline-rich motif (pppy) in the gag polyprotein of mason-pfizer monkey virus plays a maturation-independent role in virion release the pppy motif of human t-cell leukemia virus type gag protein is required early in the budding process an lypsl late domain in the gag protein contributes to the efficient release and replication of rous sarcoma virus functional characterization of the putative hepatitis b virus core protein late domain using retrovirus chimeras biological roles and functional mechanisms of arenavirus z protein in viral replication the z protein of the new world arenavirus tacaribe virus has bona fide budding activity that does not depend on known late domain motifs cells expressing the ring finger z protein are resistant to arenavirus infection arenavirus budding: a common pathway with mechanistic differences scansite . : proteome-wide prediction of cell signaling interactions using short sequence motifs kinasephos . : a web server for identifying protein kinase-specific phosphorylation sites based on sequences and coupling patterns multiple interactions between the escrt machinery and arrestin-related proteins: implications for ppxy-dependent budding assessment of the risk of ebola virus transmission from bodily fluids and fomites persistence of ebola virus in ocular fluid during convalescence a case of lassa fever: clinical and virological findings vsv strains with defects in their ability to shutdown innate immunity are potent systemic anti-cancer agents dept. of health and human services, public health service, centers for disease control and prevention, national institutes of health genomic features of attenuated junín virus vaccine strain candidate severe acute respiratory syndrome coronavirus nonstructural protein interacts with a host protein complex involved in mitochondrial biogenesis and intracellular signaling rescue of the prototypic arenavirus lcmv entirely from plasmid. virology the intracellular cargo receptor ergic- is required for the production of infectious arenavirus, coronavirus, and filovirus particles recovery of an arenavirus entirely from rna polymerase i/ii-driven cdna protein-protein interactions in lymphocytic choriomeningitis virus large-scale identification and evolution indexing of tyrosine phosphorylation sites from murine brain evaluating strategies to normalise biological replicates of western blot data strand-specific quantitative reverse transcription-polymerase chain reaction assay for measurement of arenavirus genomic and antigenomic rnas we thank the uvm immunobiology group for insightful discussions and drs. alan howard and cory teuscher for statistical support and advice. we are grateful to drs. michael buchmeier, juan carlos de la torre, margaret kielian, john hiscott, and megan shaw for providing critical reagents as described in the materials and methods. key: cord- -mlhd zbs authors: valkenburg, sophie a.; gras, stephanie; guillonneau, carole; la gruta, nicole l.; thomas, paul g.; purcell, anthony w.; rossjohn, jamie; doherty, peter c.; turner, stephen j.; kedzierska, katherine title: protective efficacy of cross-reactive cd (+) t cells recognising mutant viral epitopes depends on peptide-mhc-i structural interactions and t cell activation threshold date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: mlhd zbs emergence of a new influenza strain leads to a rapid global spread of the virus due to minimal antibody immunity. pre-existing cd (+) t-cell immunity directed towards conserved internal viral regions can greatly ameliorate the disease. however, mutational escape within the t cell epitopes is a substantial issue for virus control and vaccine design. although mutations can result in a loss of t cell recognition, some variants generate cross-reactive t cell responses. in this study, we used reverse genetics to modify the influenza np( – ) peptide at a partially-solvent exposed residue (n->a, npn a mutation) to assess the availability, effectiveness and mechanism underlying influenza-specific cross-reactive t cell responses. the engineered virus induced a diminished cd (+) t cell response and selected a narrowed t cell receptor (tcr) repertoire within two vβ regions (vβ . and vβ ). this can be partially explained by the h- d(b)npn a structure that showed a loss of several contacts between the npn a peptide and h- d(b), including a contact with his , a position known to play an important role in mediating tcr-pmhc-i interactions. despite these differences, common cross-reactive tcrs were detected in both the naïve and immune npn a-specific tcr repertoires. however, while the npn a epitope primes memory t-cells that give an equivalent recall response to the mutant or wild-type (wt) virus, both are markedly lower than wt->wt challenge. such decreased cd (+) responses elicited after heterologous challenge resulted in delayed viral clearance from the infected lung. furthermore, mice first exposed to the wt virus give a poor, low avidity response following secondary infection with the mutant. thus, the protective efficacy of cross-reactive cd (+) t cells recognising mutant viral epitopes depend on peptide-mhc-i structural interactions and functional avidity. our study does not support vaccine strategies that include immunization against commonly selected cross-reactive variants with mutations at partially-solvent exposed residues that have characteristics comparable to npn a. virus-specific cd + t cells play a critical role in host defence via the production of antiviral cytokines, the direct killing of virusinfected cells and the establishment of immunological memory [ ] . the selection of cd + t cells into an immune response requires specific interaction between the t cell receptor (tcr) and virus peptide bound to major histocompatibility complex class i (pmhc-i) molecules on the surface of infected host cells. the processing of virus proteins into short fragments generates thousands of peptides that might potentially form pmhc-i epitopes, but only a few elicit ctl responses [ ] . virus escape mutants are well documented for persistent infections and constitute a major problem for cd + t cellmediated control and vaccine design [ , , , , , , ] . with regard to the influenza a viruses, mutational changes driven by cd + cytotoxic t lymphocytes (ctls) are unlikely to result in long-term persistence within the individual, as other mechanisms (particularly antibody) can ultimately mediate virus clearance [ ] . even so, the fact that such mutants can be found in nature suggests that influenza virus-specific ctls are of protective value. perhaps this reflects that the infection of new subjects favours the selection of mutant viruses that are more slowly controlled (and thus shed for longer), particularly in the face of a seasonal ''bottleneck'' where much of the population is already immune [ ] . in humans, influenza escape variants have been observed for cd + t cell epitopes presented in context of several hlas, including hla-b , hla-b and hla-b [ , , , , , , , ] . the immunogenic peptides can be modified at an mhc anchor residue, resulting in defective binding to the mhc-i glycoprotein, or at a tcr contact site. mutations at tcr contact residues lead to partial (cross-reactive) or total (non-cross-reactive) loss of recognition by wt cd + t cells [ ] , with some variants eliciting epitopespecific cd + t cell responses that are both novel and of substantial magnitude [ ] . using influenza a virus infection of b mice [ ] , we showed previously that virus variants with mutations at critical solventexposed residues that are important for tcr binding can generate effective but non-cross-reactive cd + ctl responses to what are essentially new epitopes [ , ] . this raises the possibility that it might be worthwhile to think in terms of vaccinating against likely virus escape mutants. the present analysis focuses on the crossreactive (to wt d b np ) cd + t cell response to the mutant d b npn a epitope [ ] formed by substitution of the partially solvent exposed, and non-prominent for tcr binding, residue at position (p) of the influenza np - peptide [ , ] . the findings have implications for vaccines to combat virus mutants and tumour escape variants, and suggest that immunisation against likely cross-reactive variants would have to be carefully evaluated to see if the strategy is worthwhile. earlier analysis [ , ] using sequential alanine substitutions in the immunogenic np - peptide established that there is some cross-reactive, though diminished, stimulation following the incubation of wt d b np -specific cd + t cells with the np mutant peptide containing a single asparagine to alanine substitution at position (p) (npn a mutant). such cross-reactive cd + t cell responses after npn a stimulation are not surprising as the partially solvent exposed p n residue is unlikely to play any prominent role in contacting the tcr [ ] . the wt np - peptide binds to h- d b in an extended conformation where the p -ser, p -asn and p -met are the anchor residues, p -asp is a semi-anchor (partially buried/partially exposed), and p -glu, p -met, p -glu and p -thr are solvent exposed and available for contact by the tcr (fig. ) . this provides the basis for a defined experimental system that can be used to determine what happens when a tcr repertoire is selected to what would seem (from in vitro analysis) to be a suboptimal, cross-reactive mutant epitope. the interpretation of in vitro analysis alone should, however, be tempered with caution, as another mutant (m a) that did not cross-react at all with the wt epitope elicited a completely novel, in vivo endogenous ctl response of equivalent magnitude when expressed in an infectious influenza a virus [ ] . what would be the case for tcr responses elicited by influenza a viruses expressing the mutant npn a peptide in the native viral protein? the npn a mutation was engineered into pr (h n ) and hkx (h n ) influenza viruses (pr npn a, hknpn a) to allow cross-challenge experiments in the absence of antibody neutralisation. the b mice were immunised i.p. with the virulent pr mutant and wt viruses, while the hk viruses were used for primary ( o ) i.n. infection of naïve mice or secondary ( o ) i.n. challenge of pr -immune (. d previously) mice. naïve (primary) or pr -immune (pr , or pr npn a) mice were challenged i.n. with the homologous virus (hk, or hknpn a) and cd + t cell responses were measured using the d b np and d b npn a tetramers (fig ) . it was immediately apparent that the splenic d b npn a + cd + set elicited by the hknpn a challenge was significantly smaller on d (p, . ) than the d b np + cd + t cell response induced by the wt virus (fig b) , though there was no significant difference between d b npn a + cd + and d b np + cd + t cell numbers at the site of infection (bal, fig a) . this has been seen before [ ] and suggests that the need to clear virus from the lung results in preferential ctl localization to the site of infection when immune t cell numbers are limited. the profile of a diminished d b npn a + cd + t cell response was maintained into memory (d , fig c; p, . ), supporting the view that the relative size of persistent t cell pools reflects the extent of antigen driven proliferation during the acute anti-viral response. a high proportion of the wt d b np + cd + t cells in bal ( . % ( , d ); . ( , d ); fig a) and spleen ( . % ( , d ); . % ( , d ); fig b) bound the d b npn a tetramer and produced ifn-c when stimulated by the npn a peptide (data not figure . a stick conformation illustrating how the influenza np - interacts with h- d b . np - peptide binds to the h- d b in an extended conformation. the p -asp, p -asn and p -met are the anchor residues, whereas the p -glu, p -met, p -glu and p -thr are solvent exposed and available for contact by the tcr. doi: . /journal.ppat. .g introduction of a new influenza strain into human circulation leads to a rapid global spread of the virus due to minimal antibody immunity. established t-cell immunity towards conserved viral regions provides some protection against influenza and promotes rapid recovery. however, influenza viruses mutate to escape the protective immunity. we found that established t cell immunity can recognise influenza mutants with variations at positions that are partially involved in t cell recognition. however, an initial priming with the mutated variant decreases recognition of the original parental virus. this finding results from a markedly lower functional quality and limited structural interactions of the mutant. in terms of possible vaccination strategies for rapidly changing viruses or tumours, it appears that priming with crossreactive mutants that display such characteristics would be of no benefit as the same level of t cell immunity against such mutants can be elicited by exposure to the original virus. shown). similarly, the majority ($ %) of cd + t cells elicited by hknpn a infection bound the d b np tetramer and produced ifn-c (data not shown) at levels equivalent to those seen for the response to npn a, indicating a high level of cross-reactivity between the d b np + cd + and npn a + cd + t cell responses. staining with the d b pa tetramer was included to establish that the npn a mutation neither diminished nor enhanced other influenza-specific cd + t cell responses (fig ) . despite the decreased d b npn a + cd + t cell numbers generated following primary infection (fig a-c) , the recall response was substantial following hknpn a challenge of prnpn a-immune mice ( fig de) and the np.pa immunodominance hierarchy that has long been recognized for secondary responses to wt influenza a viruses in h b mice [ ] was maintained ( fig de) . similarly, the total cell numbers for memory d b pa -specific t cells on d were comparable for those primed and boosted within the wt and mutant virus combinations (fig f) , while the d b npn a + cd + set was -fold smaller than the wt d b np + cd + population (p, . ). again, the results following secondary challenge support the view that, at least in the earlier (d ) stages of memory, t cell numbers reflect the extent of clonal expansion during the acute phase [ ] . in our experiments, we detected d b np + cd + and npn a + cd + t cells by two techniques, tetramer staining and ifn-c ics. both techniques gave us comparable antigen-specific cd + t cell numbers, indicating that both tetramers accurately detected epitope-specific populations (fig. gh) . given that the npn a mutation was associated with a numerically diminished response following infection with either the wt or npn a influenza a viruses (fig ) , we also asked if there was any effect on cd + t cell function or phenotype, particularly for markers (cd l and cd ) that discriminate between memory t cell subsets [ , ] . more of the d b npn a + cd + t cells remained cd l hi when sampled at the peak of the response (d ) following primary challenge (fig s a) , confirming the impression from the quantitative analysis (fig a-c) that there may be less clonal expansion. on the other hand, il- r expression was comparable for the d b npn a + cd + and d b np + cd + t cell populations generated in virus-infected naïve mice (fig s cd) with both being (p, . ) lower than the values for the d b pa -specific set ( fig s c) . this suggests that il- r levels may be antigen dose-rather than magnitude-dependent. neither of these differential effects was apparent for d memory t cells specific for the mutant or wt epitopes (fig s bd) . functional analysis of cytokine production based on short term ( h) stimulation with cognate peptide in the ics assay showed no obvious differences at any stage for the d b np and d b npn aspecifc t cells, though the usual divergence [ ] from the d b pa + cd + t cell response was observed (p, . ) (fig s e-h) . these data suggest that npn a mutation leads to crossreactive, but diminished, cd + t cell responses with comparable cytokine production profiles. crystal structure and thermostability of the h db-np-n a complex can the smaller response to d b npn a be correlated with structural constraints or any decrease in stability for the pmhc-i complex? the d b npn a crystal structure containing the heavy chain of h- d b (residues - ), the b microglobulin (residues - ) and the residues of the npn a peptide was determined to a . Å resolution ( fig. and table ), with a final r factor of . % and an r free of . %. the structure of the h- d b -npn a complex was compared (fig. b , table ) to the wt d b np [ ] . as observed for the wt d b np , the mutant npn a peptide bound h- d b in an extended conformation, the p -ser, p -asn and p -met represent the anchor residues, p -ala semi-anchor residue, whereas the p -glu, p -met, p -glu and p -thr are solvent exposed and available for contact by the tcr (fig. ab) . however, the mutation of p -asn to ala leads to a loss of contacts between the peptide and the mhc molecule. in comparison to the wt np that makes contacts with the h- d b molecule, the npn a peptide achieves only contacts. interestingly, the asn ala mutation abolishes contacts between the p residue of the peptide with his and the tyr of the mhc and eliminates one hydrogen bond between the p -asn nd and the p -glu o (fig. cd ). the loss of inter-residue bonding between n and e within the npn a peptide may also be important for tcr recognition as it changes the characteristics of the wt d b np epitope (defined as type iii constraint) [ ] into an unconstrained d b npn a epitope. this change of constraint within the epitope could affect the dynamics of the peptide upon tcr ligation, and subsequently alter tcr reactivity toward the mutated epitope. in addition, the loss of contacts with the his of the mhc is of interest, as position , termed the ''gate-keeper residue'' [ ] is involved in contacting the tcrs in all tcrpmhc-i complexes solved to date [ ] . thus, the lack of interactions between the npn a peptide and the mhc through his may also affect recognition of the complex by d b np specific tcrs. the loss of contacts between the peptide and the mhc molecule could lead to decreased stability of the peptide and subsequent changes in npn a presentation. to test this hypothesis, we performed a thermostability assay on both np and npn a bound to the h- d b molecule. the np and npn a peptides are equally effective at stabilising h- d b . the pmhc-i complex with the np wt peptide had a tm of . . uc and d b npn a showed a comparable level of thermostability (tm = . uc), irrespective of the concentrations of the complex used for the assay. this suggests that the npn a mutation does not modify the stability of the pmhc-i complex when compared to the cognate epitope. naïve precursor frequency and tcr repertoire for d b npn a is the smaller d b npn a + cd + t cell response a consequence of diminished naïve ctl [ ] ? we found (fig a) similar naive ctlp counts for d b npn a ( . . ) and d b np ( . . ), indicating that the smaller response to d b npn a + cd + is not due to reduced number of naïve precursors. furthermore, assessing the extent of vb . bias (the dominant vb for the d b np + cd + set) within the naïve d b npn a + cd + population ( fig b) showed that the extent of vb . usage (mean . % . ) (fig. b ) was much the same as that determined previously [ ] for naïve d b np + cd + ctlps (mean . % . ) however, sequencing the naïve d b npn a + vb . + cd + tcr cdr b regions showed a clear difference from the comparable wt-specific set. the ''public tcr'' dominance characteristic of d b np + vb . + cd + t cells in both pre-immune [ ] and immune [ ] tcrb repertoires [ ] was not a prominent feature of the d b npn a-specific tcr repertoire. these public tcrs were found in only one (sggantgql and sgggntgql) or two (sggsntgql) of the mice tested (fig. c) . thus, although the naïve ctlp frequencies are comparable for d b np + vb . + cd + and d b npn a + vb . + cd + t cells and there is some overlap of some cross-reactive tcrs, the two repertoires are far from identical. the roughly equivalent numbers of precursors specific for the wt d b np and mutant npn a peptides were unexpected considering the lower response after infection with the mutated virus. the lower magnitude of the npn a + cd + t cell response and narrower tcrb repertoire suggest that only a proportion of naïve npn a + cd + precursors are being recruited into the immune response or that, once recruited, these cells do not expand efficiently. inefficient recruitment and/or expansion early after influenza infection, despite large naïve ctl precursor numbers, have been recently documented by our group as key determinants of subdominance for d b pb -f and k b ns -specific responses [ ] . the next step was to dissect the immune d b npn a + cd + ctl repertoire to determine how tcr diversity relates to the size of the immune d b npn a + cd + t cell response. the d b npn aspecific cd + t cells were first analysed for vb usage by staining with a panel of anti-tcrvb mabs and the d b npn a tetramer. after infection with the npn a viruses, the strong vb . bias characteristic of responding d b np + cd + t cells [ , ] was prominent for the d b npn a + cd + sets in only % of the immune mice (n = ). the mutant d b npn a + cd + t cells utilized a variable spectrum of tcrvb elements, with overrepresentation of vb , . / . , , , and after primary ( fig s a) or vb , and following secondary ( fig s b) npn a virus challenge. we analysed tcrb clonotypes within the vb . as (i) it is still a preferred region ( . % . ) of npn a + cd + t cell response) and (ii) clonotypes within this region could be highly relevant for cross-reactive cd + t cell responses between np and npn a as they are prominent in both populations. overall, the mutant and the wt immune vb . repertoires appear different. single-cell rt-pcr and sequencing of the cdr b region of tetramer + vb . + cd + t cells following either hk or hk-npn a infections showed that (table s ) ), and showed evidence of more variable cdr b loop lengths ( - aa). the reduction (relative to the wt d b np ) in vb . usage by the d b npn a + cd + t cells reproduces the relative loss of ''public'' wt tcrs found for the naïve repertoire ( fig b) . this likely reflects that the n a mutation has disrupted an ''optimal'' tcr/ pmhc fit that maximizes antigen-driven clonal expansion. indeed, the public tcrs were not a prominent feature of the d b npn a + cd + set, being found in only of the npn ainfected mice at a very low frequency, namely % in the response and . % in the response ( table ). this is in contrast to the d b np + cd + vb . + tcr repertoire [ ] that is largely ( %) comprised of high-frequency, public clonotypes found in all infected b mice [ ] . these results also establish that the naïve d b npn a + cd + tcr repertoire (fig. c) is predictive of the immune d b npn a + cd + tcrb response ( table ). the relative lack of a ''public'' response translated to a profile of reduced ''sharing'' between individual mice, and a total increase in the number of d b npn a + cd + clonotypes due to the 'private' nature of tcrb repertoire for each mouse (table ). even so, the clonotypic diversity within individual npn a virus-infected mice was reduced compared to the spectrum found following wt virus-infection. this was true whether the cells were sorted using the wt d b np or the mutant d b npn a tetramer, reflecting the significant cross-reactivity between the d b np and d b npn a-specific populations recovered from mice infected with the npn a viruses. by the measure of tetramer binding, it thus seems that the npn a virus is selecting a less diverse repertoire than the wt virus, with the repertoire being almost completely cross-reactive with that elicited by the wt d b np epitope. similarly, when the d b np + vb . + cd + t cells induced by wt hk infection were sequenced, the majority of the tcrb clonotypes were detected with both the d b np and d b npn a tetramers (table ) . however, a switch in frequency was seen for some cdr b-defined clonotypes, indicating selective binding of particular tcrs by the d b npn a tetramer. for example in m and m (table ) the 'public' sggantgql cdr b-sequence dominated the d b np + vb . + cd + t cell population, whereas this sequence was less commonly detected in the same mice by the d b npn a tetramer. the difference could, of course, reflect diversity in tcra usage. following clonotype selection with the d b np and d b npn a tetramers, sequencing of the secondary tcrb repertoire (m -m ) induced by challenge with the homologous (pr , then hk) viruses showed less divergence within the wt d b np + cd + t cell specific population. interestingly, clonotypes like kggs-ntgql were enriched by the d b npn a tetramer in some but not other mice (table ; present in m but not m ) suggesting, again, the likely importance of vb-va chain pairing for recognition of the native d b np + cd + t cells with the mutant d b npn a tetramer. thus, the analysis suggests that only a subset of the repertoire generated by wt infection is able to recognize the d b npn a epitope, though this population is more diverse than that generated in response to the mutant npn a virus. all the statistical differences ( table , table ) were confirmed when the data were standardized to a number of sequences (data not shown). we further analyzed the d b npn a + vb + cd + set that was prominent in of the hk-npn a secondarily challenged mice. an average of . tcrb clonotypes was found within this population (table s ) . again, the d b npn a + vb + cd + tcrb repertoire emerged as essentially restricted and private. however, tcrb analysis within other vbs for npn a + cd + t cells would need to be performed to compare the whole tcr repertoires. analysis of va chain usage for the mutant d b npn a + cd + and wt d b np + cd + t cells by pcr with a panel of va specific primers established that those two t cell responses indeed tend to utilise different va chains. the wt d b np + cd + t cell population [ ] (day eb, unpublished) tended to use va and va . (n = ). conversely, the d b npn a + cd + tcrs preferentially expressed va , va and va (table s ). while the sample size is small and there are at least different va chains, these results provide a snapshot of the mutant d b npn a + and wt d b np + populations and suggest that there are differences in both vb and va tcr chain usage. response characteristics following heterologous challenge and tcr/pmhc-i avidity as there was substantial cross-reactivity in vitro (fig. ) for the d b np and d b npn a-specific responses, it was important to determine whether memory t cells that cross-react for the d b np and d b npn a epitopes are preferentially recalled by secondary infection with the heterologous virus. mice that were primed with the pr npn a and then challenged with either the wt hk or mutant hknpn a viruses showed equivalent recall of d b npn a + cd + t cells during the acute phase of the secondary response. this was detected by ifn-c production ( fig a) and tetramer staining (data not shown) and is consistent with the tcr cdr b analysis (table ) . conversely, when mice were firstly primed with wt pr , then later infected i.n. with either the wt hk or mutant hk-npn a, the d b np + cd + t cells were differentially recalled indicating that (in the absence of primary selection from the naïve repertoire by the mutant epitope) only some of the d b np + cd + memory t cells that were expanded by heterologous challenge bind d b npn a (fig. a) . interestingly, wt priming and challenge ( pr -. x ) resulted in significantly higher cd + t cells numbers (fig. b ) than were found for any secondary cd + t cell responses after npn a priming ( pr -npn a-. x -npn a and pr -npn a-. x ). these results lead to two main conclusions: (i) priming with the wt virus elicits cd + t cells that respond relatively well to a subsequent infection with cross-reactive variant (ie pr -. x -npn a = pr -npn a-. x -npn a); (ii) priming with the cross-reactive variant can be detrimental as the diminished primary response may limit the full expansion of cd + t cells that are able to respond to the subsequent wt infection (ie pr -npn a-. x is lower than pr -. x ) and skew the tcr usage. thus, using npn a for either priming or the challenge gives an equally poor response. to determine whether such decreased cd + responses elicited after heterologous challenge would affect influenza virus clearance, we performed experiments to examine the protective efficacy of cross-reactive cd + t cell repertoires. we performed prime-andchallenge studies in mmt mice lacking b cells to ensure that antibody responses did not mask any possible inhibitory effects of ''suboptimal'' tcrs on viral clearance. as suggested by cd + t cell data, assessment of lung viral titres showed delayed viral clearance on d after the secondary infection in case of heterologous prime-and-boost (pr -.x -npn a and pr -npn a-.x ) compared to homologous infections (pr -.x or pr -npn a-.x -npn a) (fig. b) . these results indicate that recall of ''suboptimal'' tcrs for a single t cell specificity can lead to delayed viral clearance, despite the presence of other influenza cd + t cell responses (d b pa these patterns of complete, or partial, cross-stimulation following in vivo virus challenge were reflected in the results found for in vitro measurements of ''functional avidity'' (fig ) . pulsing immune spleen cells recovered directly ex vivo with graded doses of wt or mutant peptide in the ics assay showed comparable levels of ifn-c induction (fig ac) in every situation but one, the exposure of wt-primed t cells to the npn a peptide ( fig b) . thus, while the immune repertoire selected by d b np a shows evidence of equivalent avidity following stimulation with the npn a or np peptides, d b np induces a response that is of higher avidity for the wt than the mutant epitope. the same effect was seen even more clearly when three (all expressing the sgggntgql cdr b) t cell hybridoma lines [ ] specific for d b np were stimulated with the two peptides ( fig de) . this result is in accord with findings from both the tcr repertoire analysis of crossreactive clonotypes, assessed by tetramer binding (table ) , and the response magnitudes determined following homologous and heterologous virus challenge (fig ) . thus, priming and recall of cross-reactive cd + t cells recognising mutant viral epitopes reflects functional (defined as responsiveness to a peptide) pmhc-tcr avidity. to determine the pmhc-i avidity of the responding d b np + cd + and npn a + cd + t cells, we additionally performed tetramer dilution (fig. a -c) and tetramer dissociation (fig. d-e) assays. while tetramer dissociation assay measured the ''off-rate'' component of pmhc-i avidity, tetramer dilution technique assessed the overall pmhc-i avidity (both the ''on'' and ''off'' rates). furthermore, we also assessed cd b-dependence for functional activation (a measure of low avidity cd + t cells) of both wt d b np + cd + and the mutant npn a + cd + t cells by anti-cd b mab blocking, followed by ifn-c ics (fig. g-i) . our data obtained from those three additional measures of pmhc-i avidity confirmed the results obtained by the peptide titration combined with ics (functional avidity, fig. ) and further suggested significantly lower pmhc-i avidity of the wt d b np + cd + (generated by the wt hk infection) for npn a variant. the p n within the immunodominant influenza virus-specific d b np epitope is a partially solvent exposed, and non-prominent for tcr binding, residue that is predominantly buried within the mhc cleft [ , ] . the npn a mutation leads to both decreased recruitment of cd + t cells and a narrowed clonotype selection profile within vb . and vb regions. structurally, the mutation leads to a loss of a number of contacts between the npn a peptide and the mhc-i molecule, including a contact with the gate-keeper residue at position , and unaltered stability of the h- d b -npn a complex. the fact that the npn a mutation affects contacts with the mhc-i at his , known to play an important role in tcr-pmhc structures in general, is likely to indirectly compromise the tcr recognition. by loosing the bond with the asn , his may gain more flexibility and thus be inappropriately placed for the subsequent tcr ligation onto the npn a peptide. alternatively, it is also possible that a small part of the solvent-exposed head group of the asn residue in the wt np peptide might, to some extent, be directly interacting with the tcr following ligation. surprisingly, despite the loss of several contacts between npn a peptide and h- d b , the stability of the peptide-mhc-i complex remains constant for both np and npn a. this suggests that the asn as a secondary anchor does not play an important role in stabilizing the peptide within the mhc-i. the structural basis for the diminished recruitment of d b npn aspecific cd + t cells is thus likely to rest in the way that the partially-solvent exposed residue contacts mhc-i and modifies tcr ligation. the emerging d b npn a + cd + t cell population was characterised by different va and vb preference, distinct cdr b sequences and a lower overall tcr diversity in comparison to wt d b np + cd + t cells. these findings suggest that a very limited spectrum of cd + t cells can recognise the d b npn a mutant structure. interestingly, a similar p substitution in the influenza virus d b pa epitope had no affect on d b pa + cd + t cell recognition and recruited a diverse array of tcr clones comparable to the spectrum found for the wt response [ ] . this suggests that the partially-exposed p residue plays a greater structural and/or tcr recognition role for the ''featureless'' d b np than for the ''featured'' d b pa complex reflecting, in turn, the more limited spectrum of tcrs that bind d b np [ ] . taken together, it appears that partially-exposed residues within viral peptides can provide important contacts with the mhc-i, which can in turn cause remote effects that modify antigenicity for the tcr-pmhc-i complex and impact on both tcr repertoire selection and the magnitude of cd + t cell responses. interestingly, there were no differences in function or phenotype characteristics for the d b np + cd + and npn a + cd + t cells, although those two ctl sets had a high proportion of different tcr clones. this is in accordance with previous studies showing that the simultaneous production of antiviral cytokines [ ] and il- r [ ] expression is antigen dose-rather than magnitude-related. conversely, levels of the cd l [ , , ] activation marker differed for the d b np + cd + and npn a + cd + populations, indicating that response magnitude has some relationship to the activation status of cd + t cells [ , , ] which may, perhaps, reflect the extent of ctl proliferation. the tcr repertoires specific for d b np and d b npn a appear to be quite distinct. the response overall for wt d b np + cd + t cells is characterised by conserved, ''public'' clonotypes that constitute the majority ( . % in response and . % in response) of the selected tcr repertoire [ ] . these public clonotypes are not a prominent feature of the d b npn a + cd + set, being found only in / npn a-infected mice at very low frequency. since we know that these tcr clonotypes are present in all b mice [ ] , the difference presumably reflects the lower tcr avidity for d b npn a, as indicated by the t cell hybridoma analysis where they were shown to require times more npn a than np peptide for optimal stimulation. since the public clonotypes cannot be efficiently recruited into the immune response by the mutated n a virus, this could have created a ''hole'' in tcrs capable of recognising the mutated epitope, which subsequently can lead to a reduction in t cell immunogenicity [ ] . however, though both the d b np + cd + and d b npn a + cd + t cell responses are characterised by quite distinct tcr repertoires, the majority are bound by both the d b np and d b npn a tetramers and can be detected by stimulation with either the np or the npn a peptides, suggesting that a clonal dissection of tcr clonotypes is needed to make a valid interpretation about the truly crossreactive cd + t cell responses. these findings also raise questions concerning the true correlation between phmc-i tetramer binding in vitro and the in vivo selection of a responding tcr repertoire. overall, the results indicate that a loss of a number of contacts between the npn a peptide and the mhc-i molecule and lower functional and structural pmhc-i avidity (for wt d b np ) d b npn a selects a narrowed tcr repertoire of ''best fit'' tcrs from a spectrum of naïve clonotypes that, once activated, clonally expanded and engaged in an immune response, have sufficient avidity to be recalled by exposure to the wt d b np epitope. conversely, the ''better'' fit d b np finds a sufficient spectrum of high avidity tcrs within that available naive repertoire and does not (likely because of clonal competition) select most of the tcr ab pairs that interact optimally with d b npn a. priming with the wt virus thus establishes memory for only a very limited secondary response to the mutant. similar to our results, subtle variations within the anchor residue of h b peptide/i-e k also decreased peptide-mhc class ii affinity and the activation of responding t cells [ ] . thinking about this in terms of possible vaccination strategies for use against rapidly changing viruses or tumor epitopes, it appears that priming with cross-reactive mutants that have characteristics comparable to npn a would be of no benefit (or even could be detrimental as evidenced by delayed viral clearance) as the same level of t cell immunity against such mutants can be elicited by exposure to the wt epitope. on the other hand, changes like the non-cross-reactive np-m a mutation [ ] that induce a completely novel, high quality response might merit incorporation in an experimental vaccine or immunotherapy strategy. overall, working out the topographical constraints that determine these differential response profiles would seem eminently worthwhile. a further reason for defining the structural rules governing tcr cross-recognition is that similar effects have been found for different epitopes derived from unrelated viruses. published studies provide evidence for cross-reactive cd + t cell responses between influenza a virus and epstein-barr virus (ebv) [ ] , influenza a virus and hepatitis c virus (hcv) [ , ] or lymphocytic choriomeningitis virus (lcmv) and pichinde virus (pv) [ ] . such heterologous cross-reactive immunity can unintentionally skew tcr recruitment, result in a narrow tcr repertoire and subsequent viral escape [ ] as well as influenza disease severity [ ] . doi: . /journal.ppat. .g [ ] . the topic of tcr cross-reactivity in cd + t cell responses clearly merits more attention. all animal experimentation was conducted following the australian national health and medical research council code of practice for the care and use of animals for scientific purposes guidelines for housing and care of laboratory animals and performed in accordance with institutional regulations after pertinent review and approval by the university of melbourne animal ethics experimentation committee in melbourne. -npn a) influenza a viruses, in ml of pbs. viruses share the same internal components for np and pa from which cd epitopes are derived [ ] . virus stocks were grown in the allantoic cavity of day old embryonated chicken eggs, from which the viral titre was determined by plaque assay on monolayers of madin derby canine kidney (mdck) cells. recombinant influenza viruses with the single amino acid substitution (n a) within the np peptide, asnenmetm, were generated using the eight-plasmid reverse genetics system [ ] . the substitution was first incorporated by site directed mutagenesis using pcr primers encoding n a peptide, asaenmetm, and the opposite primer encoding np. recombinant pcr products encoding n a were digested with bsmb and ligated into the alkaline phosphatase treated phw vector. recombinant viruses (hk-npn a and pr -npn a) were rescued following transfection of mdck- t cell coculture with the eight plasmids encoding influenza segments. viruses were then amplified in the allantoic cavity of -day old embryonated chicken eggs, and the viral titre determined by plaque assay of allantoic fluid infecting monolayers of mdck cells. there was any evidence of altered fitness in vivo for the hknpn a virus, as the kinetics of virus growth and clearance following i.n. challenge of naïve b mice were found to be equivalent for the wt hk and mutant viruses (fig s a) . similarly, the levels of ctl activity found using target cells infected with hknpn a or the hk wt viruses were comparable, and the same was seen for peptide pulsed cells, suggesting that antigen presentation of npn a peptide remains constant (fig s bc) . lungs taken from mice after primary viral infection (fig. s a) or prime-and-boost approach using homologous (pr -.x and pr -npn a-.x -npn a) or heterologous (pr -.x -npn a and pr -npn a-.x ) strategy (fig. b) were homogenised and the virus-containing supernatant above the cell debris was harvested and stored at uc. titres of infectious virus in the lung supernatants were determined by plaque assay on monolayers of mdck cells. spleen and bronchoalveolar lavage (bal) samples were recovered from mice at acute phases of the primary and secondary infections (d and d respectively), and the bal samples were incubated on plastic petri-dishes for hr at c to remove macrophages. the spleens were disrupted and enriched for cd + t cells using goat anti-mouse igg and igm antibodies (jackson immunoresearch labs, west grove, pa, usa). for assessment of naïve precursor frequency of n a + cd + t cells, spleens and lymph nodes (inguinal, brachial, axillary, superficial cervical, and mesenteric) were collected from naïve mice and processed to single-cell suspensions. tetramer and phenotypic staining of cd + t cells lymphocytes from the bal and spleen were stained with tetramers conjugated to strepavidin-apc or pe (molecular probes, eugene, or, usa) at optimal staining concentrations ( mg/ml d b np , mg/ml d b n a , and mg/ml d b pa tetramers) for hr at room temperature. cells were washed twice in facs buffer, and stained with mg/ml cd -percp cy . , mg/ml cd l-fitc and mg/ml cd -apc mabs (bd biosciences) for mins on ice, washed twice and analysed by flow cytometry on the facs calibur (bd immunocytometry) and analysed by cellquest pro software (bd immunocytometry). we titrated all the batches of all the tetramers used in this study. we used tetramers at optimal concentrations ( - mg/ml) based on both the percentage of epitope-specific cd + t cells and the mean fluorescence intensity (mfi) of tetramer staining. a scatchard analysis [ ] based on the tetramer dilution assay (fig. a-c) was also plotted (fig. s ) and confirmed our observations from routine tetramer titrations that the d b np tetramer displays slightly superior pmhc binding capacities over the npn a tetramer at a concentration , mg/ml. cd + t cells from spleen were stained with the d b np and d b -npn a tetramers conjugated to streptavidin-pe (molecular probes, eugene, or) for mins at room temperature. for a tetramer dilution assay, -fold dilutions of pe-conjugated tetramers were used at a range of concentrations ( . - mg/ ml). for a tetramer dissociation assay, lymphocytes were stained at the optimal concentration of pe-conjugated tetramers as assessed by tetramer titration as determined by both the percentage of tetramer + cd + t cells and mean fluorescence intensity (mfi). cells were washed twice in facs buffer ( %bsa/ . % naaz in pbs), stained with a fitc-conjugated mab to cd a (bd biosciences pharmingen) for mins on ice, washed and analysed by flow cytometry. as a measure of pmhc avidity, splenic t cells were used in tetramer dissociation assay [ ] . after staining with tetramer, t cells were washed and incubated in the presence of anti-h d b antibody at mg/ml at c to prevent tetramer rebinding. cells were removed at intervals, stained with the fitcconjugated mab to cd a and analysed by flow cytometry. loss of tetramer + cd + t cells at particular time-points was calculated in comparison to tetramer staining at t = mins. enriched t cell populations from spleen and bal were stimulated with one of the np , n a , pa or pb peptides (auspep) for hrs at uc, % co in the presence of mg/ml golgi-plug (bd biosciences pharmingen) and u/ml recombinant human il- (roche, germany) (bd biosciences). cells were washed twice with facs buffer, stained with cd -percp cy . for mins on ice, fixed, permeabilised and stained with anti-ifn-c-fitc ( mg/ml), tnf-a-apc ( mg/ml), and il- -pe ( mg/ml) mabs (biolegend). samples were acquired using flow cytometry, and the total cytokine production calculated by subtracting background fluorescence using no peptide controls. in selected experiments, lymphocytes were stimulated with varying concentrations of peptides, three-fold dilutions ranging from nm to . nm to determine the sensitivity specific peptides, defined as 'functional avidity' [ ] . splenocytes were obtained from mice sampled on d after secondary infection. lymphocytes were pre-cultured in the presence or absence of anti-cd b antibody ( . - ) ( mg/ml). cells were then stimulated for h with peptide, il- and golgistop also in the presence or absence of anti-cd b antibody ( mg/ml). following stimulation, cells were analysed for cd and ifnc expression. shown is the percentage of cd + cells producing ifn-c after stimulation in the presence of anti-cd b blocking mab. determination of n a + cd + t cell precursor frequency naïve n a -specific cd + t cells were identified as described [ , ] . briefly, processed lymph nodes and spleen samples were resuspended in ml of sorter buffer, ml fcr block ( g and cd culture supernatant, % mouse and % rat serum) was added, and clumps of dead cells were discarded. tetramers at optimal staining concentrations (d b n a -pe at mg/ml and d b np -pe at mg/ml) were added to the cell mix and incubated for hour at room temperature in the dark. cells were washed and resuspended in ml buffer with ml anti-pe microbeads (miltenyi biotec), and incubated at uc for mins. following two washes, cells were resuspended in ml of buffer and cells that had bound the microbeads were purified on a magnetic ls column according to manufacturers instructions (miltenyi biotec). cells eluted from the column were centrifuged ( g, min, uc), supernatant carefully aspirated to leave ml buffer remaining, and ml antibody cocktail was added for min at uc. the antibody cocktail contained anti-cd -apc cy (pharmingen, bd), anti-cd -pe cy (ebiosciences), anti-b -fitc (pharmingen, bd), anti-cd b-fitc (ebiosciences), anti-cd c-fitc (ebiosciences), anti-f / -fitc (ebiosciences), anti-cd l-apc (pharmingen, bd), and anti-cd -percp cy . (pharmingen, bd) mabs. cells were washed in ml buffer, centrifuged ( g, min, uc), and supernatant aspirated leaving ml. resuspended cells were passed through mm sieve, and data acquired by flow cytometry on the lsr ii (becton dickinson) and analysed with flowjo (treestar inc.) software. in selected experiments, naïve npn a + vb . + cd + t cells were single-cell sorted for tcrb analysis. experimental details of the single cell rt-pcr designed to amplify naive npn a + vb . + cd + t cells using different sorting strategies are listed in table s . lacz-inducible t cell hybridomas specific for np peptide [ , ] were resuspended at cell/ml and aliquots ( ml) and dispensed into -well flat-bottom plates together with naïve splenocytes (apcs). cells were cultured in the presence of np or npn a peptides at concentrations ranging from m to m for h at c. the cells were then washed with pbs, fixed with ml of % formaldehyde/ . % glutaraldehyde in pbs for min on ice, washed in pbs and incubated with . mg of -bromo- -chloro- -indolyl b-d-galactoside (x-gal) for h at c. the lacz + hybridomas were then counted using a light microscope, and the number of np -specific hybridomas was calculated by subtracting the ''background'' lacz expression for cells cultured in the absence of the peptide. splenocytes were stained with mg/ml d b np or mg/ml d b n a -pe tetramer in sort buffer (pbs with . % bsa) for mins at room temperature, washed and stained with mg/ml anti-cd -allophycocyanin and mg/ml of either anti-vb . or anti-vb for mins on ice, washed twice with sort buffer. single lymphocytes were isolated by sorting with a facs aria (bd immunocytometry), into wells of an empty well twintec plate (eppendorf). mrna transcripts were reversed transcribed to cdna, using a sensiscript kit (qiagen) according to manufacturers instructions, and the cdr b region amplified by a nested hot start pcr using vb primers [ ] . positive pcr products were purified using qiagen pcr purification kit and sequenced. protein expression, purification, crystallisation and structure determination h -d b and b -microglobulin molecules were expressed in escherichia coli as inclusion bodies, refolded with the np-n a (asaenmetm) peptide and purified as previously described [ , ] . the h d b np-n a complex crystals were obtained at mg/ml by the hanging-drop vapour diffusion technique at uc. crystals were grown with a reservoir containing . m sodium citrate at ph . , % peg (w/v), . m lithium sulphate. the crystals belong to space group i and the unit cell dimensions were consistent with one molecule per asymmetric units (table s ). the crystals were flash frozen to a temperature of k before data collection in-house with a rigaku ru- rotating-anode xray generator. the data were processed and scaled with the xds [ ] . the crystal structure was solved using the molecular replacement method using the program phaser [ ] from the ccp suite of programs [ ] . the search probe used to solve the structure was the structure of mouse mhc class i h -d b minus the peptide (protein data bank accession number cpl) [ ] . the progress of refinement was monitored by the r free value with neither a sigma nor a low-resolution cutoff being applied to the data. the refinement protocol used includes several cycles of refinement with refmac [ ] followed by manual model rebuilding with o program [ ] . 'translation, liberation and screw-rotation' displacement refinement was used to model anisotropic displacements of defined domains was used during the refinement process. the electron density around the npn a peptide was unambiguous, and all the side chains were built at full occupancy. some mobile loops in the heavy chain of the h- d b molecule (residues - ; - and - ) have been removed from the final model due to missing electronic density. final refinement statistics are summarized in table i , the coordinates of the h db-np-n a complex have been deposited with the protein data bank under accession numbers ftg. thermostability measurements of recombinant class i complexes using circular dichroism (cd) circular dichroism spectra were measured on a jasco spectropolarimeter using a thermostatically controlled cuvette. a far-uv spectra was collected from nm to nm. the uv minimum was determined as nm for h db-np-n a. the measurements for the thermal melting experiments was made at the minimum for h db-np-n a, at intervals of . uc at a rate of uc/min from uc to uc. the jasco spectra manager software was used to view and smooth the traces and then the graphpad prism software was used to plot temperature versus % unfolded. the midpoint of thermal denaturation (tm) for each protein was determined as the point at which % unfolding was achieved. the measurements were done in duplicate at two concentrations ( mm and . mm) in a solution of mm tris ph , mm nacl. the atomic coordinates have been deposited in the protein data bank, www.pdb.org (pdb id code ftg). influenza and the challenge for immunology hidden epitopes emerge in secondary influenza virus-specific cd + t cell responses viral escape by selection of cytotoxic t cell-resistant virus variants in vivo evidence of hiv- adaptation to hla-restricted immune responses at a population level t cell receptor recognition motifs govern immune escape patterns in acute siv infection rapid viral escape at an immunodominant simian-human immunodeficiency virus cytotoxic t-lymphocyte epitope exacts a dramatic fitness cost dominant influence of an hla-b restricted cd + t cell response in mediating hcv clearance and evolution structural and biological basis of ctl escape in coronavirus-infected mice prevention of cytotoxic t cell escape using a heteroclitic subdominant viral t cell determinant clearance of an influenza a virus by cd + t cells is inefficient in the absence of b cells population dynamics of rapid fixation in cytotoxic t lymphocyte escape mutants of influenza a sequence variation in the influenza a virus nucleoprotein associated with escape from cytotoxic t lymphocytes sequence variation in a newly identified hla-b -restricted epitope in the influenza a virus nucleoprotein associated with escape from cytotoxic t lymphocytes antigenic drift in the influenza a virus (h n ) nucleoprotein and escape from recognition by cytotoxic t lymphocytes conservation and diversity of influenza a h n hla-restricted t cell epitope candidates for epitope-based vaccines influenza virus ctl epitopes, remarkably conserved and remarkably variable functional constraints of influenza a virus epitopes limit escape from cytotoxic t lymphocytes t-cell tolerance for variability in an hla class i-presented influenza a virus epitope hla class i molecules consistently present internal influenza epitopes establishment and recall of cd + t cell memory in a model of localized transient infection lack of prominent peptide-major histocompatibility complex features limits repertoire diversity in virus-specific cd (+) t cell populations complete modification of tcr specificity and repertoire selection does not perturb a cd + t cell immunodominance hierarchy the threedimensional structure of h- db at . a resolution: implications for antigendeterminant selection compromised influenza virus-specific cd (+)-t-cell memory in cd (+)-t-cell-deficient mice contemporary analysis of mhcrelated immunodominance hierarchies in the cd + t cell response to influenza a viruses virus-specific cd + t-cell memory determined by clonal burst size two subsets of memory t lymphocytes with distinct homing potentials and effector functions selective expression of the interleukin receptor identifies effector cd t cells that give rise to long-lived memory cells hierarchies in cytokine expression profiles for acute and resolving influenza virus-specific cd + t cell responses: correlation of cytokine profile and tcr avidity constraints within major histocompatibility complex class i restricted peptides: presentation and consequences for t-cell recognition t cell receptor recognition of a 'super-bulged' major histocompatibility complex class i-bound peptide the fidelity, occasional promiscuity, and versatility of t cell receptor recognition primary ctl response magnitude in mice is determined by the extent of naive t cell recruitment and subsequent clonal expansion conserved t cell receptor usage in primary and recall responses to an immunodominant influenza virus nucleoprotein epitope quantification of repertoire diversity of influenza-specific epitopes with predominant public or private tcr usage ctl recognition of a protective immunodominant influenza a virus nucleoprotein epitope utilizes a highly restricted vbeta but diverse valpha repertoire: functional and structural implications prominent usage of v beta . t cells in the h- db-restricted response to an influenza a virus nucleoprotein epitope lineage relationship and protective immunity of memory cd t cell subsets early establishment of diverse tcr profiles for influenza-specific cd lhi cd + memory t cells homogenization of tcr repertoires within secondary cd lhigh and cd llow virus-specific cd + t cell populations secondary memory cd + t cells are more protective but slower to acquire a central-memory phenotype stimulation history dictates memory cd t cell phenotype: implications for prime-boost vaccination junctional biases in the naive tcr repertoire control the ctl response to an immunodominant determinant of hsv- structural and functional consequences of altering a peptide mhc anchor residue cross-reactive influenza virus-specific cd + t cells contribute to lymphoproliferation in epstein-barr virus-associated infectious mononucleosis cross-reactivity between hepatitis c virus and influenza a virus determinantspecific cytotoxic t cells heterologous t cell immunity in severe hepatitis c virus infection narrowed tcr repertoire and viral escape as a consequence of heterologous immunity cellular events in the lymph node and lung of mice with influenza. consequences of depleting cd + t cells rescue of influenza b virus from eight plasmids tcr binding kinetics measured with mhc class i tetramers reveal a positive selecting peptide with relatively high affinity for tcr functional avidity maturation of cd (+) t cells without selection of higher affinity tcr naive cd (+) t cell frequency varies for different epitopes and predicts repertoire diversity and response magnitude differential antigen presentation regulates the changing patterns of cd + t cell immunodominance in primary and secondary influenza virus infections the production, purification and crystallization of a soluble heterodimeric form of a highly selected t-cell receptor in its unliganded and liganded state identification of a dominant self-ligand bound to three hla b alleles and the preliminary crystallographic analysis of recombinant forms of each complex automatic processing of rotation diffraction data from crystals of initially unknown symmetry and cell constants the ccp suite: programs for protein crystallography improved methods for building protein models in electron density maps and the location of errors in these models we thank dr john stambas for review of the manuscript, dr richard webby for help with npn a viruses, ken field and jason waithman for facs sorting. key: cord- -tdgu sr authors: reniere, michelle l.; whiteley, aaron t.; portnoy, daniel a. title: an in vivo selection identifies listeria monocytogenes genes required to sense the intracellular environment and activate virulence factor expression date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: tdgu sr listeria monocytogenes is an environmental saprophyte and facultative intracellular bacterial pathogen with a well-defined life-cycle that involves escape from a phagosome, rapid cytosolic growth, and acta-dependent cell-to-cell spread, all of which are dependent on the master transcriptional regulator prfa. the environmental cues that lead to temporal and spatial control of l. monocytogenes virulence gene expression are poorly understood. in this study, we took advantage of the robust up-regulation of acta that occurs intracellularly and expressed cre recombinase from the acta promoter and ’ untranslated region in a strain in which loxp sites flanked essential genes, so that activation of acta led to bacterial death. upon screening for transposon mutants that survived intracellularly, six genes were identified as necessary for acta expression. strikingly, most of the genes, including gshf, spxa , yjbh, and ohra, are predicted to play important roles in bacterial redox regulation. the mutants identified in the genetic selection fell into three broad categories: ( ) those that failed to reach the cytosolic compartment; ( ) mutants that entered the cytosol, but failed to activate the master virulence regulator prfa; and ( ) mutants that entered the cytosol and activated transcription of acta, but failed to synthesize it. the identification of mutants defective in vacuolar escape suggests that up-regulation of acta occurs in the host cytosol and not the vacuole. moreover, these results provide evidence for two non-redundant cytosolic cues; the first results in allosteric activation of prfa via increased glutathione levels and transcriptional activation of acta while the second results in translational activation of acta and requires yjbh. although the precise host cues have not yet been identified, we suggest that intracellular redox stress occurs as a consequence of both host and pathogen remodeling their metabolism upon infection. intracellular pathogens such as plasmodium spp., mycobacterium tuberculosis, salmonella enterica, trypanosoma cruzi, and leishmania spp. are responsible for an overwhelming amount of morbidity and mortality worldwide. successful dissemination of many of these pathogens requires complex life cycles that involve survival and replication in environmental or vector niches. to propagate within their hosts, these pathogens establish a variety of unique intracellular niches that are essential for their pathogenesis [ ] . although there is considerable understanding of how intracellular pathogens manipulate host cell biology to promote their pathogenesis, less is known about the precise mechanisms by which these pathogens sense their host cell. such an understanding may lead to targets for therapeutic intervention. in this study we used listeria monocytogenes as a model system for understanding virulence gene regulation of a facultative intracellular bacterium that transitions from extracellular to intracellular growth. l. monocytogenes is a ubiquitous environmental saprophyte capable of causing severe disease as a foodborne pathogen [ ] . l. monocytogenes is also a model system for studying bacterial adaptation to the host [ ] . the bacterial virulence program is coordinated with a life cycle that begins upon entry into a mammalian cell either by phagocytosis or bacteria-mediated internalization. to commence intracellular growth, l. monocytogenes must first escape from the hostile phagosomal environment by the expression and secretion of a cholesterol-dependent cytolysin, listeriolysin o (llo) that mediates destruction of the phagosome [ ] . upon entry into the cytosol, l. monocytogenes grows rapidly and expresses an essential determinant of pathogenesis, acta, an abundant surface protein that mediates host actin polymerization [ , ] . appropriate regulation of llo and acta is critical for l. monocytogenes pathogenesis and transcriptionally coordinated by the master virulence regulator prfa [ ] . prfa is a camp receptor protein (crp) family transcriptional regulator that is absolutely essential for l. monocytogenes virulence gene expression and pathogenesis [ ] . prfa-mediated gene expression is regulated by prfa abundance, affinity for target promoters, and activation via cofactor binding [ ] . prfa levels are controlled by three promoters. the most proximal promoter contains a site of negative regulation, while the most distal is a prfa-dependent readthrough transcript that is essential for appropriately high levels of intracellular gene expression [ ] [ ] [ ] . prfa binds a palindromic dna sequence (prfa-box) and deviations from a consensus sequence result in lower affinity dna-prfa interactions [ ] . the affinity of prfa for dna determines the degree of transcriptional activation prior to prfa allosteric activation [ ] . for example, the gene encoding llo (hly) has a high affinity prfa-box and consequently is expressed even during growth in broth when prfa is not activated. in contrast, the acta promoter contains a lower affinity prfa box and is not expressed during growth in broth [ , ] . upon entry into the host cell cytosol, prfa is over-expressed and is activated by a two-step process: first, binding of prfa to dna requires reduction of the four prfa cysteine residues while full transcriptional activation of prfa requires allosteric binding to glutathione [ ] . the requirement for glutathione can be bypassed by mutations that lock prfa in its active conformation (prfa à ) [ ] . strains with prfa à mutations constitutively express prfa-activated genes and consequently have growth defects extracellularly, demonstrating the importance of regulating virulence gene expression [ , ] . however, even prfa à strains grown in broth fail to synthesize the amount of acta observed intracellularly, which is likely attributable to translational control localized to the ' untranslated region ( ' utr) [ ] . despite these findings of exquisite gene regulation, little is known about trans-acting factors that affect expression of prfa or prfa-activated genes. in a previous study, a genetic system was designed to select for l. monocytogenes mutants that failed to express acta intracellularly [ ] . this screen led to the identification of l. monocytogenes glutathione synthase (gshf) and glutathione, a tripeptide antioxidant, as the allosteric activator of prfa. in this study we sought to further understand the host cues that are recognized by intracellular pathogens during infection. we returned to the forward genetic selection and exhaustively screened for additional mutants that failed to express sufficient acta intracellularly. this selection identified genes required at each stage of the intracellular lifecycle, including: vacuolar escape, prfa activation, and cell-to-cell spread. these data suggest a model of compartmentalized gene expression, furthering our understanding of the l. monocytogenes virulence program. the goal of this study was to identify genes involved in regulation of a principle virulence determinant in l. monocytogenes, acta. a bacterial strain was constructed that failed to replicate upon activation of the acta gene, which is specifically up-regulated during cytosolic growth and is essential for pathogenesis. this 'suicide' strain harbored loxp sites in the chromosome flanking the origin of replication (ori) and several essential genes. codon-optimized cre recombinase was expressed from the acta promoter ( fig a) . the suicide strain grew like wild type in rich media but was unrecoverable after infection of bone marrow-derived macrophages (bmms). a himar transposon library was then constructed in the suicide strain background and used to infect bmms. when bacteria were isolated at five hours post-infection (p. i.) nearly all mutants harbored transposon insertions in cre, the acta promoter driving cre expression (acta p), loxp sites, and gshf, encoding glutathione synthase. to identify additional genes required during infection, colonies were isolated at three and four hours p.i, generating a library of , transposon mutants from an initial inoculum of > million bacteria. colony pcr excluded strains with transposon insertions in cre and gshf, resulting in a collection of strains (fig a) . transposon mutants in the suicide background were screened individually for survival in bmms, narrowing the list to mutants. six transposon insertions were identified in hly and nine insertions in prfa, emphasizing that cytosolic access and prfa are absolutely required for acta activation and subsequent cre expression. saturation of the screen was further demonstrated after identification of insertions in the acta promoter driving cre and insertions in the loxp sites (which are each only nucleotides). the remaining transposon mutations were transduced into a wild type background and analyzed in a plaque assay, a highly sensitive measure of cell-to-cell spread, which is completely dependent on acta expression [ ] . using a threshold of %, mutants were identified that formed plaques significantly smaller than wild type in l murine fibroblasts (fig a and table ). with one exception, the transposon insertions were in open reading frames and likely resulted in loss-of-function mutations. the [ ] , teal arrows represent sites of transposon insertions, and numbers above these arrows correspond to mapped transposon locations (nucleotides ' of the start codon). bolded numbers denote the transposon insertions used in this study. transposon in the promoter of lmo (spxa ), a gene predicted to be essential in l. monocytogenes [ ] , resulted in a -fold decrease in spxa expression when the bacteria were grown in broth, essentially resulting in a knock-down strain (s fig). attempts to make an in-frame deletion of spxa using conventional methods were unsuccessful, consistent with a previous report [ ] . as the goal of this selection was to identify mutations that affect acta expression in vivo, we measured acta abundance during infection of bmms. four hours post-infection, cells were lysed and acta and the constitutively expressed p protein were analyzed by immunoblot. nine strains were found to express less acta than wild type after normalizing to p abundance ( fig b) . the work-flow of this selection used cre expression from the acta promoter plaque area as a percentage of wild type. data are the mean and error bars indicate the standard error of the mean (s.e.m.) for three independent experiments. p values were calculated using a heteroscedastic student's t-test and all strains are significantly different from wild type (p < . ). (b) quantification of immunoblots of acta and p during infection. acta abundance was normalized to p abundance and measured as a percentage of wild type. data are the mean ± s.e.m. of at least three independent experiments. (c) female cd- mice were infected with colony forming units (cfu) of each mutant. spleens were harvested hours post-infection and cfu were quantified. the solid lines indicate the median, and data represent three pooled experiments totaling n = mice per strain. p values were calculated using a heteroscedastic student's t-test * p < . ; ** p < . ; *** p < . . (d) gene expression of target genes measured by quantitative rt-pcr in wild type l. monocytogenes grown in broth compared to expression during infection of bmms. data are the mean ± s.e.m. of at least three independent experiments. p values were calculated using a heteroscedastic student's t-test * p < . . and plaque area as a criterion for inclusion in the core set of twelve mutants analyzed here. it was therefore unexpected that three mutants (lmo ::tn, lmo ::tn, and citc::tn) did not display a defect in acta abundance during intracellular growth. we hypothesize that these mutations may disrupt elements of bacterial physiology critical to appropriate cre activity or normal growth. the twelve mutants isolated by the genetic selection were identified based on in vitro assays for virulence. while these assays are correlated to in vivo outcomes, the importance of these genes to l. monocytogenes pathogenesis was confirmed in a murine model of infection. intravenous infection of mice revealed that four of the mutants displayed no virulence defect (lmo ::tn, rsbx::tn, lmo ::tn, and gtca::tn) while the remaining eight mutants were significantly attenuated ( fig c) . it was surprising that four mutants exhibited impaired plaque formation yet were fully virulent; it is possible that these four mutants are impaired in other aspects of pathogenesis not reflected by changes in cfu during these infection conditions. to determine if the plaque defects in these mutants were due to cell-specific defects evident only in the l murine fibroblasts used for plaque assays, cell-to-cell spread defects were also analyzed in tib- cells, a murine hepatocyte cell line (table ) . we observed consistent phenotypes between the plaque defects in tib- cells and l cells with the exception of citc::tn, p-spxa ::tn, and ohra::tn. however, these mutants were significantly attenuated during infection and thus it was unclear why they did not display a plaque defect in tib- cells. the specificity of the transposon insertion in seven of the eight attenuated strains was confirmed by expressing the disrupted gene in trans and complementing the plaque defect (s fig). attempts to complement the ppla::tn plaque defect were unsuccessful. however, ppla mutants are difficult to complement and the mutant we identified exhibited phenotypes consistent with published Δppla defects [ ] . other reports have identified genes necessary for virulence of l. monocytogenes by comparing changes in gene expression in vivo [ ] [ ] [ ] . in our analysis, only gshf was differentially transcribed between host cells and rich media (fig d) . it remains to be investigated if the activity of these genes is regulated post-transcriptionally in response to the host. in this study we focused on the following genes that were required for acta expression and pathogenesis ( fig b) . yjbh (lmo ) encodes a putative thioredoxin similar to yjbh in bacillus subtilis ( % amino acid similarity) [ ] . a transposon in l. monocytogenes yjbh was previously identified in a screen for mutants defective in llo production in vitro and was found to be attenuated in a competitive infection model [ ] . spxa (lmo ) encodes an arsc family transcriptional regulator similar to the disulfide stress regulator spx conserved in firmicutes ( % amino acid identity to b. subtilis spx) [ ] . the difference in nomenclature is due to the presence of a paralogous gene in l. monocytogenes (lmo or spxa ) that is % identical to b. subtilis spx while b. subtilis encodes only a single spx. in b. subtilis and staphylococcus aureus yjbh post-translationally regulates spx [ , ] , although it is not known if this function is conserved in l. monocytogenes. lmo encodes a hypothetical protein with a peroxiredoxin domain and is part of the organic hydroperoxide resistance (ohr) protein subfamily. it is cotranscribed with lmo , encoding a marr family transcriptional regulator which was not required for virulence, suggesting that lmo may act as a transcriptional repressor [ ] . in b. subtilis homologs of lmo and lmo are named ohra ( % amino acid similarity) and ohrr ( %), respectively, and we have adopted this nomenclature for consistency [ ] . arpj (lmo ) encodes a predicted amino acid abc transporter permease that was originally identified in a screen for genes with increased intracellular expression [ ] . however, the data presented here did not show an increase in arpj expression during infection of bmms. this may be explained by the different growth media and cell types used in the two studies. it is also possible that arpj is autoregulated, as the previous study analyzed arpj expression in an arpj transposon mutant. ppla (lmo ) encodes a lipoprotein whose secretion is increased in a prfa à mutant [ ] . the signal sequence of this lipoprotein is processed into a secreted peptide, which is required for vacuolar escape from non-phagocytic cells [ ] . finally, gshf (lmo ) encodes the only glutathione synthase in l. monocytogenes [ ] . glutathione has been demonstrated to be an allosteric activator of prfa and therefore gshf mutants are severely attenuated in vivo due to insufficient virulence gene expression [ ] . given the role of glutathione in activating prfa, we hypothesized that suppressor mutations of Δgshf might illuminate alternative pathways for prfa activation, potentially involving other genes identified. accordingly, we screened for mutations that increased the virulence of a Δgshf mutant. mice were serially infected with a high-inoculum of Δgshf, livers were harvested at hours p.i., homogenized, and diluted to inoculate naive mice. after four successive infections bacteria isolated from infected livers were analyzed by plaque assay. this approach previously identified a mutation in prfa that constitutively activates the protein (g s), known as prfa à , completely bypassing the requirement for glutathione during infection [ ] . the Δgshf prfa à suppressor forms % plaque; therefore, for these experiments we selected bacteria that formed intermediate-sized plaques, which were then subjected to genome sequencing. two suppressor mutants were isolated and found to encode a g>a mutation nucleotides ' of the prfa start codon (fig a) . this mutation lies within a previously identified site of negative regulation of prfa, the so-called "p promoter" (prfa p, fig a) and deletion of the - region of this promoter (Δp mutant) results in a - -fold up-regulation of the prfa p-dependent prfa transcript [ ] . we hypothesized that the prfa - g>a mutation also inactivated the p promoter and resulted in greater prfa abundance. indeed, the Δp gshf::tn double mutant and the Δgshf prfa - g>a suppressor mutants all formed plaques approximately % the size of wild type ( fig b) . these results did not directly implicate any of the other genes identified in our genetic selection, however these findings did highlight the impact of both prfa abundance and activation during infection. prfa expression is controlled by a feed-forward loop in which activated prfa drives its own transcription [ ] . strains expressing Δp or prfa à decouple prfa abundance and activation whereby Δp increases prfa abundance but still relies on glutathione for prfa activation; prfa à increases both the amount and activity of prfa, independent of glutathione. we next sought to determine if the other mutants identified in the screen affected prfa abundance or activation by transducing each into l. monocytogenes Δp and prfa à backgrounds and measuring the plaque size in each background ( fig c) . based on these analyses, mutants fell into three categories. the first category (yjbh::tn, p-spxa ::tn, ohra::tn, and arpj::tn) was unaffected by alterations in prfa expression or activity, indicating that these genes were required down-stream of prfa. in the second category was gshf::tn, which was partially rescued by Δp and completely rescued by prfa à , consistent with the demonstrated role for glutathione as the allosteric activator of prfa. the third category describes ppla::tn, which formed % plaques in both the Δp and prfa à backgrounds. these data suggested that the ppla mutant was capable of activating prfa (because it was rescued by Δp ) but was deficient in expression of prfadependent genes required early during infection before cytosolic access and glutathione-mediated activation of prfa. a principle difference between early and late prfa-dependent genes is that expression of early genes are less dependent on prfa activation by glutathione [ ] . the two early genes are hly (encoding llo) and plca, which share a high-affinity prfa-box and are transcribed by unactivated prfa [ , ] . the Δp mutation results in increased transcription of early genes but does not affect late gene expression, whereas prfa à increases transcription of both early and late genes. we hypothesized that strains rescued by Δp are specifically deficient in early gene expression. accordingly, we analyzed early gene expression (llo production) in broth for each mutant. several of the mutants were found to secrete less llo than wild type ( fig a) . to determine if the defect in llo production led to impaired phagosomal escape and thus a plaque defect, these mutations were transduced into a Δhly mutant over-expressing hly from a constitutive hyper promoter (ph-hly strain) [ , ] . in this background, efficiency of vacuolar escape should be equivalent in all strains, and indeed, equal llo secretion was confirmed in broth. constitutive expression of hly rescued the plaque defects of three mutants: p-spxa ::tn, ohra::tn, and ppla::tn ( fig b) . interestingly, there was discordance between llo production in broth and the defect in plaque formation one might predict from an llo deficiency. for this reason, measuring llo production in broth may be revealing aspects of bacterial physiology unrelated to llo production in vivo. the above results suggested that mutations in p-spxa , ohra, and ppla resulted in aberrant llo secretion and/or that these mutants were unable to survive in the harsh environment of the vacuole. constitutive expression of hly would likely overcome either defect. we attempted to segregate these two possibilities by analyzing sensitivity to vacuolar conditions, including reactive oxygen species which l. monocytogenes must adapt to in order to survive [ , ] . the response of each mutant to peroxide, disulfide stress, and organic hydroperoxide was analyzed by measuring their sensitivity to hydrogen peroxide (h o ), diamide, and cumene hydroperoxide (chp), respectively. knock-down of spxa and disruption of ohra or gshf significantly increased the sensitivity of l. monocytogenes to both peroxide and disulfide stress (fig c) . in accordance with its annotation and the published role of ohra in b. subtilis [ ] , the ohra::tn mutant was significantly more susceptible to chp ( fig c) . as these results suggested a role for redox control of virulence genes, we tested the hypothesis that host reactive oxygen or nitrogen species may be sensed by the bacteria during infection to activate acta. however, growth of the suicide mutant was not rescued in bmms lacking inducible nitric oxide synthase (nos -/-) or nadph oxidase (nox -/-) (s fig). therefore, l. monocytogenes may activate virulence genes in response to multiple redundant host cues or depend on yet unidentified host pathways. constitutive production of hly restored the majority of the plaque defect for p-spxa ::tn and ohra::tn, however, it did not restore the plaque to % of the parent strain ( fig b) . we hypothesized that these mutants might also be impaired in the ability to grow in the host cytosol, independently from virulence gene expression. all of the mutants identified in the screen grew similarly to wild type in bmms with the exception of p-spxa ::tn and ohra::tn (fig d) . in fact, p-spxa ::tn and ohra::tn were also the only mutants that exhibited growth defects in rich media (fig e) . these pleiotropic growth defects and sensitivity to redox stress are likely why ph-hly was only partially able to complement the plaque defect of these mutants (fig b) . previous work clearly demonstrated that glutathione was essential for transcriptional activation of virulence genes [ ] . in order to assess which factors might be independent of glutathionedependent transcriptional activation, we combined each transposon with an in-frame Δgshf mutation. the only mutation not epistatic to gshf was yjbh::tn, which produced an additive plaque defect ( fig a) . further, yjbh::tn was not rescued by constitutive activation of hly ( fig b) or prfa ( fig c) . together, these data suggested that yjbh was required for acta expression post-transcriptionally. indeed, transcript levels of acta were identical in bmms infected with wild type or the Δyjbh mutant ( fig b) . it is intriguing that arpj::tn was epistatic to gshf, yet not rescued by constitutive activation of prfa, indicating that arpj may contribute to glutathione-dependent transcriptional activation of acta through an unknown mechanism. the acta gene is preceded by nucleotides of untranslated mrna ( fig c) which is important for sufficient acta expression [ ] . a strain was constructed in which acta was expressed independent of prfa by expressing the entire acta transcript (including the ' utr) under the control of the constitutive hyper promoter in a strain deleted for acta (ph-acta strain, fig d) . acta protein abundance was then analyzed by immunoblot. in this background, acta abundance was equivalent among all strains when the bacteria were grown in broth ( fig e) . however, during infection of bmms, disruption of yjbh resulted in significant impairment in acta abundance (fig f) , indicating a failure to translationally activate acta. given that disrupting yjbh rescued the death of the suicide strain in which cre was expressed under acta p and the ' utr, these data indicate a genetic interaction between yjbh and the ' utr of acta. to further support this genetic interaction we engineered a fluorescent strain of l. monocytogenes in which rfp was expressed under the acta p promoter and ' utr (acta prfp, fig g) . during infection of bmms the Δyjbh acta p-rfp strain exhibited significantly less fluorescence than wild type acta p-rfp ( fig h) . unfortunately, we were unable to interrogate the effect of a yjbh mutation on acta abundance in the absence of its ' utr due to an inability to detect acta when the ' utr was deleted, consistent with this region being critical for acta expression [ ] . a drawback to ph-acta is that although acta is over-expressed in broth, this strain still elaborates much less acta in vivo and fails to form a plaque (fig e and f ). to analyze the role of translational activation during infection, the acta gene and ' utr were moved to a neutral locus within the l. monocytogenes chromosome [ ] . in this strain, acta was expressed only from the prfa-dependent acta p proximal promoter, eliminating read-through transcription from the distal acta p promoter (fig c) . this strain was called acta p and was only mildly impaired in plaque formation and virulence (fig i and j) . however, acta p yjbh::tn was unable to form a plaque (fig i) . the importance of acta translational activation was further underscored by a -log defect for acta p yjbh::tn in the livers of infected mice (fig j) . these data revealed a critical role for yjbh in acta activation that was less apparent in the wild type background due to redundant prfa-dependent promoters. in this study, rather than search for novel virulence factors or genes up-regulated in vivo, we screened for genes required for activation of an essential determinant of l. monocytogenes pathogenesis (acta) that is up-regulated over -fold during intracellular growth. mutants identified in the genetic selection fell into three broad categories: ( ) those that failed to reach the cytosolic compartment; ( ) mutants that entered the cytosol, but failed to activate the master virulence transcriptional regulator prfa; and ( ) mutants that entered the cytosol and activated transcription of acta, but failed to synthesize it (fig ) . this approach highlighted how and cfu were quantified. the solid lines indicate the median, and data represent two pooled experiments totaling n = mice per strain. in all panels, p values were calculated using a heteroscedastic student's t-test * p < . ; ** p < . ; *** p < . ; ns (not significant) p > . . doi: . /journal.ppat. .g once phagocytosed by a host macrophage, l. monocytogenes (light blue rods) requires the gene products of spxa and ohra to survive in the phagosome. by a mechanism that is not yet understood, ppla is required for vacuolar escape in non-phagocytic cells. yjbh and arpj are then required for cell-to-cell spread. the l. monocytogenes pathogenicity island- is pictured below. early genes (depicted in red) are those with high-affinity prfa boxes that do not require active prfa (teal) for transcription. late genes (depicted in blue) are those with relatively low-affinity prfa boxes that require activated prfa to be transcribed and these are required later during infection, in the host cytosol. the transition from unactivated to activated prfa requires glutathione (orange circles), which is synthesized by gshf. yjbh (magenta) is then required for translational activation of acta, although the mechanism is not yet understood. see text for more details, model is not drawn to scale. expression of virulence factors is spatially and temporally compartmentalized via regulation of transcription and translation during infection. one of the most striking findings of this study was that the majority of genes identified in the selection encode proteins predicted to control bacterial redox regulation, suggesting that redox changes represent one of the biological cues sensed by l. monocytogenes to regulate its virulence program. redox stress during infection can arise from endogenous by-products of bacterial metabolism and exogenously derived factors generated by the host. however, it remains to be discovered whether the redox stress that may trigger virulence gene expression is produced by the host, the bacteria, or both. yjbh, spx, ohra, and gshf have defined roles in maintaining redox homeostasis in the presence of disulfide and organic peroxide stresses in firmicutes. in b. subtilis ohra is a peroxiredoxin required during organic hydroperoxide stress [ ] . in s. aureus and b. subtilis yjbh interacts with spx to regulate the abundance and activity of spx [ , ] . specifically, yjbhbound spx is recognized by the clpxp protease and is degraded so that spx concentrations are low under steady-state conditions [ , ] . during disulfide stress the yjbh:spx interaction is disrupted by intramolecular disulfide bonds in both proteins that result in reduced proteolysis of spx. b. subtilis spx represses transcription of genes and activates transcription of genes [ ] , the majority of which are required to adapt to redox stress, including genes for production of the low-molecular weight (lmw) thiol utilized by b. subtilis, bacillithiol [ ] . l. monocytogenes spxa cannot be deleted and its regulon has not yet been characterized [ ] . similarly, in streptococcus pneumoniae simultaneous deletion of both spxa and spxa paralogues is lethal [ ] , supporting the notion that the spx regulon(s) may contain essential genes in some firmicutes. mutants exhibiting the most severe virulence phenotypes contained insertions in gshf, which encodes the sole l. monocytogenes glutathione synthase [ ] . glutathione is a tripeptide lmw thiol antioxidant present at millimolar concentrations that contributes to maintaining a reducing environment in both bacterial and host cells [ ] . not surprisingly, l. monocytogenes Δgshf mutants are more sensitive to redox stressors such as hydrogen peroxide and diamide and are -fold less virulent in mice, indicating that bacterially-derived glutathione is essential for pathogenesis [ ] . however, Δgshf mutants are fully virulent in l. monocytogenes harboring prfa à mutations that lock prfa in its constitutively active conformation. therefore, the primary role of gshf-derived glutathione during infection is to activate virulence gene expression via prfa activation, although we cannot rule out a contribution of imported host-derived glutathione [ ] . indeed, host-derived glutathione activates virulence gene expression in burkholderia pseudomallei [ ] . in the case of l. monocytogenes, gshf is transcriptionally up-regulated -fold during intracellular growth, suggesting the existence of an unidentified cue, likely redox-related, that stimulates glutathione production. the identification of many redox-related bacterial factors in this genetic selection led to our working model that specific redox changes during infection are sensed by the bacteria as a mechanism to identify their intracellular location and activate virulence genes appropriately. redox stress during infection could arise from host-derived antimicrobial factors. for example, the host generates antibacterial factors that assault invading pathogens with redox stresses, including: reactive oxygen species (ros), reactive electrophilic species (res) such as methylglyoxal, and reactive nitrogen species (rns) such as nitric oxide and peroxynitrite [ , , ] . interestingly, these redox stresses from the host are spatially compartmentalized. rns and ros are produced in the phagosome and once in the host cytosol, l. monocytogenes is confronted with res and mitochondrial-derived ros [ , ] . it is possible that the bacterial response to the redox stressors is also compartmentalized, requiring specific factors in the vacuole (such as spxa and ohra) and host cytosol (such as yjbh). eliminating host nitric oxide synthase (nos ) or nadph oxidase did not rescue growth of the suicide mutant (s fig). nos -generated nitric oxide is required for efficient l. monocytogenes cell-to-cell spread during infection, although this is due to the nitric oxide-mediated delay of phagolysosome maturation and not a direct effect on the bacteria [ ] . together, these data suggest that a combination of host factors are likely required to activate acta during infection. alternatively, the source of redox stress may come from bacterial metabolism via ros generated from incomplete reduction of oxygen during aerobic respiration [ ] . carbon source and phosphate abundance also affect the production of ros and methylglyoxal [ , ] . prfa activity has been demonstrated to be sensitive to available carbon sources [ ] . growth on plant-derived beta-glucoside sugars in the environment, such as cellobiose, represses prfa activation, whereas growth on host-derived sugars such as glucose- -phosphate stimulates prfadependent gene expression [ , , ] . therefore, entry of l. monocytogenes into the host cytosol results in a remodeling of carbon metabolism that may be linked to virulence gene regulation. glycerol is the principle carbon source used by l. monocytogenes intracellularly and growth on glycerol is a well-described stimulant of methylglyoxal production [ ] [ ] [ ] [ ] . in b. subtilis, methylglyoxal stress stimulates the spx regulon and production of bacillithiol, a low molecular weight thiol used by b. subtilis to detoxify methylglyoxal [ ] . thus, the -fold increase in gshf transcript levels in l. monocytogenes may correspond to increased methylglyoxal production during infection, which would further link metabolism of an alternative carbon source to virulence. coupling of metabolism to virulence gene regulation may allow the system to remain off in the environment while remaining poised to turn on upon entering a host. considering our finding of multiple redox factors that are required for proper virulence gene expression, we speculate that changes in carbon metabolism could alter the endogenous levels of ros and res produced, thus affecting prfa activation and leading to the "sugar-mediated repression" observed previously [ ] . appropriate up-regulation of acta at the translational level is understood to require its ' utr, although the mechanism remains unknown [ ] . the data reported here further emphasize the sensitivity of acta translation to the environment in which l. monocytogenes is growing. in broth, the prfa à strain elaborated . % the amount of acta protein as compared to constitutively expressed acta (fig e) , and increased -fold during infection (fig f) , despite the fact that transcript levels of acta are equivalent in both growth conditions [ ] . these data emphasize the importance of the translational control of this virulence factor. importantly, yjbh was required for the increased abundance of acta protein during infection. in wild type l. monocytogenes, multiple prfa-dependent promoters may compensate for loss of translational activation; however, when acta was isolated under its most proximal promoter, disruption of yjbh resulted in an attenuation of over -logs in the livers of infected animals (fig j) . it seems unlikely that the thioredoxin yjbh activates translation of acta via direct binding to the ' utr. however, yjbh may indirectly activate translation via interaction with another factor (s) or modulation of a small-molecule signal produced by the host. prfa-dependent transcription and activation are regulated redundantly at multiple levels, including: a temperature-sensitive riboswitch [ ] , allosteric activation by glutathione [ ] , multiple read-through transcripts [ , ] , positive and negative promoter elements [ , ] , and yet to be fully characterized translational control. the complexity of acta activation is likely the result of selective pressure to respond appropriately to host-derived cues. this study investigated the virulence defects associated with failure to up-regulate virulence genes; however, over-production or inappropriate regulation of virulence factors extracellularly also results in a competitive disadvantage for l. monocytogenes [ , ] . how l. monocytogenes and other intracellular pathogens regulate virulence gene expression is central to understanding their pathogenesis. results reported here suggest that redox cues are a mechanism by which intracellular pathogens recognize the host and represents an exciting new area of further investigation. this study was carried out in strict accordance with the recommendations in the guide for the care and use of laboratory animals of the national institutes of health. all protocols were reviewed and approved by the animal care and use committee at the university of california, berkeley (aup- - - ). all l. monocytogenes strains are a derivative of wild type s [ , ] and were cultivated in brain heart infusion (bhi, difco), shaking at °c unless otherwise stated. all e. coli strains were cultivated shaking in lb (miller) at °c. antibiotics (purchased from sigma) were used at the following concentrations: carbenicillin ( μg/ml), streptomycin ( μg/ml), chloramphenicol ( . μg/ml for l. monocytogenes and μg/ml for e. coli), erythromycin ( μg/ ml), and tetracycline ( μg/ml). all e. coli strains are listed in table and all l. monocytogenes strains are listed in table . bacterial broth growth curves were performed as previously described [ ] . the suicide strain was a gift from peter lauer and bill hanson (aduro biotech); details of its construction are reported elsewhere [ ] . briefly, loxp sites were inserted on either side of the origin of replication by allelic exchange into a ΔactaΔinlb strain of l monocytogenes. a transcriptional fusion of cre with acta that included the acta p promoter, ' utr, and ribosomal binding site of acta, was inserted adjacent to a loxp site. knock-in of ppl derivative plasmids was performed by standard methods [ ] . briefly, constructed ppl plasmids were transformed into chemically competent sm e. coli, selecting on chloramphenicol. donor sm and recipient l. monocytogenes were mixed at a : ratio on a non-selective bhi plate at °c for - hours, then trans-conjugation was selected for by plating bacteria on bhi containing streptomycin plus either chloramphenicol (ppl ), erythromycin (ppl e), or tetracycline (ppl t). single colonies were re-streaked for purifying selection onto bhi containing the same antibiotics as used after trans-conjugation. in-frame deletions of genes was accomplished by allelic exchange using pksv -orit and conventional methods [ ] . briefly, the constructed knock-out plasmid was transformed into sm e. coli, recovered on lb containing carbenicillin, and trans-conjugated into l. monocytogenes by mixing the donor sm and recipient l. monocytogenes at a : ratio on a non-selective bhi plate for - hours at °c, the permissive temperature for pksv -orit to replicate in gram-positive organisms. trans-conjugation was selected on bhi containing both streptomycin and chloramphenicol at °c. after isolation of a single colony of l. monocytogenes containing pksv -orit at °c, bacteria were grown at °c on bhi agar containing both streptomycin and chloramphenicol to select for chromosomal integration. colonies were restreaked onto selective media at °c two additional times for purifying selection and integrated pksv -orit. this strain was then serially passaged at °c to enrich for excision and loss of pksv -orit. mutants that lost pksv -orit were identified by sensitivity to chloramphenicol using indirect patch-plating methods. finally, allelic exchange was confirmed by pcr and, when necessary, sanger dna sequencing. preparation of electro-competent l. monocytogenes and himar transposon mutagenesis were performed as previously described [ ] , generating a transposon mutant library that was not fully characterized previously [ ] . transposon junctions were mapped as previously described [ ] . the position of each himar transposon refers to to the distance of the insertion site, ' of the first nucleotide of each gene. transposons were mapped to the s genome, however, for continuity of nomenclature the egd-e loci names have been used. for reference: lmo (lmrg_ ), lmo (lmrg_ ), rsbx is lmo (lmrg_ ), yjbh is lmo transposons in the chromosome were introduced into different genetic backgrounds by generalized transduction using the phage u , as previously described [ , ] . briefly, a transducing lysate was generated by lysogenizing approximately cfu of l. monocytogenes transposon donor with approximately pfu of phage in - ml of . % lb agar containing mgso and cacl ( mm each) on lb agar and incubated overnight at °c. phage was soaked out of the agar by incubating with ml of tm buffer ( mm tris, ph . and mm mgso ) for - hours and these recovered phage stocks were filter sterilized. with the newly generated transducing lysate, l. monocytogenes recipients were lysogenized with pfu of lysate, incubated at °c for min in lb containing mgso and cacl ( mm each), and then plated on selective bhi agar at °c. when transducing the himar transposon using erythromycin selection, colonies appeared after two days. these colonies were purified by restreaking transductants for single colonies and verified by sequencing the transposon junction. u phage stocks were propagated using l. monocytogenes strain slcc- . knock-in plasmids were constructed as previously described using primers listed in table and reagents are from new england biolabs, unless otherwise specified [ ] . briefly, vectors for complementing yjbh and spxa were constructed by amplifying each gene along with its predicted native promoters using a reverse primer that appended a dna sequence encoding a six histidine affinity tag at the c-terminus. these dna fragments and ppl [ ] were then digested with kpni and bamhi and ligated using quick ligase, according to manufacturer's instructions. the arpj and ohra complement vectors were constructed by amplifying their entire predicted operon and predicted native promoter (arpj: lmrg_ -lmrg_ , ohra: lmrg_ -lmrg_ ) without addition of affinity tags. the dna fragment was combined with linearized ppl t harboring a transcriptional terminator [ ] and assembled using in-fusion cloning (clontech) or gibson assembly ultra (synthetic genomics). the ppl t.p hyper -acta vector was constructed by amplifying both ' utr and cds of acta (lmrg_ ), and combining the dna fragment with linearized ppl t harboring a modified pspac-hy (p hyper ) [ ] sequence: "aattgtgagcgctcacaattttgcaaaaagttgttgactttatctacaaggtgtgg cataatgtgtgtaattgtgagcgctcacaatt", inserted via gblock (idt), and a transcriptional terminator for assembly using in-fusion cloning (clontech). the pksv -orit-Δyjbh vector was constructed according to methods previously described [ ] . briefly, the vector was constructed by sequentially amplifying~ kb of homology flanking the yjbh coding region using primers in table . these two fragments were joined by sequence overlap extension pcr, which included the coding region for the first six and last six amino acids of yjbh. the final pcr fragment and pksv -orit were digested with kpni and psti (rsap was also included for the vector) and ligated using quick ligase. the ligation product was transformed into xl blue e. coli and transformants were screened by pcr for the presence of the insert, followed by sanger sequencing confirmation. the plaque assay was carried out by conventional methods [ , ] . briefly, l fibroblasts (generated previously from l cells [ ] and provided as a generous gift from susan weiss in , as detailed in sun et al. [ ] ) or tib- hepatocytes (atcc tib- ) were maintained in high-glucose dmem medium plus % fbs (hyclone), mm l-glutamine (gibco), and mm sodium pyruvate (gibco). cells were plated at . x cells per well in a six-well dish and infected the next day at an moi of with l. monocytogenes grown overnight at °c, stationary. the infection was allowed to proceed for one hour before the wells were washed twice with pbs and ml of medium plus . % agarose and μg/ml gentamicin was overlaid. at hours post-infection the plaques were stained with ml of medium plus . % agarose, μg/ ml gentamicin, and μl/ml neutral red (sigma). the plaques were then imaged at hours post-infection. plaque area was quantified using imagej software [ ] . each experiment represented an average of the area of at least five plaques per strain as a proportion to wild type plaques in that experiment. data are representative of at least three experiments. macrophage growth curves were performed as previously described [ , ] . briefly, bone marrow-derived macrophages (bmms) were derived from bone marrow of c bl/ mice purchased from the jackson laboratory and were cultivated/differentiated in high-glucose dmem medium containing csf (from mouse csf- -producing t cells), % fbs (hyclone), mm l-glutamine (gibco), mm sodium pyruvate (gibco), and mm -mercaptoethanol (bme, gibco). bmms were derived as previously described and plated in mm non-tc treated dishes that contained tc-treated coverslips at x cells per dish. these dishes were then infected at an moi of . for minutes, washed twice with pbs prior to replacing media, and gentamicin was added at μg/ml one hour post-infection. three coverslips were removed from each dish at . , , , and hours post-infection and added to ml of sterile water. coverslips were rigorously mixed prior to plating on lb agar. each graph is representative of three experiments and each data point represents the average of three coverslips. to analyze virulence, female cd- mice were infected intravenously (i.v.) via the tail vein using μl of sterile pbs containing cfu of each l. monocytogenes strain as previously described [ ] . the infection was allowed to progress for hours, at which point animals were euthanized and the spleens and livers were harvested. organs were homogenized in . % np- and serial dilutions were plated on lb agar containing streptomycin. graphs represent pooled data from at least two experiments of greater than four mice each. groups were statistically compared using a heteroscedastic student's t-test. in vivo suppressors were identified similarly to previously described methods [ ] . briefly, cd- mice were infected i.v. with x cfu of Δgshf for hours and the livers were harvested, homogenized, and μl was inoculated into broth. naïve mice were then infected with these liver homogenate cultures. after four successive infections bacteria isolated from infected livers were analyzed via plaque assay and two strains with intermediate plaque phenotype were selected for genome sequencing. genomic dna was isolated from l. monocytogenes using the masterpure gram-positive dna purification kit (epicentre) according to the manufacturer's instructions. genome sequencing and dna library preparation was performed as previously described [ ] at the vincent j. coates genomics sequencing laboratory at uc berkeley. data was assembled and aligned to the s reference genome (genbank: gca_ . ) demonstrating > x coverage. snp/indel/structural variation was determined as compared to the Δgshf parent strain using clc genomics workbench (clc bio). all immunoblotting was performed as previously described [ ] . briefly, for bacteria grown in broth, overnight cultures were diluted : into bhi, incubated for five hours at °c, shaking, then the bacteria were separated from the supernatant by centrifugation. for secreted proteins, the supernatant was treated with % v/v tca for one hour on ice to precipitate all proteins. the protein pellet was washed twice with ice-cold acetone, followed by vacuum drying. the proteins were dissolved in lds buffer (invitrogen) containing % bme using a volume that normalized for od of harvested bacteria, boiled for minutes, and separated by sds-page. for surface associated proteins, bacteria were suspended in μl of lds buffer containing % bme, boiled for minutes, and proteins separated by sds-page. immunoblots of bacteria grown intracellularly within infected bmms used -well dishes with bmms at a density of cells per well and infected with an moi of . one hour postinfection the cells were washed and media containing gentamicin ( μg/ml) was added. four hours post-infection the cells were washed twice with pbs and harvested in μl lds buffer containing % bme. the samples were then boiled and separated by sds-page. primary antibodies were each used at a dilution of : , , including: rabbit polyclonal antibody against the n-terminus of acta [ ] , rabbit polyclonal antibody against llo, and a mouse monoclonal antibody against p (adipogen). p is a constitutively expressed bacterial protein used as a loading control [ ] . all immunoblots were visualized and quantified using odyssey imager and appropriate secondary antibodies from the manufacturer according the manufacturer's instructions. transcript analysis in broth was performed as previously described [ ] . briefly, bacteria were grown overnight in bhi and subcultured : into ml bhi. bacteria were harvested at an od = . . transcript analysis during infection was analyzed as previously described [ ] . briefly, bmms were plated at a density of x cells in mm tc-treated dishes and infected with an moi of . one hour post-infection the cells were washed and media containing gentamicin ( μg/ml) was added. four hours post-infection the cells were washed with pbs and lysed in ml of . % np- . after collecting the lysate, the dishes were then washed in rnaprotect bacteria reagent (qiagen), which was combined with the lysate. bacteria were isolated by centrifugation. bacteria harvested from either broth or bmms were lysed in phenol: chloroform containing % sds by vortexing with . mm diameter silica/zirconium beads (biospec products inc.). nucleic acids were precipitated from the aqueous fraction overnight at - °c in ethanol containing mm sodium acetate (ph . ). precipitated nucleic acids were washed with ethanol and treated with turbo dnase per manufacturer's specifications (life technologies corporation). rna was again precipitated overnight and then washed in ethanol. rt-pcr was performed with iscript reverse transcriptase (bio-rad) and quantitative pcr (qpcr) of resulting cdna was performed with kapa sybr fast (kapa biosystems). primers used for qpcr are listed in table . disk diffusions were performed similarly to previously described methods [ ] . briefly, approximately x cfu from overnight cultures of bacteria were immobilized using ml of molten ( °c) top-agar ( . % nacl and . % bacto-agar) spread evenly on tryptic soy agar plates. after the agar cooled, whatman paper disks soaked in μl of % hydrogen peroxide, m diamide solution, or % cumene hydroperoxide solution were placed on top of the bacteria-agar. the zone of inhibition was measured after - hours of incubation at °c. bmms were differentiated and cultivated as described for bmm growth curves. cells were plated at x cells per well in a -well dish in media without antibiotics. the following day bmms were infected at an moi of with l. monocytogenes mutants that had been incubated at °c without shaking. after minutes cells were washed once with pbs and fresh media containing gentamicin ( μg/ml) was added. six hours post infection media was removed from each well, the cells were washed with ml of pbs, and . ml of pbs was replaced for each well. rfp fluorescence was measured using a plate reader (infinite m pro, tecan) with nm excitation, nm emission, and nm band filters. each well was interrogated times on an x grid and the edge reads were excluded. data were normalized by subtracting baseline fluorescence of wild type (without rfp) infected cells and plotting data as a percentage of wild type expressing acta p-rfp. each experiment represents three infected wells per l. monocytogenes genotype and data are representative of three pooled independent experiments. bacterial pathogen manipulation of host membrane trafficking listeria monocytogenes-from saprophyte to intracellular pathogen listeria monocytogenes: a multifaceted model listeriolysin o: a phagosome-specific lysin interaction of human arp / complex and the listeria monocytogenes acta protein in actin filament nucleation monocytogenes-induced actin assembly requires the acta gene product, a surface protein coordinate regulation of virulence genes in listeria monocytogenes requires the product of the prfa gene the prfa virulence regulon regulation of listeria virulence: prfa master and commander regulation of the prfa transcriptional activator of listeria monocytogenes: multiple promoter elements contribute to intracellular growth and cell-to-cell spread dual promoters of the listeria monocytogenes prfa transcriptional activator appear essential in vitro but are redundant in vivo pleiotropic control of listeria monocytogenes virulence factors by a gene that is autoregulated sequence variations within prfa dna binding sites and effects on listeria monocytogenes virulence gene expression differential activation of virulence gene expression by prfa, the listeria monocytogenes virulence regulator intracellular induction of listeria monocytogenes acta expression expression of listeriolysin o and acta by intracellular and extracellular listeria monocytogenes glutathione activates virulence gene expression of an intracellular pathogen a gly ser substitution in the transcriptional activator prfa causes constitutive overexpression of virulence factors in listeria monocytogenes prfa regulation offsets the cost of listeria virulence outside the host constitutive activation of prfa tilts the balance of listeria monocytogenes fitness towards life within the host versus environmental survival evidence implicating the ' untranslated region of listeria monocytogenes acta in the regulation of bacterial actin-based motility isolation of listeria monocytogenes small-plaque mutants defective for intracellular growth and cell-to-cell spread identification in listeria monocytogenes of meca, a homologue of the bacillus subtilis competence regulatory protein identification of a peptide-pheromone that enhances listeria monocytogenes escape from host cell vacuoles five listeria monocytogenes genes preferentially expressed in infected mammalian cells: plca, purh, purd, pyre and an arginine abc transporter gene intracellular gene expression profile of listeria monocytogenes the listeria transcriptional landscape from saprophytism to virulence yjbh is a novel negative effector of the disulphide stress regulator, spx, in bacillus subtilis development of a marinerbased transposon and identification of listeria monocytogenes determinants, including the peptidylprolyl isomerase prsa , that contribute to its hemolytic phenotype spx-rna polymerase interaction and global transcriptional control during oxidative stress wachenfeldt von c. the yjbh adaptor protein enhances proteolysis of the transcriptional regulator spx in staphylococcus aureus ohrr is a repressor of ohra, a key organic hydroperoxide resistance determinant in bacillus subtilis identification of novel listeria monocytogenes secreted virulence factors following mutational activation of the central virulence regulator a multidomain fusion protein in listeria monocytogenes catalyzes the two primary activities for glutathione biosynthesis allosteric mutants show that prfa activation is dispensable for vacuole escape but required for efficient spread and listeria survival in vivo prfa mediates specific binding of rna polymerase of listeria monocytogenes to prfa-dependent virulence gene promoters resulting in a transcriptionally active complex the ' untranslated region-mediated enhancement of intracellular listeriolysin o production is required for listeria monocytogenes pathogenicity in vivo effects of sporulation kinases on mutant spo a proteins in bacillus subtilis the role of the activated macrophage in clearing listeria monocytogenes infection localized reactive oxygen and nitrogen intermediates inhibit escape of listeria monocytogenes from vacuoles in activated macrophages construction, characterization, and use of two listeria monocytogenes site-specific phage integration vectors yjbh-enhanced proteolysis of spx by clpxp in bacillus subtilis is inhibited by the small protein yirb (yuzo) the yjbh protein of bacillus subtilis enhances clpxp-catalyzed proteolysis of spx a regulatory protein that interferes with activator-stimulated transcription in bacteria regulation of bacillus subtilis bacillithiol biosynthesis operons by spxa , a novel transcriptional regulator involved in x-state (competence) development in streptococcus pneumoniae the many faces of glutathione in bacteria host cytosolic glutathione sensing by a membrane histidine kinase activates the type vi secretion system in an intracellular bacterium inducible nitric oxide synthase and control of intracellular bacterial pathogens cells producing their own nemesis: understanding methylglyoxal metabolism how mitochondria produce reactive oxygen species nitric oxide increases susceptibility of toll-like receptor-activated macrophages to spreading listeria monocytogenes bacterial adaptation to oxidative stress: implications for pathogenesis and interaction with phagocytic cells life of listeria monocytogenes in the host cells' cytosol bacterial production of methylglyoxal: a survival strategy or death by misadventure? carbon-source regulation of virulence gene expression in listeria monocytogenes glucose- -phosphate utilization by listeria monocytogenes is prfa dependent and coordinately expressed with virulence factors carbon metabolism of listeria monocytogenes growing inside macrophages carbon metabolism of intracellular bacterial pathogens and possible links to virulence lethal synthesis of methylglyoxal by escherichia coli during unregulated glycerol metabolism methylglyoxal resistance in bacillus subtilis: contributions of bacillithiol-dependent and independent pathways an rna thermosensor controls expression of virulence genes in listeria monocytogenes dual roles of plca in listeria monocytogenes pathogenesis negative regulation of prfa, the key activator of listeria monocytogenes virulence gene expression, is dispensable for bacterial pathogenesis comparative transcriptomics of pathogenic and non-pathogenic listeria species adoptive transfer of immunity to listeria monocytogenes. the influence of in vitro stimulation on lymphocyte subset requirements comparison of widely used listeria monocytogenes strains egd, s, and egd-e highlights genomic variations underlying differences in pathogenicity cyclic di-amp is critical for listeria monocytogenes growth, cell wall homeostasis, and establishment of infection a broad host range mobilization system for in vivo genetic-engineeringtransposon mutagenesis in gram-negative bacteria. bio-technology the pamp c-di-amp is essential for listeria monocytogenes growth in rich but not minimal media due to a toxic increase in (p) avoidance of autophagy mediated by plca or acta is required for listeria monocytogenes growth in invasive extravillous trophoblasts restrict intracellular growth and spread of listeria monocytogenes functional impact of mutational activation on the listeria monocytogenes central virulence regulator prfa. microbiology (reading, engl) generalized transduction of serotype / and serotype b strains of listeria monocytogenes a prl mutation in secy suppresses secretion and virulence defects of listeria monocytogenes seca mutants enhanced growth of a murine coronavirus in transformed mouse cells nih image to imagej: years of image analysis role of hemolysin for the intracellular growth of listeria monocytogenes sting-dependent type i ifn production inhibits cell-mediated immunity to listeria monocytogenes constitutive activation of the prfa regulon enhances the potency of vaccines based on live-attenuated and killed but metabolically active listeria monocytogenes strains expression of the iap gene coding for protein p of listeria monocytogenes is controlled on the posttranscriptional level listeria monocytogenes is resistant to lysozyme through the regulation, not the acquisition, of cell wall-modifying enzymes mutations of the listeria monocytogenes peptidoglycan n-deacetylase and o-acetylase result in enhanced lysozyme sensitivity, bacteriolysis, and hyperinduction of innate immune pathways the authors would like to thank nancy freitag (university of illinois at chicago college of medicine), pete lauer, and bill hanson (aduro biotech) for strains and helpful discussions. the authors would also like to thank nicholas garelis and sonya john for technical assistance, gabriel mitchell for assistance with microscopy, chen chen for assistance with flow cytometry, and brittany ruhland and eric d. lee for critical reading of the manuscript. key: cord- -zn tm ww authors: sokoloski, kevin j.; nease, lauren m.; may, nicholas a.; gebhart, natasha n.; jones, claire e.; morrison, thomas e.; hardy, richard w. title: identification of interactions between sindbis virus capsid protein and cytoplasmic vrna as novel virulence determinants date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: zn tm ww alphaviruses are arthropod-borne viruses that represent a significant threat to public health at a global level. while the formation of alphaviral nucleocapsid cores, consisting of cargo nucleic acid and the viral capsid protein, is an essential molecular process of infection, the precise interactions between the two partners are ill-defined. a clip-seq approach was used to screen for candidate sites of interaction between the viral capsid protein and genomic rna of sindbis virus (sinv), a model alphavirus. the data presented in this report indicates that the sinv capsid protein binds to specific viral rna sequences in the cytoplasm of infected cells, but its interaction with genomic rna in mature extracellular viral particles is largely non-specific in terms of nucleotide sequence. mutational analyses of the cytoplasmic viral rna-capsid interaction sites revealed a functional role for capsid binding early in infection. interaction site mutants exhibited decreased viral growth kinetics; however, this defect was not a function of decreased particle production. rather mutation of the cytoplasmic capsid-rna interaction sites negatively affected the functional capacity of the incoming viral genomic rnas leading to decreased infectivity. furthermore, cytoplasmic capsid interaction site mutants are attenuated in a murine model of neurotropic alphavirus infection. collectively, the findings of this study indicate that the identified cytoplasmic interactions of the viral capsid protein and genomic rna, while not essential for particle formation, are necessary for genomic rna function early during infection. this previously unappreciated role of capsid protein during the alphaviral replication cycle also constitutes a novel virulence determinant. a a a a a alphaviruses are positive-sense rna viruses that exhibit a broad host range; and as evidenced by the emergence of chikungunya virus (chikv), represent a significant burden on the public health systems of developed and underdeveloped communities [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . alphaviral disease is broadly classified based on the symptomology associated with clinical infection. arthritogenic alphavirus infections, such as those involving chikv, ross river virus (rrv), and sindbis virus (sinv), induce debilitating arthritis in infected individuals [ ] . in contrast to the arthritogenic alphaviruses, infection with encephalitic alphaviruses may result in severe neurologic disease and significant mortality, primarily in young children [ , ] . regardless of disease symptomology the members of the genus alphavirus exhibit highly similar single-cell replication cycles [ , ] . mature infectious alphavirus particles are approximately nm in diameter, and consist of two concentric protein shells divided by a host derived lipid envelope [ , ] . the arrangement of the outer protein shell, consisting of the viral glycoproteins e and e , and the inner protein shell, comprised of capsid protein, are arranged with icosahedral symmetry [ , ] . the innermost icosahedral structure is the nucleocapsid core, which consists of the viral capsid protein and the rna cargo. however, the assembly of mature alphavirus particles is a highly selective process as, to date, many characterizations of alphaviral particles have agreed that the viral genomic rna is the predominant rna molecule within the nucleocapsid core [ ] [ ] [ ] [ ] [ ] . several studies have identified a region of the genomic rna associated with the selective packaging of the viral genome during the assembly of infectious particles. this element, termed the packaging signal, consists of a highly-structured region found within the open reading frame of the nonstructural polyprotein [ ] [ ] [ ] [ ] [ ] [ ] . while these studies provided an excellent definition of the cis-acting elements involved in the selection of the cargo rna, they did not identify nor describe the interaction between the capsid protein and the viral rna cargo [ ] . the interactions between the alphaviral capsid protein and the viral genomic rna is an under characterized, yet vitally important, rna:protein interaction. alphaviral in vitro assembly systems have largely indicated that the assembly of capsid:nucleic acid complexes is a nonspecific process [ ] [ ] [ ] [ ] [ ] . however it should be noted that the direct interaction between the viral capsid protein and viral rna has not been exhaustively characterized in the cytoplasm of infected cells, or in mature viral particles leaving our understanding of the molecular interactions between these essential components of the virus incomplete. this report details our efforts to identify and characterize the sites of interaction between the viral capsid protein and the genomic rna using the model alphavirus sindbis virus (sinv). a clip-seq approach was used to screen for potential sites of capsid:rna (c:r) interaction within sindbis virus (sinv) particles and cytoplasmic nucleocapsid complexes. interestingly, while the c:r interactions of purified viral particles are relatively evenly distributed across the genome, the c:r interactions in cytoplasmic rna:protein complexes exhibit a discrete binding profile. further characterization of these candidate c:r interaction sites showed a decrease in capsid interaction when the sites were mutated, and indicated that the sites are essential for efficient viral growth as mutation of individual c:r interaction sites significantly reduces the infectivity of the mature viral particles. further analysis indicated that the c:r interactions are necessary at an early stage of virus infection and stabilize incoming viral genomic rna. moreover, the cytoplasmic c:r interaction sites represent a series of novel pathogenicity determinants as c:r interaction site mutants are significantly attenuated in a neurotropic mouse model of infection. collectively, the results presented indicate a new role for capsid protein during the viral replication cycle and identify a novel determinant of viral pathogenesis expanding our understanding of how capsid:rna interactions regulate viral infection beyond particle assembly. to identify candidate site(s) of interaction between the viral genomic rna and the capsid protein we utilized a clip-seq method to develop cdna libraries of c:r interactions [ ] [ ] [ ] . briefly, either purified viral particles or infected tissue culture cells were irradiated with shortwavelength uv radiation to form covalently cross-linked rna:protein complexes. the rna components of cross-linked complexes were then fragmented via rnase digestion. the fragmented c:r complexes were then selectively immunoprecipitated from the lysates using anticapsid polyclonal antibodies (supporting information, s fig) . from the immunoprecipitated materials, cdna libraries were generated using an adaptor ligation process and deep sequenced. in addition to anti-capsid clip-seq libraries, derived from purified virions and infected cells, nonspecific control libraries were also developed. for all cdna libraries the sequencing data were aligned and clustered to a reference genome ( fig a, and supporting information s data), consisting of the input sinv toto parental sequence. the c:r interactions in mature virions are dispersed and nonspecific. clip-seq analysis of purified sinv particles indicates that the contacts between the viral capsid proteins and the genomic rnas are extensive, and likely nonspecific in nature. as shown in fig b, the interactions between the encapsidated genomic rna cargo and the viral capsid protein in purified viral particles are widely dispersed across the genome. nonetheless, two regions of the genome exhibited decreased c:r interaction relative to the entirety of the genomic rna. interestingly, one of these sites corresponds to the previously identified packaging signal [ ] . the second region of low coverage corresponds to the subgenomic promoter and the nucleotides corresponding to the 'utr of the subgenomic rna [ , ] . the c:r interactions of cytoplasmic nucleocapsid complexes exhibit specificity. in contrast to the sequence coverage observed with purified virions, c:r complexes derived from infected cell lysates exhibit remarkable specificity. co-precipitation of viral rna and capsid protein was dependent on crosslinking and the use of antibodies specific for capsid (s fig) . subtractive analysis of nonspecific control and anti-capsid cdna library sequencing data revealed several discrete regions of significant enrichment in the anti-capsid library (fig c) . the most highly enriched regions correspond to the coding sequence of the structural orf, in particular the e and e genes. nonetheless, several minor peaks are found in other regions of the genomic rna, including the ' and ' utrs and the nonstructural and structural coding regions. interestingly, these peaks do not correlate with the previously described packaging signal or other known cis-acting features of the viral rna [ , ] . to prioritize which peaks would be further characterized we utilized a statistical method to examine the relative enrichment of individual sequences against the entire subtractive data set. as depicted in fig d, statistical analysis of these data indicated that several of the regions with substantial depth-ofcoverage were statistically enriched (as determined by z-score) relative to other sequence clusters. collectively, these data indicate that the intracellular c:r interactions occur at discrete interaction sites on the viral genome and that the c:r interaction sites found within the structural orf are the most significantly enriched. for reference the graphs in panels b through d are aligned with this schematic. b) coverage of anti-capsid clip-seq libraries derived from purified, mature infectious viral particles. depth of coverage is represented by the y-axis, with the xaxis representing nucleotide position. c) depth of coverage for anti-capsid libraries derived from cytoplasmic fractions. plotted on the y-axis is the depth of coverage of the anti-capsid libraries following subtractive analysis, with the x-axis representing nucleotide position. d) statistical analysis of the data presented in panel c, with the y-axis indicating the zscore of all represented sequences. the x-axis represents the nucleotide position, with all panels above being aligned with the indicated nucleotide numbering intervals. as described earlier, several of the intracellular c:r interaction sites were statistically enriched relative to others. these sites, henceforth referred to as nt , nt , and nt based on their approximate nucleotide locations in the viral genome, were prioritized for further characterization. initially, we sought to determine if the intracellular c:r interaction sites exhibited similar rna secondary structures, or primary sequence motifs. bioinformatic analysis of the nt , nt , and the nt regions (including up to flanking nucleotides) using mfold failed to identify any rna secondary structures that were energetically favorable [ ] . similarly, analysis of the nt , nt , and nt c:r interaction sites did not reveal primary sequence motifs, and did not overlap with known subgenomic promoter elements [ ] . nonetheless, bioinformatic analysis of the individual intracellular c:r interaction sites indicates that the regions identified as highly enriched c:r interaction sites are well conserved across sinv strains. as shown in fig a, the prevalence of single nucleotide polymorphisms, as determined by the sequence alignment of independent sinv strains (supporting information s table) , indicated that the regions of interest exhibited few snps. numerous residues within the c:r interaction sites were absolutely conserved; furthermore, the overall incidence of snps was on average comparable to or below neighboring sequences that were not identified as c:r interaction sites. mutation of the candidate interaction sites reduced capsid binding confirming the relevance of the rna sequence for the interaction with capsid. analysis of the individual c:r interaction site mutants by quantitative immunoprecipitation following cross-linking indicated that mutation of each of the candidate c:r interaction sites reduced capsid binding. as shown in fig c, the retention of the individual c:r interaction sites following capsid immunoprecipitation was significantly reduced relative to parental virus for each of the c:r interaction sites. these data support the identification of the c:r interaction sites as bona fide capsid: rna interaction sites and indicate that the mutational approach is capable of reducing capsid interaction at these specific sites. following the identification of the cytoplasmic sinv c:r interaction sites we next sought to determine their biological importance to viral infection. to this end we developed a series of c:r interaction site mutants. due to the c:r interaction sites being present in open reading frames it was necessary to maintain amino acid identity during mutation. therefore, mutations were limited to substitutions of the wobble-base position of each codon ( fig b) . importantly, the mutants were designed to maintain codon usage rarity to prevent changes in translational efficiency. in some instances no alternative codons were available (such as for methionine), and as a result the corresponding codons were not altered. the one-step growth kinetics of the c:r interaction site mutants were characterized in mammalian tissue culture cells. as shown in fig a, mutation of each of the individual sinv c:r interaction sites significantly reduced the number of infectious particles produced during infection. on average, a reduction of~ -fold in the yields of infectious virus at hpi was detected for the c:r nt , nt , and nt , compared with wildtype sinv. since a potential consequence of disrupting the interactions between the viral capsid protein and the genomic rna could be a reduced production of viral particles, we next sought to determine if particle production was negatively affected in c:r interaction mutant viruses. interestingly, as shown in fig b, the total number of viral particles produced (as measured by genome equivalents per unit volume) by both wildtype sinv and c:r interaction site mutants were numerically, and statistically, equivalent. therefore, the production of viral particles was not perturbed by mutation of the c:r interaction site mutations. however, the capacity of the fig . the right y-axis indicates the prevalence of single nucleotide polymorphisms (snps) across a curated set of sinv genomic rnas; higher values indicate increased base identity variation at a particular nucleotide but are not informative as to relative base conservation. nucleotide position is reported on the x-axis. b) the individual nucleotide sequences of the c:r interaction sites are described in regards to parental sequence (top) and mutant sequence (bottom). the mutated nucleotides are highlighted in red. c) quantitative analysis of the individual c:r interaction sites following mutation indicates that capsid:rna binding is significantly reduced relative to parental virus. data shown is the mean of three independent biological replicates, with the error bar representing the standard deviation of the mean. to extend our understanding of the molecular impact of c:r interaction site mutation we assayed viral rna synthesis and viral gene expression. as demonstrated in fig a mutation of the c:r interaction sites did not significantly alter either the accumulation, or relative ratios of the individual sinv rna species. for each of the c:r interaction site mutants, the accumulation of the minus-strand rna was slightly increased relative to wildtype levels. while this was consistent over multiple biological replicates, the observed differences in minus-strand accumulation are relatively minor. analysis of viral gene expression by metabolic labeling of hek cells at and hours post infection indicates that viral gene expression is similarly unperturbed by c:r interaction site mutation ( fig b) . additionally, host cell shutoff, as indicated by the relative intensity of the actin band, is equivalent at and hpi for the c:r interaction site mutants and wildtype sinv at equal moi in terms of infectious units per cell. the data reported above indicates that the c:r interaction sites identified by clip-seq analysis of cytoplasmic complexes are not directly involved with nucleocapsid assembly and particle release. moreover, the lack of an apparent defect in viral rna synthesis or structural gene expression indicates that the sinv gene products are functioning normally, and that the c:r interaction site mutants are, late during infection, equivalent to wildtype virus. however the infectivity of sinv c:r interaction site mutants is significantly diminished relative to parental virus. hence, these data suggest that mutation of the c:r interaction sites negatively affects an early event of the viral lifecycle prior to viral rna synthesis. the data acquired indicted that while the c:r mutants did not inhibit particle production the infectivity of the particles was decreased. however, once infection was established the synthesis of viral rnas and viral gene expression was unaffected. these data implied that the c:r mutations disrupted particle infectivity at an early stage of infection and that the cytoplasmic c:r interactions were less essential at later stages of infection. previously, we demonstrated that alphaviral infectivity is largely determined by the functional capacity and stability of the genomic rna immediately following viral entry [ ] . on this basis we determined the genomic rna half-lives for each of the individual c:r interaction site mutants at very early times postinfection during a synchronized infection of hek cells. to this end, hek cells cultured in the presence of the uridine analogue -thiouridine ( su) were infected at an moi of infectious units per cell at ˚c to allow for viral adsorption but not entry. after the initial adsorption period, the cell monolayers were extensively washed to remove unbound particles and pre-warmed media containing su was added to release the block to viral entry. at the indicated times post-infection the total cellular rna was harvested and the incoming genomic rnas purified from the nascent transcribed rnas. the viral genomic rnas were then quantified using qrt-pcr as described in the materials and methods. as shown in fig a, the stability of the c:r interaction site mutant viral rnas was decreased in hek cells. b) quantitative analysis of the sinv c:r interaction site mutants and parental wild type virus in regards to the number of infectious units (left y-axis), and the total number of viral particles produced as measured by qrt-pcr (right y-axis). c) the infectivity of the individual sinv c:r interaction site mutants and parental wild type virus as reported as the ratio of total particles per infectious unit as determined using bhk- cells. all quantitative data in this figure represents the mean of three independent biological replicates, the error bar representing the standard deviation of the mean. statistical significance, as indicated on the individual panels above, are the p-values obtained from student's t-test. compared with the stability of wildtype sinv rna. importantly, the rna half-life observed during these studies is very similar to that previously reported [ ] . however, and interestingly, while wildtype sinv exhibits a steady monophasic decay profile, the individual c:r interaction site mutants exhibit multi-phasic decay, with an initial period of notable instability followed by a prolonged period of stability. calculation of the individual half-lives for each of the viruses used in this study indicates that mutation of c:r interaction sites destabilizes the incoming viral genomic rnas significantly, decreasing the mean half-life by -fold on average ( fig b) . collectively, these data indicate that mutation of the individual c:r interaction sites results in destabilization of the incoming genomic rnas early during infection. since efficient function of the incoming viral genomic rna is essential to the establishment of viral infection, it is, a priori, understandable that failure of the genomic rna early in infection, such as rna instability, would result in reduced viral infectivity. aside from persisting in the host cytoplasm, another important molecular function of the incoming genomic rna is to act as an mrna for the synthesis of the viral replication machinery. moreover, since the translational capacity of a transcript often correlates with the relative stability of an rna it is likely that the earliest translation events of c:r interaction site mutants are also perturbed [ ] . as such, we next sought to determine if the translational activity of the incoming viral genomic rnas differed amongst wild type and c:r interaction site mutant viruses. to this end we utilized a reporter sinv strain that expresses nanoluciferase internal to the nsp protein. this construct enables highly sensitive detection of gene expression early during infection, similar to previously described. to assess the translational capacity of the c:r interaction site mutants we developed a series of individual c:r interaction site mutants in the nsp nanoluciferase reporter backbone. unfortunately, despite many attempts we were unable to recover sinv nsp .nanoluc nt mutant virus as the resulting mutant was so severely attenuated. the translational activity of the parental wild type sinv nsp nanoluciferase construct and the nt and nt c:r interaction site mutants was assessed in hek cells. briefly, hek cells were infected at an moi of infectious units per cell. after the removal of unbound viral particles the cells were harvested at min, hr, and hrs post infection and assayed for nanoluciferase activity. as shown in fig c , wild type parental virus exhibited steady expression during early infection. however, both nt and nt exhibited significantly reduced translational activity early during infection. indeed, at -minutes post infection the normalized nanoluciferase activity was reduced . -fold and . -fold for the nt and nt mutants, respectively. at -hour post-infection the nanoluciferase activity of the nt mutant was further reduced, approximately -fold relative to wild type levels. however, at the same time point, the nt mutant exhibited an increase in nanoluciferase activity to more or less wild type levels, indicating that the defect imparted by the nt mutant is short lived. moreover, the nanoluciferase expression levels at two-hours post-infection for the nt mutant are~ -fold less than that of wild type sinv; however, the nt mutant has surpassed that observed for wild type sinv by approximately . -fold. to ensure that the mutations had not compromised the inherent translatability of the genomic rnas we checked translation using an in vitro system (supporting information, s fig). no differences in translation from the mutant and wt genomic rnas were observed in vitro. interaction site mutants and parental wild type virus were determined by qrt-pcr analysis as described in the materials and methods. plotted is the relative abundance of the incoming viral genomic rnas (y-axis) with regards to time (x-axis). regression analysis was utilized to determine the rna decay profile (as shown with the solid line) and the dashed lines represent the % confidence intervals of the aforementioned regression. b) the half-lives of the individual genomic rnas as determined using the calculations reported in dolken et al., as determined by the first point at which the relative abundance has reached . . c) the levels of nanoluciferase activity for wild type parental virus and the nt and nt c:r interaction site mutants were determined as reported in the materials and methods at the indicated times post infection. all quantitative data in this figure represents the mean of at least three independent biological replicates. comparative analysis was performed using variable bootstrapping, as described in the materials and methods, with the error bar representing the standard deviation of the mean. statistical significance, as indicated on the individual panels above, are the p-values obtained from student's t-test. https://doi.org/ . /journal.ppat. .g together, these data support the conclusion that the c:r interaction sites are important to early genomic rna function during viral infection. indeed, mutation of the individual c:r interaction sites reduces the rna stability of the incoming genomic rna; and, at least for nt and nt , reduces or delays the translational activity early during infection. however, the deficit created by the mutation of the c:r interaction sites appears to be specific to the early stages of the viral molecular lifecycle. this notion is supported by the observation that, at late stages of infection, the steady state levels of the three viral rna species are unperturbed, and the viral gene expression profiles are more or less equivalent. therefore, we posit that the c:r interactions are critical for molecular events at the earliest stages of viral infection, and that the c:r interaction sites represent a means by which the incoming viral genomic rna is stabilized, and perhaps licensed for translation. once this initial critical event is surpassed, the biological role of the c:r interaction sites become less essential for reasons currently unclear. regardless, diminished viral genomic rna function early during the viral lifecycle appears to significantly impact the progression of infection [ , ] . previously, we demonstrated that the rapid rna decay of alphaviral genomic rnas correlated with an increased elicitation of a type-i ifn response [ ] . to determine if the sinv c:r interaction site mutants induced a more robust ifn response we quantified the soluble type-i ifn produced during infection using a tissue culture model [ , , ] . as reported in fig a , the individual c:r interaction site mutants produced on average -fold more soluble type-i ifn relative to wildtype sinv infection. the induction of a robust ifn response is undoubtedly a significant consequence of the mutation of the cytoplasmic c:r interaction sites. indeed, the instability of the viral rna and apparent reduction of translational capacity early during infection likely contributes to the inability of the virus to successfully limit the induction of a soluble type-i ifn response. together, these findings indicate that the c:r interaction site mutants are liable to be highly restricted in ifn competent systems [ , [ ] [ ] [ ] [ ] [ ] [ ] . interactions between sindbis virus capsid protein and cytoplasmic vrna neurotropic sindbis virus is significantly attenuated in a mouse model the ifn findings described above suggested that c:r interaction site mutants would be attenuated, at least in regards to replication, in ifn competent models of infection, including immunocompetent wt mice. to test this hypothesis, we characterized the sinv c:r interaction site mutant viruses in a mouse model of infection. as shown in fig , wt c bl/ mice infected interactions between sindbis virus capsid protein and cytoplasmic vrna with parental wild type ar sinv, which uniquely among sinv strains remains neurovirulent in adult wt mice [ ] , displayed significant mortality and weight loss, with a median time to death of~ . days post infection (fig a and b ). in contrast, the nt c:r interaction site mutant virus was significantly attenuated relative to wild type infected mice, with only a fraction of the animals infected with nt mutant virus succumbing to disease. analysis of sinv.nt infected mice indicated that the disease associated with infection was mild compared with wild type ar -infected mice as indicated by the timing and severity of weight loss. similarly, but to a much greater extent, the sinv.nt mutant virus was also attenuated in wild type mice as none of the sinv.nt infected animals succumbed to infection, or demonstrated overt signs of disease as indicated by the absence of weight loss. it is important to note that these animals were productively infected with sinv, as infectious units were recovered from central nervous system tissue. as shown in fig c, the viral load detected in the brains of sinv.nt infected mice was decreased greater than -fold relative to wild type ar infected mice. in contrast to sinv.nt and sinv.nt , mice experimentally infected with sinv. nt exhibited mortality comparable to wild type ar -infected animals; however, disease progression was delayed with a median time to death of~ days post infection ( fig a) . indeed, as indicated by animal weight loss and health monitoring, the disease progression of sinv.nt was distinct from that observed in mice infected with wild type ar as sinv. nt infected animals exhibited delayed weight loss and experienced severe rapid onset paralysis at day , requiring the animals to be humanely euthanized (fig b) . collectively, these data suggest that the sinv c:r interaction sites identified via clip-seq are biologically important to infection in tissue culture models of infection and in vivo. given the behavior of the sinv c:r interaction site mutants in tissue culture cells, diminished viral replication in an in vivo model was anticipated. the obvious difference in disease phenotype for the and c:r mutants versus the c:r mutant indicates there is an unappreciated degree of complexity in the regulation of viral processes associated with the c:r interaction. the alphaviral capsid protein is a~ kda protein which surrounds the viral genomic rna in viral particles and in the cytoplasm in the form of nucleocytoplasmic cores. architecturally, the alphaviral capsid protein is globular in nature with an n-terminal domain that is implicated in rna binding and dimerization [ , ] . the alphavirus capsid protein is expressed as part of the structural polyprotein, which due to the serine-like protease activity of the c-terminal domain of the capsid protein, is autoproteolytically processed into free capsid protein [ ] . in addition to its well-known roles in particle assembly, the alphavirus capsid of new world alphaviruses, in particular venezuelan equine encephalitis virus (vee), is also directly involved in the shutoff of host macromolecular synthesis via the interruption of nuclear export [ , ] . nonetheless, a larger role for the alphavirus capsid protein in the regulation of alphaviral infection has not been previously described; and, hence, the observations described in this report represent a novel contribution of the c:r interaction during alphaviral infection and pathogenesis. positive-sense rna virus capsid proteins: more than just packaging in many positive-sense rna viruses the viral capsid proteins serve additional roles for the viral capsid protein / rna interactions outside of the context of particle assembly. these functions include the regulation of viral translation and rna synthesis. for instance, the ms coat protein dimer binds to the viral genomic rna of the ms bacteriophage to regulate the expression of the viral rna-dependent rna polymerase [ ] [ ] [ ] . similarly, the core protein of hepatitis c virus (hcv) binds to the ' ires during infection where it modulates the level of translation in a seemingly concentration dependent context [ ] [ ] [ ] [ ] [ ] . furthermore, a similar mechanism of translational regulation has been reported for other members of the alphaviruslike superfamily, in particular members of the bromoviridae, including brome mosaic virus (bmv) [ , ] . however, unlike these previous reports where the capsid protein expressed during the course of infection serves to regulate viral gene expression, the observations reported here indicate that the incoming capsid protein's association with the sinv genomic rna is necessary for viral rna translation early during infection. therefore, this phenomenon appears to be more similar to that previously described for alfalfa mosaic virus (amv), where the association of the amv capsid protein with distinct elements of the 'utr is required for rna function [ ] [ ] [ ] . nonetheless, in contrast to amv, the regulatory binding sites for sinv are found within the structural coding region of the viral rnas. it is worth mentioning that this orf, in the context of the genomic rna, is indeed part of a large non-translated region. examples of capsid protein / rna interactions that regulate rna synthesis can readily be found in bmv, amv, and with members of the coronaviridae. binding of the capsid proteins of amv and bmv to rna regulatory elements have been implicated in the regulation of viral rna synthesis and promoter recognition [ , ] . additionally, members of the coronaviridae require functional n protein, likely via the association of the highly charged n-terminal domain with the transcription-regulating sequences, to achieve efficient viral replication [ ] [ ] [ ] [ ] . nonetheless, from the data presented here we are unable to assign a role for an alphaviral c:r interaction in regards to viral rna synthesis. for, as reported above, the accumulations of the viral rna species is similar between the individual c:r interaction mutants and wild type parental virus. this implies that rna synthesis / promoter utilization is not negatively affected. however, it remains possible that degeneracy between the individual c:r interaction sites is able to compensate for this activity. unfortunately, the development of combined c:r mutants has, so far, been unsuccessful. several studies have described interactions between the alphaviral capsid protein and host ribosomal rnas. indeed, co-sedimentation studies indicated that the host s rrnas interact with viral capsid proteins in vertebrate cells during infection [ , ] . this interaction has been proposed to be a leading mechanism in particle disassembly [ ] . nonetheless, these interactions have not been exhaustively characterized and further examination is needed to complete the mechanistic understanding of alphaviral nucleocapsid disassembly. collectively, the observations reported here are indicative of a novel role for the c:r interactions in regard to the function of the sinv genomic rna early during viral infection. as demonstrated by the data in this report, mutation of the individual candidate c:r interaction sites identified via clip-seq screen significantly reduced the function of the genomic rna early during infection. a key novel observation of these studies is that the mutation of the c:r interaction sites reduced the rna stability of the incoming viral genomic rna. a role for a viral capsid protein in the stabilization of viral rnas, to our knowledge, has not yet been described for positive sense rna viruses. the data above leads to the conclusion that the interaction sites of the sinv capsid protein with the viral genomic rna identified in this study by clip-seq, while not required for rna function late during infection, are vital to early genomic rna stability, and function. moreover, mutation of the c:r interaction site diminished translation of the viral genomic rna early during infection, likely resulting in increased type-i ifn production. currently the precise mechanism of c:r-mediated regulation is unclear; however, the c:r interaction sites are clearly involved in the stabilization of the incoming viral genomic rna and the regulation of early viral translation. we suggest a model in which capsid protein of incoming nucleocapsid complexes remains associated or re-associates with specific sites in the genome following nucleocapsid disassembly enhancing rna stability and facilitating translation (fig ) . this is a working model, whether the effects on rna function are due only to capsid binding, or could be affected by the binding of other rna binding proteins at proximal or overlapping sites is unknown. ongoing studies in the sokoloski and hardy laboratories have indicated that the c: r interaction sites may be common sites of virus and host protein binding. the precise role(s) and contributions of these interactions are currently being characterized. further potential mechanisms include direct or indirect roles in the recruitment of the translational machinery, or beneficial host rna-binding proteins to the viral rna; or perhaps the evasion of toll-like receptors and rna-helicase sensors such as rig-i and mda , or the eluding of the host cellular rna surveillance machinery such as the host nonsense-mediated rna decay pathway. while our data cannot completely rule out this possibility, we do not believe that the decreased rna stability observed in the c:r interaction site mutants is due to nonspecific recruitment of cellular endonucleases (for instance rnase l), as mutation of off-target sites does not result in an altered phenotype ( supplementary information, s fig) . whereas, the mutation of the c:r interaction sites reduced viral growth kinetics, likely as a result of decreased infectivity due to impaired genomic rna function early during infection. in addition to their molecular role(s), the c:r interaction sites represent a set of previously unidentified pathogenicity determinants in that mutation of the c:r interaction sites significantly attenuates viral infection in vitro and in vivo. as shown in this study mutation of the sinv c:r nt and nt interaction sites reduced the viral load, pathology, and morbidity of a neurotropic sinv infection. these findings have great potential as a means by which viruses may be attenuated for the purpose of rational vaccine development. given that c:r interaction site mutation limits viral rna function early during infection, but not late during infection, implies that viral dissemination but not immunogenicity would be limited. however, further exploration is necessary, as not all c: r interaction sites resulted in complete attenuation; as the sinv.nt mutant exhibited significant mortality and morbidity in experimentally infected mice. currently, the precise mechanism behind this phenotype is unclear. one possibility being investigated is that this site is also bound by a host factor and this contributes to the phenotype observed with this particular mutant. it should be noted that while the c:r interaction at obviously is important in maintaining genome stability following infection the site may also play a role in the function of the viral subgenomic mrna that this may be affected by the binding of a host factor. several studies have indicated that the association of the alphaviral capsid protein with viral rna is nonspecific in regards to primary nucleotide sequence and secondary structure, and is likely driven by electrostatic interactions between the n-terminus of capsid and the phosphodiester rna backbone [ ] [ ] [ ] [ ] [ ] . while these studies primarily focused on the interactions of nucleic acids and the viral capsid proteins in vitro, it is highly likely that these observations are valid during bona fide infections as evidenced by the distributive nature of the capsid:rna interactions observed in particles. however, collectively the data presented in this report indicate that the interactions, or at least the strength thereof, between the sinv capsid protein and the genomic rna may be context dependent. the disseminated pattern of binding of the viral genomic rna to capsid observed in purified viral particles supports a model where the c:r interactions are largely nonspecific, and perhaps based on charge-charge interactions [ , , ] . however, from this data set it is impossible to identify if the interactions between the capsid protein and cargo rna are mutually saturating (where each capsid monomer interacts with~ nt of genomic rna), or unsaturated (where not every nt is associated with capsid monomer) but nonspecifically distributed along the length of the genome. further examination of the pattern of binding observed in purified viral particles reveals an interesting phenomenon-there are two regions of the sinv genome where sequence coverage is greatly decreased. these regions roughly correspond to the previously described packaging signal, and the subgenomic promoter region [ , , ] . importantly, the decreased coverage of these regions cannot be simply explained by bias in the library generation (as is the case for the extreme ' and ' termini of the genome). in these instances, the decrease in coverage was due to genomic rna having a ' cap and ' poly(a) tail, which effectively prevented the linkage of the ' and ' adaptors as these rna features were not modified prior to library construction. nonetheless, decreased sequence coverage at the packaging signal could be potentially explained due to the presence of a high degree of secondary structure within the element. the nuclease fragmentation approach utilized in these studies is selective for single-stranded rna, therefore regions with a high degree of dsrna may be excluded from nuclease digestion. the net result would be the formation of rna fragments that lay outside the region of interest / selection during the preparation of the cdna libraries. alternatively, if the capsid interaction is exceptionally avid at this location the fragmentation of the viral rna may be similarly diminished, resulting in decreased library coverage at the packaging signal. further analysis of the decreased coverage at the cis-acting sites is separate and ongoing research focus. examination of the interactions between the capsid protein and the viral genomic rna in a cytoplasmic context indicated that several discrete interaction sites were readily observable. however, from our data we cannot conclude as to the molecular context of these interactions, as the milieu of capsid interactions likely expands from monomers (or as some models suggest dimers) to complete intact nucleocytoplasmic cores. from our current data it is impossible to determine the molecular nature of the interaction in regards to protein:rna stoichiometry and molecular context. unfortunately, given the promiscuous interaction of alphaviral capsid proteins with nucleic acids (including dna) deciphering the supramolecular nature of the interaction through a reductionist in vitro approach presents significant challenges [ , , ] . moreover, it is unclear if these interactions are exclusive of other c:r binding events, or simply the cytoplasmic c:r interactions with high relative affinities. it should be noted that the method of cross-linking used in these studies is inefficient, which increases the specificity but reduces overall signal intensity during the clip-seq analyses; it is therefore likely that the observed cytoplasmic c:r interaction sites are exceptionally stable relative to other c:r interactions. tissue culture cells bhk- (gift from charles rice, rockefeller university), hek , and l cells (both cell lines were gifts from pranav danthi, indiana university) were cultured in minimal essential media (mem, cellgro) supplemented with % fetal bovine serum (fbs, atlanta biologicals), x antibiotic / antimycotic solution (cellgro), x nonessential amino acids (cellgro) and l-glutamine (cellgro). all cell lines utilized in this study were cultured at ˚in the presence of % co . wild type sinv toto , sinv p (a toto derived strain containing gfp in frame with nsp ), sinv ptoto -naluc (a toto derived strain containing nanoluciferase in frame with nsp ), wild type sinv ar , and all derivatives thereof (described below), were prepared by electroporation as previously described [ ] . briefly, ug of in vitro transcribed rna were electroporated into bhk- cells via a single pulse from a gene pulser xcell system (bio-rad) at . kv, ma and ohms. after the development of cytopathic effect (typically to -hours post-electroporation) the tissue culture supernatants were collected and clarified via centrifugation at , xg for minutes at ˚c. the resulting stocks were aliquoted and either used immediately or stored at - ˚c for later use. sinv particles were purified via a two-step process [ ] . first, supernatants from x hek cells infected with sinv toto at an moi of pfu/cell were harvested at -hours post infection. the viral particles were concentrated via pelleting through a % (m/ v) sucrose cushion prepared in hne buffer ( mm hepes [ph . ] / mm nacl / mm edta) by centrifugation at , xg for . hours in a ti rotor. second, the pelleted viral particles were then resuspended in hne buffer and applied to a % / % (m/v) sucrose step gradient prepared in hne. the sinv particles were then banded via centrifugation at , xg for . hours in an sw rotor at ˚c. the purified sinv particles were collected via needle aspiration, aliquoted and stored at - ˚c for later use. per clip-seq library, either purified sinv particles or infected tissue culture cells were crosslinked via exposure to shortwave ( nm) uv irradiation. briefly, approximately sinv toto particles in a volume of μl, or x hek cells infected with sinv toto at an moi of pfu/cell at hpi, were irradiated with x μjoules per square centimeter in a stratalinker. after cross-linking the rna-protein complexes were solubilized in ripa buffer ( mm tris [ph . ] / mm nacl / . % np- / . % sodium deoxycholate / . % sds) and frozen at - ˚c for later use. prior to immunoprecipitation the lysates were thawed on ice, vortexed and clarified via centrifugation at , xg for minutes at ˚c. the clarified supernatants (~ μl) were transferred to a fresh tube. the cross-linked lysates were then incubated with ul packed volume of protein g sepharose beads for minutes at ˚c prior to being clarified via centrifugation at , xg for five minutes. the resulting pre-blocked lysates were then immunoprecipitated using a polyclonal anti-capsid antibody, or control rabbit igg sera. the cross-linked rna:protein complexes were incubated in the presence of antibody at ˚c for a period of hours under constant agitation. after antibody binding, ul packed volume of protein g sepharose was added to each sample and further incubated for another hours. after resin binding the rnas were fragmented via the addition of rnase t (thermoscientific) to each immunoprecipitation. the fragmentation reactions were allowed to incubate for minutes at room temperature. after fragmentation, the immunoprecipitated complexes were purified via centrifugation at , xg for minutes and washed three times with ripa buffer and twice more with sterile xpbs. the resulting purified, fragmented rna: protein complexes were treated with proteinase k for minutes at ˚c to release the rna fragments from the immunoprecipitated complexes. the purified rna fragments were then extracted using trizol and resuspended in a minimal volume. the rna fragments were then used as the input materials for cdna library generation via the nebnext sequencing kit, as according to the manufacturer's instructions. the resulting cdna libraries were sequenced using the miseq platform. the specificity of the immunoprecipitation protocol described above was confirmed independently using small scale analytical replicates and metabolic labeling. hek cells were either mock treated, or infected with sinv at an moi of pfu/cell, and allowed to incubate for hours under normal conditions. thirty minutes prior to the labeling period the infected monolayers were washed twice with xpbs and the media was replaced with methionine / cysteine free dmem supplemented with % dialyzed fbs. after the depletion period the media was removed and replaced with methionine/cysteine free dmem supplemented with l-azido-homoalanine (l-aha) at a concentration of um. the cells were allowed to incubate under normal conditions for a period of two hours prior to removal of the culture media. prior to harvesting the monolayers were washed three times with xpbs. whole cell lysates were generated via the addition of ripa buffer. protein concentration was quantified via bradford assay and equivalent amounts of protein were precipitated via methanol / chloroform treatment. the precipitated proteins were resuspended in xpbs containing um dibo-alexafluor and incubated for hour at room temperature while shielded from light. after the labeling period, the samples were diluted in x volumes of ripa buffer and immunoprecipitated identically to that described for the clip-seq experiments. these treatments only differed on the basis of scale-as the total materials used were reduced -fold relative to those used for library generation. after immunoprecipitation x laemmli buffer was added to a final concentration of x and the samples analyzed via sds-page. protein species were detected via a pharos molecular imager using the appropriate excitation and emission detection settings. as shown in supporting information (s fig) , the anti-capsid sera specifically immunoprecipitated the sinv capsid protein from infected extracts. in addition, no other host proteins were enriched during immunoprecipitation with anti-capsid sera in either infected or mock infected lysates. furthermore, the nonspecific antibody control did not enrich for any host, or viral, protein. collectively these data are indicative of the specificity and purity of the immunoprecipitations used for cdna library generation. the cdna libraries were trimmed to remove indices and adaptors prior to alignment to a reference genome consisting of the parental toto strain of sinv. alignments were performed using lastz with standard parameters. only reads corresponding to the positive-sense genomic rna were mapped to the reference genome sequence. sequence coverage was clustered at the nucleotide level of resolution from the aligned sequence reads. analysis of the clustered sequence data relied on a subtractive method whereby the sequence coverage of capsidspecific libraries was compared to nonspecific control libraries. for these analyses the coverage of both the capsid and nonspecific control libraries were normalized internally by dividing each base by the total number of represented nucleotides to gain a percentage which was then multiplied by an arbitrary amount to generate a standardized measure of each nucleotides relative representation amongst independent library sets. the resulting values from the control libraries were averaged and subtracted from the capsid-specific libraries to generate a difference map (as shown in fig c) . z-scores for each individual nucleotide were calculated from the statistical mean and standard deviation from the subtractive analysis data sets. for these studies statistical significance was rigorously defined as nucleotide clusters with differential coverage values greater than standard deviations from the mean, which correlates to a zscore of~ − . the sequencing data directly relevant to the studies described herein are available accompanying this document as s data. this supplemental information also includes a statistical summary of the entire rna sequencing dataset and analysis of read size for the regions of interest and a control region. the entire rna sequencing dataset has been deposited in the national center for biotechnology information (ncbi) gene expression omnibus, and can be accessed using the following url: https://urldefense.proofpoint.com/v /url?u= http- a__www.ncbi.nlm.nih.gov_geo_query_acc.cgi- facc- dgse &d=awieag&c= sgmrq dbjbgx e zsshgezx a iaf so aj bnrhlk&r=oeul-bhg_ jju s ftlcvxycn jpesq pjohuawmnty&m=s zyvrm qbr me xeyir dqzkoyopa rz m xm q&s=m w fvnsvbtk bssvmljiua pxourfnbzukiy xy jg&e= following the identification and prioritization of the nt , nt , and nt c:r interaction sites mutant sinv strains were developed using the q site directed mutagenesis kit (neb). briefly, pcr amplification of the parental toto , or ar , infectious cdna clones were performed using the sequences indicated in fig . q reaction products were confirmed visually using standard agarose electrophoresis and diagnostically digested to confirm product size. the individual c:r interaction site mutant sinvs were completely sequenced to confirm the presence of the mutation and sequence relative to wild type virus prior to being utilized as templates for the production of infectious virus as described above. the quantitative assessment of the capsid:rna interactions of the individual c:r interaction sites for parental and c:r interaction site mutants utilized small scale extracts generated identically to that described above for the clip-seq process. briefly, hek cells were infected at an moi of prior to cross-linking via uv irradiation. after the preparation of whole cell lysates, the capsid:rna complexes were immunoprecipitated and the bound rnas fragmented with rnase t . the immunoprecipitates were washed extensively and the retained rna fragments were release via proteinase k digestion. the rna fragments were extracted via trizol according to the manufacturer's instructions. the precipitated rnas and paired input controls, were used as the input materials for reverse transcription using random hexamer. the resulting cdnas were assessed quantitatively via qrt-pcr as described below using the following primer pairs: sinv.nt f '-gcaccgccatcaagcaatgtgtggc- '; sinv.nt r '-caatttc ccttgggccgtgtggtcg- '; sinv.nt f '-tgttccaaatgtgccacagataccg- '; sinv.nt r '-aatgt actcttggttggtggaaggc- '; sinv.nt f '-cagcagattgcgcgtctgaccatgc- '; sinv.nt r '-gactcc gttcacgtacacatctagg- '. the viral growth kinetic assays performed in these studies were essentially as previously described [ ] . briefly, hek cells were infected with either wildtype or c:r interaction site mutant viruses at a multiplicity of infection of pfu/cell. the infected monolayers were washed with xpbs to remove unbound virus and fresh whole media was added. at regular times post infection the tissue culture supernatant was harvested and replaced with fresh growth media. all samples were frozen at - ˚c until completion of the time course. viral titers were determined using the standard plaque assay protocols involving bhk- cells and a % agarose overlay. plaque assays were allowed to incubate until plaques were readily visible prior to being fixed with . % formaldehyde and stained with crystal violet. at the indicated times post infection, infected hek cells were washed twice with xpbs and harvested into trizol. total rna was extracted according to the manufacturer's directions, with carrier glycogen being added to the precipitations. equal amounts of total rna ( . μg) were used as template for the synthesis of cdna and assessed quantitatively via qrt-pcr using the method and oligonucleotide primers previously described [ , , ] . briefly, the positive and negative sense viral rnas were selectively reverse transcribed using specific rt primers. the absolute quantities of the genomic and total positive sense viral rnas (consisting of the genomic and subgenomic rnas) were determined using paired standard curves; and the absolute quantity of the subgenomic rna itself was determined via subtraction of the number of genomes from the total positive sense rnas. the minus strand was detected in isolation from the positive sense rnas. all qrt-pcr reactions were normalized to the level of s rrna present as previously described [ , , ] . to determine the relative infectivity of each sinv c:r interaction site mutant the number of total viral particles per unit volume, as measured by the genome equivalents per ml, was determined using qrt-pcr, as previously described [ ] . briefly, the viral genomic rnas from bhk- tissue culture cell supernatants were utilized as the source template for cdna synthesis. the resulting cdnas were then assessed using qrt-pcr and the absolute quantity of viral particles per unit volume determined using standard curve analysis. the number of infectious units was determined using standard plaque assay on bhk- cells, and the infectivity was determined by comparing the total number of particles per infectious unit. the quantitative analyses of viral gene expression described in these studies was performed via one of two methods-gross viral and cellular gene expression was determined via metabolic labeling of infected cells [ , ] . briefly, hek cells were infected with sinv at an moi of pfu/cell and allowed to incubate for the indicated times under normal conditions. thirty minutes prior to the labeling period the infected monolayers were washed twice with xpbs and the media was replaced with methionine / cysteine free dmem supplemented with % dialyzed fbs. after the depletion period the media was removed and replaced with methionine/cysteine free dmem supplemented with s-labeled methionine and cysteine at a specific activity of μci/ml. the cells were further incubated under the labeling conditions for the indicated time periods. at the end of the labeling period the cells were washed three times with xpbs prior to lysis in ripa buffer. equal volumes of cell lysates were analyzed by sds-page and radiolabeled proteins were detected using standard phosphorimaging practices. quantitative analysis of genomic rna gene expression early during infection was performed using a process previously described, with one notable exception [ , ] . specifically, the reporter construct, and hence detection system used, consisted of an in-frame fusion of the nanoluc reporter gene with nsp at a position identical to that of ptoto -fluc [ , ] . nanoluciferase signal was assayed according to the manufacturer's instructions. all analysis methods were identical to that previously described [ ] . viral rna half-lives were assessed as previously described [ ] . hek cells, cultured in media supplemented with um -thio uridine (sigma), were infected with either parental sinv toto or the individual sinv c:r interaction site mutant sinvs at an moi of pfu per cell. at the indicated times post infection the tissue culture supernatant was removed and the cell monolayers were washed three times with pbs prior to trizol (invitrogen) extraction. a total of μg of total rna was biotinylated using hpdp-biotin (pierce). the biotinylated rnas were bound to ultralink streptavidin resin (pierce) to remove newly transcribed viral rnas. the unbound rnas were then phenol extracted and ethanol precipitated prior to use as a template for cdna synthesis via reverse transcription using random hexamer. the resulting cdnas were assayed via qrt-pcr to determine the relative abundances of the incoming sinv genomic rnas normalized to the cellular s rrna. rna half-lives were calculated as reported in dolken et al from data pertaining to the initial phase of decay [ ] . the production of soluble type-i ifn was assayed as previously described [ ] . briefly, the ifn competent l cell line was infected with the c:r interaction site mutant derivatives or parental wildtype sinv ar at an moi of pfu/cell for hour at room temperature. prior to further incubation the infected tissue culture cells were washed three times with xpbs and fresh media was added. the tissue culture supernatants were harvested hpi and clarified via centrifugation. infectious viral particles in the supernatant were inactivated via acidification and uv irradiation. the inactivation of samples was confirmed via standard plaque assays. the inactivated supernatants were then serially diluted and tittered onto fresh l cells in a -well format. after a period of hours of treatment the media was removed and the cells were challenged with a fluorescent chikungunya mcherry reporter strain at an moi of pfu/cell. viral gene expression was detected at hpi via a typhoon phosphorimager and quantified via densitometry. after hpi the cells were fixed and assayed for cell death via crystal violet staining; the formation of cpe was highly consistent with viral mcherry expression. relative ifn production was calculated as a function of the dilution needed to attain a % reduction in viral gene expression. four-week old wt c bl/ j mice were obtained from the jackson laboratory. mice were inoculated in the left rear footpad with virus in diluent (phosphate-buffered saline [pbs] supplemented with % fbs) in a volume of μl. on the termination day of each experiment, mice were sedated with isoflurane and euthanized by thoracotomy, blood was collected, and mice were perfused extensively by intracardiac injection of pbs. serum was obtained by collecting blood in serum separator tubes (bd). pbs-perfused tissues were removed by dissection, placed into pbs- % fbs and homogenized using a magna lyser (roche). the amounts of infectious virus in tissues were quantified by standard plaque assays using bhk- cells. this study was conducted in accordance with the recommendations in the guide for the care and use of laboratory animals and the avma guidelines for the euthanasia of animals. all animal experiments were performed with the approval of the institutional animal care and use committee at the university of colorado school of medicine (assurance number: a - ) under protocol b- ( ) e. experimental animals were humanely euthanized at defined endpoints by exposure to isoflurane vapors followed by thoracotomy. unless otherwise noted, the quantitative data reported in this study are the means of a minimum of three independent biological replicates. where appropriate, the statistical analysis of comparative samples was performed using variable bootstrapping, as previously described [ ] . the error bars indicate the standard deviation from the mean. when indicated, the p-values associated with individual data sets are the result of the student's t-test for the corresponding data. supporting information s table. sinv genomes used for single nucleotide polymorphism analysis. (pdf) s dataset. sequence data used for clip-seq analyses. statistical summary of sequencing data. sinv-specific sequence reads. read size analysis across the identified capsid interaction sites and a control region in the nsp coding sequence. qc was performed using trimmomatic. quality scores were averaged over a sliding window of nucleotides and nucleotides with values less than were removed. (xlsx) s fig. the immunoprecipitation of sinv capsid protein is specific. a) metabolically labeled mock control and infected hek cells cell lysates were immunoprecipitated with either non-specific control sera or anti-capsid sera using conditions identical to those used for the preparation of the cdna libraries used in clip-seq. the input lane represents / th of the starting material for the ip. data shown is representative of several biological replicates. b) sinv infected hek cells were either mock-or uv-crosslinked via shortwave uv irradiation at hpi. the cell monolayers were harvested via gentle scraping and solubilized in ripa buffer to form whole cell lysates. the cell lysates were then precipitated with antibodies specific for either sinv capsid, or control rabbit igg, as indicated on the figure. all purification conditions were identical to those described for the development of the clip-seq cdna libraries utilized in this study, with the only exception being that rna fragmentation was omitted. after purification, cdna was generated from the immunoprecipitated materials, and the presence of the nsp coding region was detected via rt-pcr and agarose gel electrophoresis. data shown is representative of three biological replicates. in vitro transcribed genomic rnas for parental sinv, and each of the individual c:r interaction site mutants were assessed for translation using rabbit reticulocyte extracts according to the manufacturer's directions. the amount of translation was detected using nanoluciferase detection. data shown is the mean of three independent biological replicates, with the error bar representing the standard deviation of the mean. (tif) the one-step growth kinetics of parental and a non c:r interaction site mutant as observed in hek cells. briefly, a region of the sinv genomic rna detected, but identified as statistically insignificant, by clip-seq analysis was mutated using identical parameters to the bona fide c:r interaction site mutants. this region corresponds to nt - of the viral genomic rna, which includes the genuine start site of nsp . the quantitative data in this figure represents the mean of three independent biological replicates, the error bar representing the standard deviation of the mean. nowcasting the spread of chikungunya virus in the americas centers for disease c, prevention. notes from the field: transmission of chikungunya virus in the continental united states-florida, . mmwr morbidity and mortality weekly report reemergence of an unusual disease: the chikungunya epidemic. seminars in pediatric infectious diseases morbidity and impaired quality of life months after chikungunya infection: comparative cohort of infected and uninfected french military policemen in reunion island epidemiology of chikungunya infection on reunion island, mayotte, and neighboring countries a major epidemic of chikungunya virus infection on reunion island post-epidemic chikungunya disease on reunion island: course of rheumatic manifestations and associated factors over a -month period clinical infectious diseases: an official publication of the infectious diseases society of america arrival of chikungunya virus in the new world: prospects for spread and impact on public health the alphaviruses: gene expression, replication, and evolution. microbiological reviews equine encephalitis in massachusetts medically important arboviruses of the united states and canada alphavirus rna synthesis and non-structural protein functions nucleocapsid and glycoprotein organization in an enveloped virus . a cryo-em structure of an enveloped alphavirus venezuelan equine encephalitis virus packaging signals in alphaviruses venezuelan equine encephalitis virus nsp protein regulates packaging of the viral genome into infectious virions conservation of a packaging signal and the viral genome rna packaging mechanism in alphavirus evolution encapsidation of hostderived factors correlates with enhanced infectivity of sindbis virus nucleotide-resolution profiling of rna recombination in the encapsidated genome of a eukaryotic rna virus by next-generation sequencing venezuelan equine encephalitis virus variants lacking transcription inhibitory functions demonstrate highly attenuated phenotype the efficient packaging of venezuelan equine encephalitis virus-specific rnas into viral particles is determined by nsp - synthesis deletion mapping of sindbis virus di rnas derived from cdnas defines the sequences essential for replication and packaging sindbis virus nucleocapsid assembly: rna folding promotes capsid protein dimerization self-assembly of an alphavirus corelike particle is distinguished by strong intersubunit association energy and structural defects role of sindbis virus capsid protein region ii in nucleocapsid core assembly and encapsidation of genomic rna in vitro-assembled alphavirus core-like particles maintain a structure similar to that of nucleocapsid cores in mature virus in vitro assembly of sindbis virus core-like particles from crosslinked dimers of truncated and mutant capsid proteins identification of rna-protein interaction networks using par-clip transcriptome-wide identification of rna-binding protein and microrna target sites by par-clip par-clip (photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation): a step-by-step protocol to the transcriptome-wide identification of binding sites of rna-binding proteins mfold web server for nucleic acid folding and hybridization prediction a cis-acting mutation in the sindbis virus junction region which affects subgenomic rna synthesis noncapped alphavirus genomic rnas and their role during infection connections underlying translation and mrna stability modulation of type i ifn induction by a virulence determinant within the alphavirus nsp protein differential induction of type i interferon responses in myeloid dendritic cells by mosquito and mammalian-cell-derived alphaviruses variation in interferon sensitivity and induction among strains of eastern equine encephalitis virus early events in alphavirus replication determine the outcome of infection roles of nonstructural protein nsp and alpha/beta interferons in determining the outcome of sindbis virus infection role of alpha/beta interferon in venezuelan equine encephalitis virus pathogenesis: effect of an attenuating mutation in the ' untranslated region characteristics of alpha/beta interferon induction after infection of murine fibroblasts with wild-type and mutant alphaviruses alpha/beta interferon protects adult mice from fatal sindbis virus infection and is an important determinant of cell and tissue tropism complete nucleotide sequence and full-length cdna clone of s.a.ar a south african alphavirus related to sindbis analysis of venezuelan equine encephalitis virus capsid protein function in the inhibition of cellular transcription the old world and new world alphaviruses use different virus-specific proteins for induction of transcriptional shutoff nucleotide sequence at the binding site for coat protein on rna of bacteriophage r sequence-specific interaction of r coat protein with its ribonucleic acid binding site control mechanisms for phage-specific syntheses hepatitis c virus core protein acts as a trans-modulating factor on internal translation initiation of the viral rna interaction of hepatitis c virus core protein with viral sense rna and suppression of its translation amino acids - of the hepatitis c virus (hcv) core protein specifically inhibit hcv ires-dependent translation in hepg cells, and inhibit both hcv ires-and capdependent translation in huh and cv- cells. the journal of general virology down-regulation of the internal ribosome entry site (ires)-mediated translation of the hepatitis c virus: critical role of binding of the stem-loop iiid domain of ires and the viral core protein hepatitis c virus internal ribosome entry sitemediated translation is stimulated by cis-acting rna elements and trans-acting viral factors brome mosaic virus capsid protein regulates accumulation of viral replication proteins by binding to the replicase assembly rna element rna binding by the brome mosaic virus capsid protein and the regulation of viral rna accumulation specific rna binding by amino-terminal peptides of alfalfa mosaic virus coat protein. the embo journal coat protein binding sites on rna of alfalfa mosaic virus removal of the n-terminal part of alfalfa mosaic virus coat protein interferes with the specific binding to rna and genome activation rna-binding proteins that inhibit rna virus infection the nucleoprotein is required for efficient coronavirus genome replication selective replication of coronavirus genomes that express nucleocapsid protein coronavirus n protein n-terminal domain (ntd) specifically binds the transcriptional regulatory sequence (trs) and melts trs-ctrs rna duplexes functional transcriptional regulatory sequence (trs) rna binding and helix destabilizing determinants of murine hepatitis virus (mhv) nucleocapsid (n) protein. the journal of biological chemistry role of ribosomes in semliki forest virus nucleocapsid uncoating semliki forest virus capsid protein associates with the s ribosomal subunit in infected cells promoter for sindbis virus rna-dependent subgenomic rna transcription sequence requirements for sindbis virus subgenomic mrna promoter function in cultured cells the ' untranslated region of sindbis virus represses deadenylation of viral transcripts in mosquito and mammalian cells sindbis virus usurps the cellular hur protein to stabilize its transcripts and promote productive infections in mammalian and mosquito cells heterogeneous nuclear ribonuclear protein k interacts with sindbis virus nonstructural proteins and viral subgenomic mrna role for subgenomic mrna in host translation inhibition during sindbis virus infection of mammalian cells expression of the zinc-finger antiviral protein inhibits alphavirus replication fluorophore-nanoluc bret reporters enable sensitive in vivo optical imaging and flow cytometry for monitoring tumorigenesis high-resolution gene expression profiling for simultaneous kinetic parameter analysis of rna synthesis and decay sindbis virus infectivity improves during the course of infection in both mammalian and mosquito cells we would like to thank the members of the sokoloski, hardy, mukhopadhyay, and danthi labs for their input regarding the development of this project and preparation of this manuscript. key: cord- - j qqi w authors: du, wenjuan; guo, hongbo; nijman, vera s.; doedt, jennifer; van der vries, erhard; van der lee, joline; li, zeshi; boons, geert-jan; van kuppeveld, frank j. m.; de vries, erik; matrosovich, mikhail; de haan, cornelis a. m. title: the (nd) sialic acid-binding site of influenza a virus neuraminidase is an important determinant of the hemagglutinin-neuraminidase-receptor balance date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: j qqi w influenza a virus (iav) neuraminidase (na) receptor-destroying activity and hemagglutinin (ha) receptor-binding affinity need to be balanced with the host receptor repertoire for optimal viral fitness. nas of avian, but not human viruses, contain a functional (nd) sialic acid (sia)-binding site ( sbs) adjacent to the catalytic site, which contributes to sialidase activity against multivalent substrates. the receptor-binding specificity and potentially crucial contribution of the sbs to the ha-na balance of virus particles is, however, poorly characterized. here, we elucidated the receptor-binding specificity of the sbs of n na and established an important role for this site in the virion ha-na-receptor balance. nas of h n / pandemic virus with or without a functional sbs and viruses containing this na were analysed. avian-like n , with a restored sbs due to an amino acid substitution at position , was more active than human n on multivalent substrates containing α , -linked sias, corresponding with the pronounced binding-specificity of avian-like n for these receptors. when introduced into human viruses, avian-like n gave rise to altered plaque morphology and decreased replication compared to human n . an opposite replication phenotype was observed when n was combined with avian-like ha. specific bio-layer interferometry assays revealed a clear effect of the sbs on the dynamic interaction of virus particles with receptors. the absence or presence of a functional sbs affected virion-receptor binding and receptor cleavage required for particle movement on a receptor-coated surface and subsequent na-dependent self-elution. the contribution of the sbs to virus-receptor interactions depended on the receptor-binding properties of ha and the identity of the receptors used. we conclude that the sbs is an important and underappreciated determinant of the ha-na-receptor balance. the rapid loss of a functional sbs in pandemic viruses may have served to balance the novel host receptor-repertoire and altered receptor-binding properties of the corresponding ha protein. a a a a a influenza a virus (iav) particles contain hemagglutinin (ha) and neuraminidase (na) glycoproteins. ha functions as a sialic acid (sia)-binding and fusion protein. na has receptordestroying activity by cleaving sias from sialoglycans. the ha and na protein functionalities are critical for host tropism, and need to be well balanced in relation to the host receptor repertoire for optimal in vivo viral fitness [ ] [ ] [ ] . however, there is no standard assay and unit for measuring a functional balance and the precise mode by which ha-and na-receptor interactions contribute to the balance at the molecular level remains mostly unexplored. an optimal ha-na balance is hypothesized to allow virions to penetrate the heavily sialylated mucus layer, to attach to host cells prior to virus entry, and to be released from cells after assembly [ ] [ ] [ ] [ ] . aquatic birds constitute the natural reservoir of iavs. occasionally iavs from birds cross the host species barrier and manage to adapt to non-avian species, including humans. the human receptor repertoire differs from avians and requires adaptations in the sia-interacting ha and na proteins for optimal interaction. the ha protein of avian iavs prefers binding to terminally located sias linked to the penultimate galactose via an α , -linkage. human iavs preferentially bind to α , -linked sialosides [ ] [ ] [ ] [ ] . internal sugars and their linkages as well as glycan branching have been shown to determine fine specificity of ha-receptor binding [ ] [ ] [ ] [ ] [ ] [ ] . changes in the receptor-binding properties of the ha proteins are achieved by mutations in the receptor binding site, which have been well documented for several ha subtypes [ , , , ] . much less is known about the adaptations in na required to match the corresponding ha proteins. na is a type ii transmembrane protein that forms mushroom-shaped homotetramers. tetramerization is essential for its enzymatic activity [ , ] . the enzyme active site is located in the globular head domain that is linked to the endodomain via a thin stalk. the active site is made up by catalytic residues that directly contact sia and by framework residues that keep the active site in place [ , ] . the catalytic and the framework residues are extremely conserved between avian and human iavs [ ] . nevertheless, although both avian and human na proteins preferentially cleave α , -linked sias, human viruses appear relatively better at cleaving α , -linked sias [ ] [ ] [ ] [ ] . adjacent to the catalytic site, na contains a nd sia-binding site ( sbs; also referred to as hemadsorption site) (s fig) [ ] [ ] [ ] [ ] . the sbs is made up by three loops, which contain residues that interact with sia. mutations in these loops in n , n and n affected na binding of erythrocytes [ , [ ] [ ] [ ] [ ] or sialosides [ , ] and enzymatic cleavage of multivalent substrates [ , ] but not of monovalent substrates [ , , ] . a detailed analysis of the receptor binding properties of the sbs of most nas is lacking. n and n proteins bind to α , -as well as α , -linked sias based on binding of resialylated erythrocytes [ , ] whereas n and n proteins mainly bind, via their sbs, to α , -linked sialosides present on glycan arrays [ ] or in biolayer interferometry assays [ ] . interestingly, the high conservation of sia-contact residues in the sbs of avian iav is lost in n and n of human iavs [ , , , ] that, supposedly, all lack a functional sbs. for n of avian viruses, the conservation of the sbs is only lost in viruses of the h n subtype, which mainly infect galliformes species, in contrast to other n -containing viruses, which mainly infect non-galliformes species [ ] (s fig). conservation of the sia-contact residues in the sbs of n is also lost in canine and not restored in swine viruses, the latter of which are generally derived from human viruses (s fig). it is tempting to hypothesize that the loss of a functional sbs in pandemic viruses is part of a required adaptation of the ha-na balance in order to deal with the altered receptor repertoire in the novel human host [ , ] . at first, to test this hypothesis, a detailed analysis of the contribution of (mutations in) the sbs to receptor binding and cleavage in the context of iav particles is necessary as the interplay with ha proteins binding to either avian-or human-type receptors needs to be taken into account. we define the ha-na balance as the balance between the activities of ha and na in virus particles in relation to their functional receptors on cells and decoy receptors present e.g. in mucus. we have recently established novel kinetic assays based on biolayer interferometry (bli) with which, in the context of virus particles, ha binding, na cleavage and their balance can be monitored in real time using synthetic glycans and sialylated glycoproteins [ ] . multivalent iav-receptor binding is established by multiple low affinity interactions of several ha trimers and sialosides [ , ] . this enables a dynamic binding mode in which individual interactions are rapidly formed and broken without causing dissociation of the virus but providing access of na to temporarily free sias. cleavage by na results in reduced sia-receptor density, in virus movement and ultimately in virion dissociation [ ] . how fast this occurs depends on the ha-na-receptor balance governing the dynamics of virus-glycan interactions. in the present study, we applied these novel bli assays to study the ha-na-receptor balance of viruses that have a single amino acid substitution in the sbs. we first performed a detailed analysis of the functional importance of the sbs in n for substrate binding and cleavage by comparing na of the pandemic h n virus from , containing a mutated nd sia-binding site, with an avian-like na, in which the nd sia binding site was restored. preferred binding to α , -linked sialosides was shown to result in enhanced cleavage of substrates containing these glycans. analysis of the ha-na-receptor balance of viruses containing these n proteins in combination with h proteins that prefer binding to avian or human-type receptors clearly demonstrated a role for the sbs in the complex and dynamic interplay between ha, na and receptor, which has been largely overlooked until now. the functional importance of the sbs for the ha-na-receptor balance may explain the conservation and loss of this site in avian and human iavs, respectively. we first analysed the receptor binding and cleavage activity of n na with and without a functional sbs using purified recombinant soluble na expressed in hek t cells [ ] . in na of a/singapore/ / (h n ) pandemic virus (referred to as human n [hn ]) one of the siacontact residues in the sbs is mutated compared to the avian consensus sequence (s n, s fig) . introduction of the reciprocal mutation (n s) in this na restored the sbs (referred to as avian-like n [an ]) [ ] . hn and an displayed similar specific activities when using the monovalent munana [ '-( -methylumbelliferyl)-α-d-n-acetylneuraminic acid] substrate ( fig a, s a and s b fig) , indicating that mutation of the sbs did not affect the catalytic activity of the n proteins per se. similar results were obtained previously using membrane-associated proteins [ ] , indicating that the activity of the recombinant soluble proteins accurately reflects the activity of their membrane-bound counterparts as concluded earlier for n [ ] . cleavage of sias from fetuin and transferrin sialoglycoproteins was quantified by enzyme-linked lectin assay (ella), by analysing the increase or decrease in binding of lectins depending on their binding specificities (s enzymatic activity of n proteins assayed using different substrates. (a) specific activity of hn and an was determined by munana assay and ella using different glycoprotein-lectin combinations (fetuin-eca, fetuin-pna, fetuin-mal i, fetuin-sna, transferrin-eca and transferrin-sna) and normalized to the specific activity of hn for munana and each glycoprotein-lectin combination. (b) specific activity of hn and an is graphed normalized to the specific activity as determined for each protein by the fetuin-eca combination. mean values and standard deviations from two independent experiments performed in triplicate are shown. stars depict p values calculated using an unpaired two-tailed student t test ( �� , p< . ; ��� , p< . ). (c and d) bli kinetic assay of na enzymatic activity. streptavidin biosensors were coated with biotinylated synthetic glycans ( 'slnln, 'slnln or lnln). subsequently, the sensors were incubated in buffer containing μg an or hn in the absence or presence of μg eca or eca alone. eca binding to sensors coated with 'slnln or 'slnln is a measure for sia cleavage from these receptors by na. experiments were independently performed three times. representative experiments are shown. specifically binds glycans containing terminal galα , glcnac corresponding to non-sialylated n-linked sugars [ ] , while pna (peanut agglutinin) binds to terminal galβ , galnac, which generally corresponds to non-sialyated o-linked sugars [ ] . na activity thus results in increased binding of these lectins. mal i (maackia amurensis lectin i) and sna (sambucus nigra lectin) specifically bind α , -or α , -linked sias, respectively [ , ] . binding of sna and mal i is decreased by na activity. for all lectins analysed, an was more active than hn using fetuin, containing α , -and α , -linked sias (fig a) [ ] . in contrast, no statistically significant difference was observed using transferrin that only contains α , -linked sialoglycans ( fig a) [ , ] . plotting the specific activities of the na proteins relative to their specific activities as determined by the fetuin-eca combination resulted in similar activity profiles (fig b) , which mimic those determined previously for n and n [ , ] . both hn and an preferred cleavage of α , -(determined with fetuin-mal i) over α , -(determined with fetuin-sna) linked sias (fig b) . in agreement herewith, the specific activities were higher when determined with the fetuin-eca than with the transferrin-eca combination as fetuin, but not transferrin, contains α , -linked sias (fig b) . these results show that an avian-like sbs in n contributes to cleavage of the sialoglycoprotein fetuin containing α , -and α , -linked sias. we next used bli to study the kinetics of na activity on a multivalent surface coated with either an avian receptor ( 'slnln: neuacα - galβ - glcnacβ - galβ - glcnac) or a human receptor ( 'slnln: neuacα - galβ - glcnacβ - galβ - glcnac). na activity can be directly monitored in real-time by the specific binding of the lectin eca to terminal galβ - glcnac glycotopes that become available upon removal of sia by na (fig c and d , red and black lines). note that cleavage of the small sia moiety is not detected directly by bli (fig c and d , dashed red and black lines). binding of eca to a sensor coated with lnln (galβ - glcnacβ - galβ - glcnac, fig c and d blue lines) rapidly reaches the maximum eca binding signal (representing % de-sialylation) assuring that eca binding during the relatively slow accumulation of desialylated glycans by na activity (red and black lines) reflects the cleavage kinetics of the n proteins. both hn and an more efficiently cleaved 'slnln over 'slnln. especially the an protein displayed much more efficient cleavage of 'slnln. we conclude that restoration of the sbs in hn to the avian consensus sequence results in enhanced cleavage of substrates containing α , -linked sias. the increased cleavage by an of substrates containing α , -linked sias is expected to result from specifically increased binding to α , -linked sias due to the presence of an avian sbs, although n proteins were reported to bind both α , -and α , -linked sias by using resialylated erythrocytes [ ] . we observed hemagglutination for recombinant soluble an but not for hn (s a fig). however, no specific binding to synthetic α , -and α , -linked sialoglycans by bli could be observed for the recombinant soluble n proteins, which could be due to low affinity of the sbs. by embedding the n proteins in membrane vesicles highly multivalent receptor interactions may increase receptor-binding avidity. to this end, full length n proteins were expressed in t cells. n virus-like particles (vlps) [ ] were directly harvested from the culture supernatant, while cells were treated with hypotonic and hypertonic buffers, resulting in the release of n protein-containing vesicles [ ] . preparations containing similar amounts of n , based on munana activity ( fig a) were used to determine the receptor specificity of the sbs by bli [ ] . negligible binding was obtained for hn vlps (fig a and b ) or vesicles (s d and s e fig) to α , -or α , -linked sias, regardless of the presence of the na inhibitor oseltamivir carboxylate (oc), which binds the na catalytic site. in contrast, highly 'slnln-specific, binding was observed for an vlps and vesicles (fig a and b ; s d and s e fig) in the presence of oc leading to the conclusion that an has much higher lectin activity than hn due to the presence of a functional sbs. the observed α , -linked sia specificity is in agreement with the particularly enhanced cleavage of substrates containing α , -linked sias (fig ) . no binding of an vlps to 'slnln was observed in the absence of oc, which is likely explained by immediate self-elution of vlps carrying active na proteins. to examine the contribution of the sbs to the ha-na balance of virus particles we examined the replication phenotype of recombinant viruses containing either an or the hn in the background of the pandemic virus a/hong kong/ / (h n ) (referred to as hh an and hh hn ) [ ] . the hh hn virus, lacking a functional sbs, produced large and clear plaques on vero cells (fig a and b , s a and s b fig) as compared to the smaller, fuzzy plaques of the hh an virus with a functional sbs. staining of plaques at h post infection indicated that all cells within the plaques of hh hn virus were infected, whereas many noninfected cells could be observed in the hh an plaques. this could be due to the more active an , which may destroy receptors on cells before the virus can enter into the cells. hh an reached lower titres than the hh hn virus at and h post infection when vero cells were used (fig c) , while no significant differences were observed for replication in mdck cells ( fig d) . differences in cell surface sialosides and their distribution may explain differences between replication in vero and mdck cells. although the sialylation patterns of mdck and vero cells are poorly characterized, both cell lines can be infected with human and avian iavs and express α , -and α , -linked sias [ ] [ ] [ ] . from these results we conclude that the absence or presence of a functional sbs in n may affect virus replication kinetics in a cell type-dependent manner. using a recently established bli-based kinetic binding assay [ ] an enhanced initial binding rate to 'slnln but not 'slnln ( fig a and b ), was observed for hh an virus containing role for nd sia-binding site of iav na in ha-na-receptor balance a functional sbs in comparison to hh hn . as a result the hh an virus displayed a higher initial binding-rate ratio 'slnln/ 'slnln than hh hn (fig c, red and [ , ] ) were used in bli as recently described for recombinant fetuin [ ] . lamp and glycophorin a mimic the presumed functional and decoy receptors found on cells (lamp ) and on mucins (glycophorin a) that are rich in n-or o-glycans, respectively. analysis of the glycans on these glycoproteins by lectin binding using bli confirmed the presence of sialylated n-linked glycans (both α , -and α , -linked) on both proteins, while only glycophorin a was shown to contain sialylated o-glycans (s fig). again, a functional sbs (present in an ) contributed to virus plaque assays were performed for hh hn (a) and hh an (b) viruses using vero cells followed by crystal violet dye staining (left panels) or by immunostaining of infected cells (right panels). vero (c and e) or mdck (d and f) cells were infected with hh hn or hh an (c and d), or with ah hn or ah an (e and f). virus in the cell culture supernatants at the indicated times post infection was titrated, and the titres were expressed as log (ffu/ml). standard deviations are indicated. significant differences were analysed using an unpaired two-tailed student t test ( � , p< . ; �� , p< . ; ��� , p< . ; n.s., not significant). https://doi.org/ . /journal.ppat. .g role for nd sia-binding site of iav na in ha-na-receptor balance binding (fig d and e ). this contribution was larger for binding to glycophorin a than to lamp as judged from the initial binding rates (fig f) . the ha of the pandemic h n virus (referred to as hh ) prefers binding to terminal α , -linked sias [ , ] . the results above implicate that, besides adaptations in ha, also adaptations in the sbs may contribute to a specificity-switch when an avian iav adapts to humans. we therefore studied the effect of the sbs in na when combined with an avian-type role for nd sia-binding site of iav na in ha-na-receptor balance ha preferring binding to α , -linked sias. we generated the corresponding recombinant a/ hong kong/ / (h n ) viruses containing amino acid substitutions in the ha (see s a fig) . these substitutions reverted the ha back to the avian consensus sequence (referred to as avian-like ah ), including the crucial substitutions q l and g s, which enable ha preferential binding to avian-type receptors [ , ] . the resulting viruses are referred to as ah hn and ah an , depending on the absence and presence of the functional sbs, respectively. we confirmed the receptor-binding specificities of soluble hh and ah proteins by solid phase fetuin-and transferrin-binding assays and bli (s b and s c fig). as expected, ah displayed higher binding levels to fetuin, containing α , -and α , -linked sias, than hh , while hh bound better than ah to transferrin, which only contains α , -linked sialoglycans. bli analysis using h -containing vesicles obtained from cells expressing full-length versions of hh or ah confirmed the different receptor-binding properties of these h proteins to 'slnln and 'slnln (s d and s e fig) . in contrast to viruses containing hh , the presence of a functional sbs in an enhanced replication of viruses with ah both on vero ( fig e) and mdck (fig f) cells. differences in virus replication were smaller for mdck than for vero cells. we next analysed receptor-binding properties of ah hn and ah an viruses using bli. as observed before for the hh -containing viruses (fig c; red and black bars), a functional sbs enhanced binding to 'slnln but not 'slnln when n was combined with ah ( fig c) . however, viruses containing ah displayed similar binding kinetics in the presence of oc regardless of the presence of a functional sbs for both lamp and glycophorin a (fig g, h and i ). from these results we conclude that a functional sbs site in na contributes to virion-receptor binding in a ha-and receptor-dependent manner. the na enzymatic activity of the different recombinant viruses with and without a functional sbs was analysed using the monovalent soluble substrate munana, by ella and by bli. the different viruses displayed a similar na activity per particle using the monovalent soluble substrate munana ( fig a) . as also the na proteins do not differ in their munana activity regardless of the presence or absence of a functional sbs (fig a) , we conclude that similar amounts of na are incorporated into virions of the four viruses. the viruses differed, however, in their specific activities when the multivalent glycoprotein fetuin was used as substrate in an ella (fig b) . hh hn virus was less active compared to viruses containing an and/or ah , indicating a contribution of receptor binding via ha and the sbs to na enzymatic activity in the context of virus particles. in agreement with the results obtained with the recombinant proteins (fig b) , cleavage of α , -linked sia found on transferrin was less efficient and did not appear to differ significantly between the different viruses (s fig) . the ellas (fig b and s fig) indicate that both receptor binding via ha and the sbs of na contribute to the sialidase specific activity of virus particles. these endpoint assays do not, however, elucidate the ha-na balance of these viruses, for which kinetic bli assays are required [ ] . preliminary experiments showed inefficient cleavage of the synthetic glycans by the recombinant viruses. kinetic assays to determine the ha-na balance of these viruses were therefore performed with the glycoprotein receptors (lamp or glycophorin a). in the absence of oc, that is, with active na proteins, no appreciable binding of hh -containing viruses could be detected indicating efficient receptor cleavage by na (fig c and d ). limited binding could be detected, however, for the ah -containing viruses in the absence of oc. the binding curve of the virus with a functional sbs (ah an ) bended earlier and had a smaller area under the curve than that of the virus without a functional sbs (ah hn ) for both lamp and glycophorin a. this bending of the curves is explained by ongoing cleavage of sias by viruses attached to the sensor-attached glycoproteins, resulting in release of bound role for nd sia-binding site of iav na in ha-na-receptor balance virus particles [ ] . the earlier bending and smaller area under the curve observed for the ah an virus is indicative of more efficient cleavage of the sensor-attached receptors by this virus than by ah hn , lacking a functional sbs. the effect of receptor binding via na and ha on na activity of virions was analysed further by na-dependent virion self-elution from a receptor-coated bli sensor after prior binding of the virions in the presence of oc. self-elution of iav particles requires na activity and self-elution is not observed when na activity is blocked by oc [ ] . after binding of the four recombinant viruses to lamp and glycophorin a in the presence of oc, oc was removed by repeated short washes in dulbecco's phosphate buffered saline (pbs) with calcium and magnesium and virus self-elution was monitored. clearly, viruses with an proteins eluted faster from the sensors than the viruses with hn (compare hn an with hn hn and ah an with ah hn ; fig e and f ), for both glycoprotein receptors. of note, na-depended self-elution of virus particles is often preceded by an apparent increase in virus binding [ ] represented here as negative self-elution, particularly in the case of ah an and ah hn (fig f) . the larger negative area of self-elution for ah hn reflects the reduced na activity of this virus compared to ah an . also the identity of ha affected the virus self-elution rate. viruses with hh eluted faster than corresponding viruses with ah (e.g. compare hh an with ah an ). for hh an , self-elution was faster from glycophorin a than from lamp . for ah -containing viruses, the opposite was observed. differences in virion self-elution observed for different ha-receptor combinations could be due the different receptor repertoires present on the two proteins (s fig). the results indicate that receptor binding via the sbs of na contributes to enzymatic cleavage by na in virions and to virion self-elution from a receptorcoated surface. virion self-elution was also shown to depend on the identity of the ha and the glycoprotein receptor used. the s n mutation in the sbs of n was rapidly obtained after emergence of the h n pandemic virus in and was observed in human h n viruses until . most viruses isolated thereafter did not contain the s n mutations but rather contained the s l mutation, which also results in loss of a sia-contact residue in the loop (s fig) and hemadsorption activity [ ] . both single mutations had a similar negative effect on catalytic activity of the na [ ] . these results indicate that there was not a selection against s per se, but rather against a functional sbs, which is achieved by either mutation. however, several additional mutations accumulated in time in the three loops of the sbs. n from the a/hong kong/ (h n ) (referred to as hk n ) contains five mutations (s l, n s, n d, w r, p k) in the sbs compared to the avian consensus sequence (fig a) . to analyse the contribution of the sbs to the enzymatic activity of these different n proteins, a comparative analysis of recombinant proteins and viruses using monovalent and multivalent substrates was performed. the hk n protein was - fold less active than hn both on the monovalent substrate munana and the multivalent substrate fetuin. cleavage of sialoglycans attached to transferrin was not significantly affected (fig b) . we also compared the na activity of recombinant h n viruses only differing in their na segment. hk h n virus, containing the hk n protein, displayed -fold lower na activity per virus particle than the hh hn virus, containing the n protein, as determined by munana cleavage (fig c) . similarly, the time required for % self-elution of virions from the multivalent receptor lamp , was -fold longer for hk h n than for hh hn (fig d) . thus, hn from and hk n differ to a similar extent in their catalytic activity both when monovalent or multivalent substrates are used. as receptor-binding via the sbs only increases na activity for multivalent, but not monovalent substrates, we conclude that these differences do not result from differences in receptor-binding by their (non-functional) sbs. moreover, the difference observed when comparing the two recombinant proteins is similar to the difference in activity of the two nas in the virus context. since the discovery of hemadsorption activity in na [ ] and the structural evidence of the sbs in n [ ] , only few studies have addressed sbs-mediated receptor binding and the functional consequences thereof for na activity [ , , , , ] . we now show that the sbs is an important factor in the complex interplay between ha, na and receptors, referred to as the ha-na-receptor balance. a functional sbs in n was shown to prefer binding to α , -linked sialosides similarly to n [ ] and n [ ] . in agreement herewith, it enhances catalytic activity against substrates carrying α , -linked sias. the contribution of the sbs to the ha-na-receptor balance of virus particles was shown to be receptor-and ha protein-dependent as demonstrated by kinetic analysis of receptor-binding and -cleavage of virions using bli. the sbs was shown to contribute to receptor binding also when na was role for nd sia-binding site of iav na in ha-na-receptor balance combined with a receptor-binding ha in iav virions, as well as to cleavage of receptors by virions and to virion self-elution from a receptor-coated surface. the absence or presence of a functional sbs also affected virus replication in a cell type-and ha-dependent manner. our results indicate that mutation of the sbs as observed in early human pandemic viruses negatively affects the catalytic activity of na and may serve to restore the ha-na-receptor balance of viruses carrying ha proteins with altered receptor-binding properties in relation to a novel host sialome. conservation of the sbs in most avian strains, with the notable exception of h n viruses, is lost in human [ , , , ] , swine and canine variants (s fig). strong conservation usually reflects a critical function. it would be very interesting to investigate in depth whether a critical function for the sbs in avian strains, for instance related to the ha-nareceptor balance, is not required for efficient replication and transmission of human, canine and swine strains. n prefers binding of α , -over α , -linked sias via its sbs. the specificity of the n sbs correlates with the enhanced cleavage of substrates carrying α , -linked sias compared to substrates carrying only α , -linked sialosides. of note, enhanced activity was also observed for α , -linked sias at least when these sialosides were linked to substrates additionally carrying α , -linked sias (fig a; fetuin-sna combination). these results indicate that the sbs enhances catalytic activity by bringing sialosides on multivalent substrates close to the catalytic site and that, depending on the substrate used, the enhanced cleavage of sias not necessarily matches the specificity of the sbs. preferred binding of avian-type receptors via its sbs was previously also observed for n [ ] and n [ ] , suggesting that this is a conserved feature for nas of different subtypes. we cannot exclude, however, that the sbs of different na subtypes may differ in their receptor-binding fine specificity, as structural differences were observed in the interactions between ligands and the sbs for different na subtypes [ ] . in n , the conserved k residue in the sbs forms a hydrogen bond with sia [ ] and mutation k e in n has a large negative effect on the cleavage of multivalent substrates [ ] . in contrast, several other avian na subtypes, including n , contain a q or e residue at this position, which does not form a hydrogen bond with sia in the few available crystal structures [ ] . previously, it was shown that n and n nas bound with similar efficiency to both avian and human type receptors sias [ , ] . this discrepancy is probably explained by the different methods used to analyse the receptor specificity of the sbs. in the previous reports, a red blood cell binding assay was employed, in which desialylation of erythrocytes was followed by resialylation using α , -or α , -sialyltransferases. binding to resialylated erythrocytes might be affected by prior incomplete desialylation. alternatively, a higher receptor density on erythrocytes compared to the bli sensor surface might allow for binding of α , -linked sias. the ability of the sbs to bind human-type receptors to some extent is also suggested by the modestly increased or decreased cleavage of sias from substrates only containing α , -linked sias upon the introduction of mutations in the sbs (this study and [ , ] ). the sbs contributed to receptor-binding also when na was combined with a receptorbinding ha in iav virions. in combination with ha preferring binding to α , -linked sias (hh ), the sbs enhanced binding for all receptors analysed, except 'slnln, to which the recombinant an protein did not bind. binding to glycophorin a, carrying many o-linked sugars also found on mucins, was more enhanced by the sbs than binding to lamp , which carries mostly sialylated n-glycans. the functional significance of this difference remains to be determined. when combined with ha that prefers binding to α , -sialosides (ah ), the enhancing effect of the sbs was not observed for the glycoprotein receptors analysed. thus, the contribution of na to virion-receptor binding depends on the specificity/affinity of the corresponding ha and the receptors present. previously it was shown that the active site of na contributes to virion-receptor binding in case of a low-activity catalytic site [ ] , a characteristic which is also appears to be displayed by recent h n viruses [ , ] . as we now show that a functional sbs in na can also contribute to virion-receptor binding, two mechanisms exist by which na can assist in binding of virions to host cells. a complex interplay between ha, na and receptor determines the attachment of virus particles to and release from a receptor-containing surface. this ha-na-receptor balance can be experimentally determined using kinetic bli assays by analysis of virus binding in the absence or presence of na inhibitors and self-elution from different receptors (this paper and [ ] ). the ha-na-receptor balance determines the residence time of a virus on a sialylated surface and the speed by which it moves over this surface. we assume that an optimal balance is important for virions to efficiently pass the heavily sialylated mucus layer, while still allowing virion attachment to host cells resulting in endocytic uptake. the complexity of the ha-nareceptor balance is exemplified by the contribution of na to receptor binding [ ] and of ha to the apparent catalytic activity of na (this paper) [ , ] . we now show that the ha-nareceptor balance as reflected for example in virion self-elution (fig ) is affected by a functional sbs, depending on the particular ha with which na is combined and the receptors used. changes in the sbs of na should thus be considered in the context of mutations affecting the receptor-binding site of ha and the catalytic site of na. the sbs of n appears to accumulate more mutations than other surface exposed parts of the na protein (s fig). while the n protein has a single substitution in the sbs, the n protein contains five mutations in this site. the accumulation of several mutations in the sbs was found to have no further negative effects on the enzyme-enhancing function of sbs as compared to a single mutation of a sia contact residues in the sbs of an early pandemic virus from . although we cannot exclude that the accumulation of mutations in the sbs of n indicates ongoing adaptation of na to the human host or serves to restore subtle deviations in the ha-na-receptor balance resulting from other mutations in ha and/or na, it seems more likely that it rather results from continuous immune pressure on this site [ , ] in combination with loss of functional importance of the sbs in human viruses. an important role for the na sbs in iav replication in vivo is suggested by the conservation of this site among na subtypes of most avian viruses, the rapid loss of this site in human pandemic viruses ( [ , , , , ] and s fig) , the important role of this site in ha-nareceptor balance (this study) and observations that this site affects virus replication in vitro ([ , , ] and this study). of note, we now show that the presence or absence of a functional sbs affected virus replication depending on the receptor-binding properties of ha, with which na was combined. replication of viruses with a human or avian-like ha is enhanced by the absence or presence of a functional sbs, respectively, although some cell-dependent differences were observed. the absence or presence of a functional sbs was reported not to affect influenza viral replication in ducks [ ] . however, in this latter study recombinant viruses were used containing ha from a h n and na from a h n virus. this may have resulted in a mismatched ha-na combination in which the presence of the sbs might be of minor influence on replication. alternatively, the sbs may be important for virus transmission rather than for replication in ducks per se. clearly, additional experiments are needed to demonstrate the importance of the sbs for iav replication and transmission in vivo. interestingly, both for h n and h n viruses, the well-known q l mutation in the receptorbinding site of ha, resulting in a shift from avian to human receptor specificity, is associated with mutations in the sbs that negatively affect receptor binding [ , ] . these avian viruses thus display a striking parallel with the changes observed in the receptor-binding sites of ha and na of avian-origin pandemic viruses. we propose that mutations in the sbs of avian viruses may be indicative of an as of yet underappreciated, increased potential of avian viruses to cross the host species barrier. of note, also upon introduction of coronavirus oc into humans, the lectin function of the receptor-destroying hemagglutin-esterase protein was lost through progressive accumulation of mutations resulting in reduced cleavage of multivalent substrates [ ] . thus, both coronaviruses and iavs appear to adapt to the sialoglycome of the human respiratory tract by tuning the virion receptor-binding and cleavage functions, the latter among others by mutation of the lectin domain of the receptor-destroying na. human-codon optimized cdnas (genescript) encoding the n ectodomain of a/singapore/ / (h n ) (genbank accession no. ay . ; referred to as human n [hn ]) and a variant thereof containing the n s mutation (referred to as avian-like n [an ]) were cloned into a pfrt expression plasmid (thermo fisher scientific) in frame with sequences encoding a signal sequence derived from gaussia luciferase, a strep tag and a tetrabrachion tetramerization domain, similarly as described previously [ ] . the corresponding full length (fl) nacoding plasmids were generated by replacement of the non-na coding sequences by sequences encoding the na transmembrane domain and cytoplasmic tail of n of a/singapore/ / (h n ). human-codon optimized cdnas encoding fl h or the h ectodomain of a/hong kong/ / (h n ) (genbank accession no. cy ; referred to as human h [hh ]) or of an variant thereof containing amino acid substitutions, which revert the ha back to the avian consensus sequence [ ] (referred to as avian-like h [ah ]) were cloned in pcd expression vectors similarly as described previously [ ] . codon optimized glycoproteins lamp and glycophorin a ectodomain-encoding cdnas (genescript) were genetically fused to fc-tag, for protein-a based purification, and a bap tag [ ] , for binding to octet sensors, and cloned in a pcaggs vector, similarly as described previously for fetuin [ ] . na and glycoprotein expression plasmids were transfected into hek t (atcc) cells using polyethylenimine (polyscience) [ ] . an expression vector encoding bira ligase was cotransfected with the lamp -and glycophorin a-coding vectors [ ] . five days post transfection, cell culture media containing soluble na proteins and glycoproteins were harvested and purified using strep tactin or protein a containing beads [ , ] . purified na proteins were quantified by quantitative densitometry of gelcode blue (thermo fisher scientific)-stained protein gels additionally containing bovine serum albumin (bsa) standards. the signals were imaged and analysed with an odyssey imaging system (li-cor). hek t cells were transfected with full-length na constructs to obtain membrane vesicles. to this end, cells were vesiculated as described previously [ , ] . vlps and membrane vesicle preparations were purified using capto core beads (ge healthcare life sciences) according to the manufacturer's instructions and as detailed previously [ ] to remove proteins smaller than kda. the amount of na protein in the vlps and vesicle preparations was determined using the munana assay described below. generation of recombinant virus hk h n , which harbours all genes from the pandemic virus a/hong kong/ / (h n ) has been described before [ ] . also the generation of hh hn and hh an viruses, which carry the n gene of the pandemic a/singapore/ / (h n ) in the background of a/hong kong/ / (h n ) has been described before [ ] . the hh an virus contains substitution n s in the n protein. ah hn and ah an viruses were generated as described previously [ ] in the background of a/hong kong/ / (h n ). these latter viruses carry the h protein of a/hong kong/ / (h n ) containing amino acid substitutions in ha which revert the ha back to the avian consensus sequence [ ] combined with the n protein of a/singapore/ / (h n ) with (ah an ) or without (ah hn ) the n s substitution. virus stocks were grown in mdck-ii cells (ecacc). viruses were inactivated by uv radiation using uv stratalinker (stratagene) on , μjoules prior to their use in the binding and cleavage assays. uv inactivation did not affect the enzymatic activity of na as determined with the munana assay. the na enzymatic activity was determined by using a fluorometric assay [ ] in combination with '-( -methylumbelliferyl)-α-d-n-acetylneuraminic acid (munana; sigma-aldrich) as described previously [ ] . enzymatic activity of the na proteins towards multivalent glycoprotein substrates was analysed using a previously described enzyme-linked lectin assay (ella) [ ] . the binding of eca, pna, sna and mal i was detected using horseradish peroxidase (hrp)conjugated streptavidin (thermo fisher scientific) and tetramethylbenzidine substrate (tmb, biofx) in an elisa reader el- (biotek) by measuring the optical density (od) at nm. the data were fitted by non-linear regression using the prism . software (graphpad). the resulting curves were used to determine the amount of na protein corresponding to half maximum munana cleavage or lectin binding. the inverse of this amount is a measure of specific activity (activity per amount of protein) and was graphed relative to other na proteins or substrate-lectin combinations. plaque assays were performed in vero cells (atcc) as described previously [ ] . one hour after infecting the cell monolayers with - plaque forming units of the virus in ml of maintenance medium, the virus inoculum was removed and cells were covered the avicel rc- overlay medium and cultures were incubated at ˚c in % co atmosphere. after three days of incubation, the overlay was removed by suction and the cells were fixed with % formalin and stained with % crystal violet solution in % methanol in water. for immunostaining, cells were fixed with % paraformaldehyde solution for min at ˚c, washed with pbs and permeabilized by incubation for - min with buffer containing . % triton-x- and mm glycine in pbs. cell layers were incubated with monoclonal antibodies specific for the influenza a virus nucleoprotein (kindly provided by dr. alexander klimov at centers for disease control, usa) for hour followed by another hour incubation with peroxidaselabeled anti-mouse antibodies (dako, denmark) and min incubation with precipitateforming peroxidase substrates true blue. stained plates were washed with water to stop the reaction, scanned on a flatbed scanner and the data were acquired by adobe photoshop . software. to characterize replication kinetics of different recombinant viruses, two replicate cultures of vero or mdck cells in -well plates were infected with each virus at moi . (vero cells) or . (mdck cells). inocula were removed hpi, fresh medium was added, and cultures were incubated at ˚c. samples of culture supernatant were taken , and hpi and stored frozen. they were titrated together using focus formation assay in mdck cells as described previously [ ] . numbers of infected cells per well were counted for the virus dilution that produced from to infected cells per well and recalculated into numbers of focus forming units (ffu) per ml of the original undiluted virus suspensions. for the full length protein-containing vesicles and vlps, similar amounts of na activity, and thus na protein, were applied in the bli assays using the octet red (fortebio). inactivated virus preparations were analysed using nanoparticle tracking analysis (nanosight ns , malvern) as detailed below in order to use similar number of virus particles in the bli assays. bli assays were performed as described previously [ ] . all experiments were carried out in dulbecco's pbs with calcium and magnesium (lonza) at ˚c and with sensors shaking at rpm. streptavidin biosensors were loaded to saturation with biotinylated synthetic glycans , -sialyl-n-acetyllactosamine-n-acetyllactosamine ( 'slnln), , -sialyl-n-acetyllactosamine-n-acetyllactosamine ( 'slnln), n-acetyllactosamine-n-acetyllactosamine (lnln), lamp or glycophorin a glycoproteins. synthetic glycans were synthesized at the department of chemical biology and drug discovery, utrecht university, utrecht, the netherlands. for the na kinetic cleavage assay, the sensors loaded with synthetic glycans were incubated in μl buffer containing μg recombinant soluble an or hn in the absence or presence of μg eca. as controls, sensors were also incubated with eca in the absence of n . association of the n vlps, vesicles and virus particles was analysed for minutes in the absence or presence of μm oc (roche). for viruses, the virus association phase in the presence of oc was followed by three s washes and a dissociation phase in the absence of oc. initial binding rates were determined similarly as previously described [ ] . for lectin binding, the sensors loaded with recombinant glycoproteins were incubated with the different lectins ( μg/ μl) for minutes. nta measurements were performed using a nanosight ns instrument (malvern) following the manufacturer's instructions. the uv-inactivated virus preparations were diluted with pbs to reach a particle concentration suitable for analysis with nta. all measurements were performed at ˚c. per analysis, the nanosight ns recorded five second sample videos, which were then analysed with the nanoparticle tracking analysis . software, resulting in quantitative information on particle number and particles sizes (s fig). each virus preparation was analysed twice and mean values were used. nta measurements were validated by analysis of virus stocks quantified earlier by silver staining of viral proteins after electrophoresis on polyacrylamide gels [ ] . results obtained via both methods correlated well (less than % deviation). sequence logos were generated for the three loops ( , and loop) that constitute the sbs using dnastar lasergene software (megalign pro ) . the overall height of the stack indicates the sequence conservation at that position, while the height of symbols within the stack indicates the relative frequency of each amino acid at that position. all sequences available for avian viruses containing n excluding h n (indicated by avian hxn ), avian h n , dog h n , human h n , human h n until , swine h n and swine h n from the influenza research database (https://www.fludb.org/) were used. sia-contact residues were highly conserved in avian hxn , but not in h n viruses. avian h n viruses were mainly (> %) found in galliformes species (chicken, turkey and quail), while avian hxn viruses were isolated mainly from non-galliformes species (> %). dog h n viruses generally contain a s l mutation in the loop, which is known to affect functionality of the sbs [ ] , while in addition the identity of the residue deviates from those found in avian viruses. please note that the phylogenetic analysis shown in s fig indicates that human h n viruses either have a mutated sia-contact residue at position or at position , both of which are known to disrupt the sbs [ ] . swine viruses containing n , which are generally derived from human viruses [ ] , also contain a mutated sbs. sia-contacting residues were labelled with asterisks in the sequence logo of the avian hxn viruses. the grey asterisk indicates an additional sia-contact residue in the loop of n . numbering of the start and end residues of the three loops is indicated. (tif) s fig. phylogenetic analysis of n of human h n and h n viruses from until . all full-length and unique n protein sequences of human h n and h n viruses between - were downloaded from the genbank and gisaid databases. n protein trees were constructed by using the phylip neighbor-joining algorithm with the mpam distance matrix. this tree was used as a guide tree to select n sequences representing all main branches of the tree. the selected n proteins were used to construct a summary tree with topology similar to that of the guide tree. mutations that became fixed along the trunk of the tree are indicated as well as sbs residues that differ between different branches. on the right site the residues of the , and loops that make up the sbs are shown. sia-contact residues in the n protein are indicated by the red shading. mutations in n relative to the avian consensus sequence are shown in red. (tif) (a) the enzymatic activity of hn and an proteins for a monovalent substrate was determined using the munana fluorometric assay. to this end, limiting dilutions of the different n proteins were subjected to the assay and the fluorescence generated upon cleavage of munana was measured using a plate reader (in relative fluorescent units [rfu] ). the data were fitted by non-linear regression using the prism . software (graphpad). the resulting curves were used to determine the amount of na protein corresponding to half maximum munana cleavage (indicated by the arrow). the inverse of this amount is a measure of specific activity (activity per amount of protein) and was graphed relative to hn in (b). ellas were used to determine the relative specific activities of the n proteins for multivalent substrates (c-f). the od nm values correspond to lectin binding upon incubation of the glycoprotein with different dilutions of the na preparations. in the examples shown, removal of sias from fetuin was probed using the lectins eca (c) and mal i (e). increasing dilutions of the na preparations resulted in reduced cleavage of sias as indicated by the reduced binding of eca, which (just as pna) binds to desialylated glycans. the opposite was observed for mal i (and sna) which binds to sialylated glycans. the data were fitted by non-linear regression using the prism . software (graphpad). the resulting curves were used to determine the dilution (or amount) of na protein corresponding to half maximum lectin binding (indicated by the arrow). this value was used to determine the relative specific activity (activity per amount of protein) for a specific glycoprotein-lectin combination (d and f). what adaptive changes in hemagglutinin and neuraminidase are necessary for emergence of pandemic influenza virus from its avian precursor? the interplay between the host receptor and influenza virus hemagglutinin and neuraminidase functional balance between neuraminidase and haemagglutinin in influenza viruses hemagglutinin-neuraminidase balance confers respiratory-droplet transmissibility of the pandemic h n influenza virus in ferrets functional balance of the hemagglutinin and neuraminidase activities accompanies the emergence of the h n influenza pandemic influenza type a in humans, mammals and birds: determinants of virus virulence, host-range and interspecies transmission influenza a penetrates host mucus by cleaving sialic acids with neuraminidase host-range barrier of influenza a viruses avian and human receptor binding by hemagglutinins of influenza a viruses receptor specificity in human, avian, and equine h and h influenza virus isolates early alterations of the receptor-binding properties of h , h , and h avian influenza virus hemagglutinins after their introduction into mammals glycan microarray analysis of the hemagglutinins from modern and pandemic influenza viruses reveals different receptor specificities effects of sialic acid modifications on virus binding and infection recent h n viruses have evolved specificity for extended, branched human-type receptors, conferring potential for increased avidity only two residues are responsible for the dramatic difference in receptor binding between swine and new pandemic h hemagglutinin highly pathogenic influenza a (h nx) viruses with altered h receptor-binding specificity receptor specificity of influenza viruses from birds and mammals: new data on involvement of the inner fragments of the carbohydrate chain a two-amino acid change in the hemagglutinin of the influenza virus abolishes transmission steps in maturation of influenza a virus neuraminidase identification of residues that affect oligomerization and/or enzymatic activity of influenza virus h n neuraminidase proteins influenza virus neuraminidase: structure and function influenza other respir viruses the structure of h n avian influenza neuraminidase suggests new opportunities for drug design the n neuraminidase of human influenza virus has acquired a substrate specificity complementary to the hemagglutinin receptor specificity high-throughput neuraminidase substrate specificity study of human and avian influenza a viruses substrate binding by the second sialic acid-binding site of influenza a virus n neuraminidase contributes to enzymatic activity amino acid residues contributing to the substrate specificity of the influenza a virus neuraminidase functional significance of the hemadsorption activity of influenza virus neuraminidase and its alteration in pandemic viruses structural evidence for a second sialic acid binding site in avian influenza virus neuraminidases structure of influenza virus n : the last piece of the neuraminidase "jigsaw" puzzle a secondary sialic acid binding site on influenza virus neuraminidase: fact or fiction? transfer of the hemagglutinin activity of influenza virus neuraminidase subtype n into an n neuraminidase background mutation of the second sialic acid-binding site, resulting in reduced neuraminidase activity, preceded the emergence of h n influenza a virus neuraminidase hemadsorption activity, conserved in avian influenza a viruses, does not influence viral replication in ducks n neuraminidase of influenza virus a/fpv/rostock/ has haemadsorbing activity h n influenza a viruses from poultry in asia have human virus-like receptor specificity kinetic analysis of the influenza a virus ha/na balance reveals contribution of na to virus-receptor binding and na-dependent rolling on receptor-containing surfaces hemagglutinins from two influenza virus variants bind to sialic acid derivatives with millimolar dissociation constants: a -mhz proton nuclear magnetic resonance study a surface plasmon resonance assay for the binding of influenza virus hemagglutinin to its sialic acid receptor differential affinities of erythrina cristagalli lectin (ecl) toward monosaccharides and polyvalent mammalian structural units molecular basis of recognition by gal/galnac specific legume lectins: influence of glu on the specificity of peanut agglutinin (pna) towards c -substituents of galactose the elderberry (sambucus nigra l.) bark lectin recognizes the neu ac(alpha - )gal/galnac sequence structure of the complex oligosaccharides of fetuin studies on glycoconjugates. lxiv. complete structure of two carbohydrate units of human serotransferrin development of a pharmaceutical apotransferrin product for iron binding therapy formation of virus-like particles from human cell lines exclusively expressing influenza neuraminidase production of plasma membrane vesicles with chloride salts and their utility as a cell membrane mimetic for biophysical characterization of membrane protein interactions enhanced expression of an alpha , -linked sialic acid on mdck cells improves isolation of human influenza viruses and evaluation of their sensitivity to a neuraminidase inhibitor african green monkey kidney (vero) cells provide an alternative host cell system for influenza a and b viruses patterning of lectins of vero and mdck cells and influenza viruses: the search for additional virus/cell interactions: crc assignment of o-glycan attachment sites to the hinge-like regions of human lysosomal membrane glycoproteins lamp- and lamp- isolation and characterization of human lysosomal membrane glycoproteins, h-lamp- and h-lamp- . major sialoglycoproteins carrying polylactosaminoglycan structures of the asparagine-linked sugar chains of glycophorin a a single-sample method for determination of carbohydrate and protein contents glycoprotein bands separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis role of substitutions in the hemagglutinin in the emergence of the pandemic influenza virus avian-virus-like receptor specificity of the hemagglutinin impedes influenza virus replication in cultures of human airway epithelium. virology influenza virus neuraminidase with hemagglutinin activity role of neuraminidase in influenza a(h n ) virus receptor binding neuraminidase receptor binding variants of human influenza a(h n ) viruses resulting from substitution of aspartic acid in the catalytic site: a role in virus attachment? neuraminidase-mediated haemagglutination of recent human influenza a(h n ) viruses is determined by arginine flanking the neuraminidase catalytic site neuraminidase activity and specificity of influenza a virus are influenced by haemagglutinin-receptor binding antigenic structure and variation in an influenza virus n neuraminidase betacoronavirus adaptation to humans involved progressive loss of hemagglutinin-esterase lectin activity a single immunization with soluble recombinant trimeric hemagglutinin protects chickens against highly pathogenic avian influenza virus h n in vivo site-specific biotinylation of proteins within the secretory pathway using a single vector system novel high-throughput approach for purification of infectious virions fluorometric assay of neuraminidase with a sodium ( -methylumbelliferyl-alpha-d-n-acetylneuraminate) substrate new low-viscosity overlay medium for viral plaque assays influenza neuraminidase operates via a nucleophilic mechanism and can be targeted by covalent inhibitors evolutionary history and phylodynamics of influenza a and b neuraminidase (na) genes inferred from large-scale sequence analyses we thank robert webster (st. jude children's research hospital, memphis, tn, usa) for phw reverse genetics plasmid. we thank hanno müller for technical assistance in part of the study. key: cord- -eenaixp authors: brennan, greg; kitzman, jacob o.; rothenburg, stefan; shendure, jay; geballe, adam p. title: adaptive gene amplification as an intermediate step in the expansion of virus host range date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: eenaixp the majority of recently emerging infectious diseases in humans is due to cross-species pathogen transmissions from animals. to establish a productive infection in new host species, viruses must overcome barriers to replication mediated by diverse and rapidly evolving host restriction factors such as protein kinase r (pkr). many viral antagonists of these restriction factors are species specific. for example, the rhesus cytomegalovirus pkr antagonist, rhtrs , inhibits pkr in some african green monkey (agm) cells, but does not inhibit human or rhesus macaque pkr. to model the evolutionary changes necessary for cross-species transmission, we generated a recombinant vaccinia virus that expresses rhtrs in a strain that lacks pkr inhibitors e l and k l (vvΔeΔk+rhtrs ). serially passaging vvΔeΔk+rhtrs in minimally-permissive agm cells increased viral replication - to -fold. notably, adaptation in these agm cells also improved virus replication - to , -fold in human and rhesus cells. genetic analyses including deep sequencing revealed amplification of the rhtrs locus in the adapted viruses. supplying additional rhtrs in trans confirmed that amplification alone was sufficient to improve vvΔeΔk+rhtrs replication. viruses with amplified rhtrs completely blocked agm pkr, but only partially blocked human pkr, consistent with the replication properties of these viruses in agm and human cells. finally, in contrast to agm-adapted viruses, which could be serially propagated in human cells, vvΔeΔk+rhtrs yielded no progeny virus after only three passages in human cells. thus, rhtrs amplification in a minimally permissive intermediate host was a necessary step, enabling expansion of the virus range to previously nonpermissive hosts. these data support the hypothesis that amplification of a weak viral antagonist may be a general evolutionary mechanism to permit replication in otherwise resistant host species, providing a molecular foothold that could enable further adaptations necessary for efficient replication in the new host. there are at least described zoonotic microbial pathogens, % of which are capable of human to human transmission [ ] . recent viral zoonoses have led to some of the most devastating and medically relevant outbreaks in modern history, including sars coronavirus, pandemic influenza, and hiv/aids, highlighting the urgent need to understand how viruses adapt to infect new species. at a population level, factors influencing the transmission of zoonotic pathogens to humans include increasing population density, greater contact with wildlife, increased travel, and poor public health infrastructure [ , ] . however, these factors only allow the microbe increased access to new hosts; they do not directly enable it to adapt to and replicate in the new species. intermediate hosts, animals that are not the natural host of a virus but are still permissive or semi-permissive for viral replication, play a critical role in cross-species transmission. these hosts can facilitate increased contact between a virus and a new host, and drive adaptive changes that may improve virus replication (reviewed in [ ] ). for example, spill-over of nipah virus from fruit bats into pigs, the intermediate host, increased human exposure to the virus and resulted in eventual human outbreaks in malaysia [ , ] . in another example, lentiviral adaptation through intermediate chimpanzee hosts led to both increased contact with humans, and adaptive genetic changes permitting the virus to inhibit the human versions of several host restriction factors (reviewed in [ ] ). at a molecular level, the initial success of a virus after entry into a new host cell depends on its ability to overcome cellular host restriction factors. a subset of these proteins inhibits specific virus families, such as the restriction of retroviruses mediated by trim a [ ] . however, other restriction factors, including protein kinase r (pkr), block the replication of multiple virus families. pkr is activated by binding to double-stranded rna (dsrna), a common byproduct of both rna and dna virus replication, followed by dimerization and autophosphorylation. activated pkr then phosphorylates the a-subunit of eukaryotic initiation factor (eif a), ultimately arresting translation initiation [ ] . in response to the broad and potent barrier to viral infection imposed by pkr, most virus families have evolved at least one mechanism to inhibit the pkr pathway [ ] . this conflict between host restriction factors and their viral antagonists results in an ''arms race'' leading to rapid evolution of both sets of genes [ ] . the extraordinarily high rate of positive selection (dn/ds) among primate pkr genes reflects the evolutionary pressure on pkr to evade virus antagonists [ , ] . to productively infect new host species, virus antagonists must rapidly adapt to escape these differences in pkr. in the current study, we modeled the process of viral adaptation through an intermediate, minimally permissive host. we experimentally evolved a recombinant vaccinia virus expressing the rhesus cytomegalovirus pkr antagonist rhtrs in african green monkey cells expressing rhtrs -resistant pkr. we demonstrate that amplification of the exogenous rhtrs locus was an early adaption that is sufficient to rescue virus replication in minimally-permissive agm cells. this amplification of the rhtrs locus was also sufficient to expand the species tropism of these viruses, enabling them to infect both human and rhesus cells substantially better than the initial virus. importantly, rhtrs amplification did not occur when the initial virus was passaged through human fibroblasts, suggesting that amplification in agm cells was a necessary intermediate step to expand the virus host range. gene amplification is a universal mechanism of rapid adaptation occurring in eukaryotes [ , ] , prokaryotes [ ] , and viruses [ , ] , enabling diverse adaptations including evasion of host restriction factors (reviewed in [ ] ). our results suggest that gene amplification in an intermediate host may be a risk factor for broad cross-species transmission independent of other adaptive events. the rhesus cytomegalovirus (rhcmv) pkr inhibitor trs (rhtrs ) can block pkr activation and rescue replication of a vaccinia virus mutant lacking the pkr inhibitor e l (vvde l+rhtrs ) in several african green monkey (chlorocebus aethiops, agm) cell lines [ ] . however, we discovered that a recombinant vaccinia virus expressing rhtrs and lacking both of the known vaccinia pkr antagonists, e l and k l, (vvdedk+rhtrs ) produced to -fold less virus in agm-derived pro cells relative to vv-bg (which contains both e l and k l) although it replicated almost as efficiently as vv-bg in agm-derived bsc cells (fig. a) . thus, rhtrs varies in its ability to support vvdedk replication in different agm cells. the spread of microbes from animals to humans has been responsible for most recently emerging human infectious diseases, including aids, bird flu, and sars. therefore, understanding the evolutionary and molecular mechanisms underlying cross-species transmission is of critical importance for public health. after entering a new host cell, the success of a virus depends on its ability to overcome antiviral factors in the cell, such as protein kinase r (pkr). to investigate the process of virus transmission between species, we employed a recombinant vaccinia virus (vvdedk+rhtrs ) expressing the rhesus cytomegalovirus pkr antagonist rhtrs . this protein inhibits some african green monkey (agm) pkrs; however, it does not inhibit human or rhesus variants of pkr. serial passaging vvdedk+rhtrs in rhtrs -resistant agm cells resulted in rhtrs duplication in the viral genome, which improved vvdedk+rhtrs replication in agm cells. remarkably, rhtrs duplication also enhanced virus replication in human and rhesus cells. in contrast, passage of vvdedk+rhtrs in human cells, without prior adaptation in agm cells, did not improve vvdedk+rhtrs replication. these results support the hypothesis that amplification of a weak viral antagonist of a host defense protein in one species may enable cross-species transmission into new hosts that are nonpermissive to the initial virus. this vvdedk+rhtrs replication defect in pro cells may be due to incomplete inhibition of pkr in these cells by rhtrs . to test this hypothesis, we generated pro cells stably expressing either a pkr-specific shrna (pro -pkr kd), which resulted in a % reduction of pkr expression, or a scrambled control shrna (pro -ctrl kd) (fig. b) . similarly, pkr-specific rt-qpcr demonstrated a % reduction in pkr mrna from pro -pkr kd cells relative to pro -ctrl kd cells ( copies or copies pkr/ng total rna respectively) but little difference between pro and pro -ctrl kd cell pkr levels (fig. s ). in pro -pkr kd cells, vvdedk+rhtrs replication was almost completely rescued to vv-bg levels (fig. c) . we detected a similar increase in vvdedk+rhtrs replication after transiently transfecting pro cells with a sirna specific for pkr, but not a control sirna (data not shown). sequence analysis of pro pkr identified three non-synonymous mutations relative to a previously reported agm pkr (genbank # eu ) that is sensitive to rhtrs (fig. s , [ ] ). interestingly, one of these single nucleotide variants is heterozygous in pro cells, and changes a residue (t m) that is evolving under positive selection in primates [ ] . although additional studies will be needed to determine whether one or more of these pro pkr polymorphisms is responsible for increased resistance to rhtrs , the results shown in fig. demonstrate that the block to vvdedk+rhtrs replication in pro cells is mediated by pkr. to determine whether vvdedk+rhtrs could adapt to overcome the pkr-mediated block to replication in pro cells, we utilized a system of experimental evolution. we infected pro cells with vvdedk+rhtrs at a low multiplicity of infection (moi = . ). hours post infection (hpi) we lysed the infected cells, titered the resulting virus, and infected new pro cells, repeating this cycle multiple times. after four passages we observed a -to -fold increase in viral replication that remained stable for at least three subsequent passages in each of three independent lineages (fig. ) . we next performed a competition assay to assess the relative fitness of the passaged virus in comparison to the initial vvdedk+rhtrs virus. we coinfected pro cells with either vvdedk+rhtrs or vv-a, both of which express egfp, and the same competitor, vvde l +rhtrs , which expresses b-gal (moi = . for each virus). virus produced hpi was titered on permissive bsc cells and the specific progeny viruses were enumerated by detecting b-gal (vvde l+rhtrs ) and egfp (vvdedk+rhtrs and vv-a) expression in the plaques (fig. s ). vvdedk+rhtrs replicated , -fold better than vvde l+rhtrs , whereas vv-a replicated -fold better than vvde l+rhtrs , confirming that serial passage through pro cells increased the fitness of passaged viruses approximately -fold relative to the initial vvdedk +rhtrs . since the passaged viruses replicated more efficiently in a minimally permissive cell line, we investigated the ability of the passaged viruses to replicate in primary cells from more divergent primates. we have shown that rhtrs does not inhibit human or rhesus pkr in the context of vvde l [ ] . to determine whether the adaptations that evolved during serial passage in pro cells affected the virus species tropism, we infected primary human foreskin fibroblasts (hff) or primary rhesus fibroblasts (rf) with vvdedk+rhtrs , each of the three passaged virus pools or vv-bg at an moi of . (fig. ). as expected, vvdedk+rhtrs replicated poorly and vv-bg replicated efficiently in each cell type. remarkably, all three passaged pools replicated between -to , -fold better than vvdedk+rhtrs in both hff (fig. , center) and rf (fig. , right). for each pfu of vv-a, vv-b and vv-c used to infect the cells, . , . , and . pfu of progeny emerged from hff and . , . , and . emerged from rf, respectively, suggesting that these viruses were sufficiently well adapted to enable continuous propagation in these cells (see below). however, these virus pools still replicated -to -fold better in pro than in either human or rhesus cells (fig. , left) . thus, adaptation of vvdedk+rhtrs in minimally permissive agm fibroblasts also provides a substantial replication benefit in human and rhesus cells expressing distantly related pkr proteins. to elucidate the underlying mechanism for this gain of fitness, we harvested dna from passage viruses for genetic analyses and then passaged the viruses once more in pro to generate viral stocks for biochemical and infectivity analyses. gene amplification as a mechanism of rapid adaptation in vaccinia virus has been well documented [ , , ] . to determine whether gene amplification could account for the broadly improved replication of passaged vvdedk+rhtrs we performed paired-end illumina based deep sequencing (short read archive #srp ). based on read depth, we detected duplication of the rhtrs locus in all three passage virus pools but not in vvdedk+rhtrs (fig. a) . each of the passaged pools contained between . and . copies of rhtrs per genome, although these numbers reflect averages of a heterogeneous population of viral genomes. confirming this estimate of rhtrs copy number, the frequency of reads in which we detected a recombination site near the rhtrs locus increased as a percentage of total reads in viruses predicted to have more copies of rhtrs (fig. b) . we used pcr to confirm that the rhtrs locus was amplified, using externally directed primers specific to rhtrs that only amplify a product if there is a tandem duplication of the gene (fig. s a ). we detected kb products in all three virus pools, and . kb and . kb products only in the vv-a virus pool (fig. s b ). we were unable to obtain enough of the . kb band for further analysis, but we did characterize the kb and . kb products by sanger sequencing. the larger product was identical in all three passaged virus pools, and represents a recombination between the vaccinia virus gene l r upstream of rhtrs with j r downstream of rhtrs . in the smaller product j r recombined with the neor gene, which was introduced as a selection marker during construction of vvdedk+rhtrs (fig. s c ). these two sites represented the predominant recombination sites ( . % and . % respectively) identified by illumina deep sequencing. however, we found additional minor recombination sites by illumina deep sequencing, including a kb duplication in vv-b. the presence of an identical recombination site in all three passaged virus pools suggests that duplication may have been present at a very low frequency in the initial virus population even though we did not detect it in the illumina sequencing data. regardless, taken together these data demonstrate that the copy number of rhtrs in the viral genome was substantially increased by passage through pro cells. unlike a previous study which identified adaptive point mutations arising after locus expansion [ ] , we did not detect any point or indel mutations in rhtrs in any of the passaged virus pools. however, we identified vaccinia virus gene mutations present at greater than % frequency in at least one of the pools ( table ). all three passaged pools had one or more of four different single nucleotide deletions within the a r gene at frequencies ranging from to %. transition mutations affecting the a r and a r genes were present at . % frequencies in vv-a and vv-b respectively, but were rare or absent in the other viral pools. none of the other mutations were detected in all three pools or occurred at . % frequency in any pool, so are unlikely to account for the improved replication of the passaged viruses. however, the presence of these vv gene mutations raised the question of whether the expanded species tropism we observed was due to the vv gene mutations or to rhtrs amplification. if rhtrs amplification alone is sufficient for the observed increase in fitness, we reasoned that overexpression of rhtrs in trans might rescue vvdedk+rhtrs replication. to investigate this possibility, we stably transduced rhtrs into hff (hff+rhtrs ), and confirmed rhtrs expression by immunoblot (data not shown). we also prepared a control cell line (hff-lhcx) by transducing the empty vector, lhcx, into hff. in the control cells, vvdedk+rhtrs replicated approximately -fold less efficiently than vv-bg (fig. a ). in hff+rhtrs , vvdedk+ rhtrs replication increased more than -fold. thus, combined expression of rhtrs from genes in both the cell and the infecting virus potentiated vvdedk+rhtrs replication, supporting the hypothesis that rhtrs amplification alone is sufficient to expand the species tropism of vvdedk+rhtrs . although rhtrs amplification provided a substantial growth benefit in hff and rf, the passaged viruses still replicated -to -fold less efficiently than vv-bg (fig. ) . to determine whether this incomplete rescue in hff was due to incomplete pkr inhibition or represented a second block to replication, we infected hff stably transduced with either a pkr specific shrna (hff-pkr kd) that reduces pkr expression . %, or a nonspecific shrna (hff-ctrl kd) [ ] . knocking down pkr increased vvdedk+rhtrs replication , -fold, indicating that pkr is a major barrier to replication in these cells (fig. b ). all three passaged virus pools replicated , -fold better in hff-pkr kd cells than in hff-ctrl kd cells, suggesting that rhtrs amplification, which fully inhibits pro pkr, only partially inhibits human pkr. however, these viruses all replicated , fold less well than vv-bg in the hff-pkr kd cells. this remaining replication defect may be due to incomplete pkr knockdown in these cells [ ] , although it is also possible that an additional host factor inhibits vvdedk+rhtrs replication in hff. unlike many known pkr inhibitors that block pkr phosphorylation [ , ] , rhtrs inhibits the pkr pathway after pkr phosphorylation but prior to eif a phosphorylation [ ] . however, it is possible that rhtrs amplification inhibits pkr through an alternative mechanism, such as dsrna sequestration. to determine whether rhtrs amplification altered the mechanism of pkr inhibition, we infected pro with vv-bg, vvde l, vvdedk+rhtrs , and vv-a at an moi of , and collected cell lysates hpi. s metabolic labeling demonstrated that vvdedk+rhtrs expressed vaccinia virus proteins in pro cells, though in much lower quantities than vv-bg, consistent with the former virus being unable to inhibit pkr completely (fig. , top left panel). in contrast, vv-a produced abundant vaccinia virus proteins, similar to vv-bg, confirming that this virus efficiently inhibits the pkr pathway. we used immunoblot analyses to determine the stage of the pkr pathway inhibited by each virus (fig. , lower panels). rhtrs expression was noticeably higher in the vv-a infected cells compared to those infected with vvdedk+rhtrs , consistent with the better replication of vv-a in pro cells (fig. , lanes and ). pkr phosphorylation was elevated in all virus infected cells except vv-bg, consistent with our previous report [ ] that even when rhtrs blocks the pkr pathway, it does not block pkr autophosphorylation (fig. , lanes - ) . phospho-eif a levels in vvdedk+rhtrs infected cells were intermediate between mock and vvde l infected pro cells, suggesting that a single rhtrs gene weakly inhibits the pkr pathway (fig. , lane ) . infection with vv-a resulted in low levels of eif a phosphorylation, similar to that detected in vv-bg infected pro cells (fig. , compare lanes and ), indicating that rhtrs amplification is sufficient to completely inhibit pkr-mediated translational shutdown at a stage after pkr phosphorylation. together, these data suggest that pkr-mediated pressure in pro cells selected for rapid amplification of the rhtrs locus, and that this amplification was sufficient to enable the virus to block pkr-mediated defenses in pro cells without altering the mechanism of rhtrs -mediated pkr inhibition. in previous studies, rhtrs alone was insufficient to inhibit human pkr [ ] ; however, the passaged viruses reported here replicate substantially better in hff than vvdedk+rhtrs , suggesting that amplification of rhtrs is able to inhibit pkr at least partially in these cells. to test this hypothesis and determine whether the mechanism of pkr inhibition was the same in hff as it is in pro , we infected hff (moi = ) and prepared cell lysates hpi. in contrast to infection of pro cells, infection of hff with vvdedk+rhtrs resulted in nearly complete shut off of protein synthesis by hpi (fig. , top right panel) and produced only trace amounts of rhtrs . compared to vvdedk+rhtrs , vv-a infection of hff resulted in detectable, though still low overall levels of s labeled proteins, and much more rhtrs (fig. , lanes and ) . pkr phosphorylation and eif a phosphorylation were elevated after infection with both vvdedk+rhtrs and vv-a compared to mock or vv-bg controls (fig. , compare lane to lanes and ). these data suggest that a single copy of rhtrs was insufficient to inhibit the translational shutoff mediated by human pkr, but amplification of this weak antagonist resulted in partial inhibition of human pkr allowing enough protein synthesis to support a modest level of virus replication. because selection in agm cells resulted in a broad expansion of viral species tropism, we investigated whether passage of vvdedk+rhtrs directly in hff would similarly select for mutants, such as rhtrs amplification, that improved replication in hff. we therefore serially infected pro cells and hff with vvdedk+rhtrs in parallel (fig. ) . in pro cells, viral fitness again increased after four passages. in contrast, virus replication was strongly inhibited in hff, and we were unable to detect any viral replication after three rounds of infection in all three pools. these data suggest that, under these experimental conditions, adaptation in pro cells was a necessary intermediate step for improved replication in hff. finally, we evaluated whether the improved replication of the viruses that had been passaged in pro cells was sufficient to enable stable propagation in hff. we therefore serially infected hff with vv-a, vv-b and vv-c at low multiplicity of infection (moi = . at each passage). we were able to propagate these viruses in hff for at least four passages. moreover, replication increased between -to -fold after only two passages, suggesting further adaptation occurred in hff cells (fig. b) . to define mutations that may have evolved during serial passage in hff, we performed paired-end illumina based deep sequencing on viral dna isolated after the fourth round of passage in hff. again, we did not find any mutations in rhtrs ; however, we detected an average rhtrs copy number of . , . , and . for the virus pools derived from vv-a, vv-b and vv-c, respectively, representing an average expansion of the rhtrs locus by approximately one additional copy relative to the pro -adapted viruses. in addition, two vv gene mutations (in f l [indel] and j r) that arose during pro adaptation were lost after hff adaptation, and two new mutations (in f l [missense] and h l) of uncertain significance appeared during hff adaptation (table s ) . taken together, our study suggests the hypothesis that gene amplification acts broadly to increase replication in a variety of hosts, and may provide a molecular foothold that allows for continued speciesspecific adaptation of the virus in more resistant hosts. cross-species pathogen transmissions have been responsible for more than % of all emerging infectious diseases in humans during the past years [ ] . human immunodeficiency viruses, avian influenza viruses, and the recently described middle east respiratory syndrome coronavirus exemplify the ongoing threat and potential of animal viruses to spread to and among humans, highlighting the urgent need to understand the mechanisms underlying cross-species transmission and adaptation to new hosts. one such mechanism, genetic locus amplification in response to selective pressure, has been observed in both viruses and bacteria [ ] [ ] [ ] ] . here we demonstrate that amplification of the exogenous gene rhtrs is sufficient to block potent pkr-mediated inhibition and improve vvdedk+rhtrs replication in agmderived pro cells. this adaptation also expanded the species tropism of the virus, enabling markedly improved replication in otherwise resistant human and rhesus monkey cells. importantly, vvdedk+rhtrs failed to replicate in hff to a level sufficient to sustain transmission upon serial passage, demonstrating that adaptation in pro was a critical intermediate step to expand the viral species tropism. thus, the process of adaptation in one host may increase the likelihood of virus transmission to a variety of divergent species. under pkr-mediated selective pressure, the rhtrs locus amplified during serial passage of vvdedk+rhtrs in minimally permissive pro cells (fig. ) . it is not clear whether the initial duplication(s) occurred during preparation of the vvdedk+rhtrs stock in bsc cells or during the first few passages in the pro cells. the observation of a faint pcr product from the starting virus, vvdedk+rhtrs using outward directed rhtrs primers in one of three experiments (fig. s ) , and the detection of an identical recombination break point in all independently passaged virus pools suggest the amplification may have been present at a very low level in the starting virus. however, amplification of the locus in vvdedk+rhtrs occurred below the level of illumina deep sequencing detection (fig. ) , supporting the idea that if rhtrs duplications are present they are rare in the initial vvdedk+rhtrs stock. additionally, we detected recombination between j r and neor (fig. s ) only in the vv-a pool, suggesting that recombination events did arise during serial passage. regardless of when they arose, amplifications of the rhtrs locus were substantially enriched during virus passage under selective pressure (figs. and ) . a previous study demonstrated amplification of k l as an adaptive mechanism against human pkr with strikingly similar kinetics to the current study [ ] . together, these two studies support the hypothesis that preexisting or frequently arising de novo gene duplications enable vaccinia virus to adapt rapidly to selective conditions imposed by relatively resistant host restriction factors. the ''accordion hypothesis'' of rapid evolution posits that gene amplification provides a replication benefit to the virus and those extra copies of a weak viral antagonist of host defenses provide additional templates to acquire potentially adaptive mutations. indeed, elde, et al. detected such an adaptive mutation in k l (h r), apparently arising after amplification of the locus [ ] . therefore, we were surprised that no mutations arose in rhtrs , although it may be that additional rounds of replication would reveal such mutations. we did, however, detect mutations in endogenous vaccinia virus genes that occurred at . % frequency (fig. ) . while our data suggest that none of these mutations are necessary to expand the species tropism of vvdedk+rhtrs (fig. ) , we have not ruled out the possibility that they may provide some replication benefit. the presence of mutations in a r, a r, and a r are the most intriguing, as they were either present at . % frequency in one pool (a r and a r) or detectable in all three pools (a r). none of these genes has been previously implicated as a pkr antagonist. a r is conserved across multiple poxvirus families (http://www.poxvirus.org), but its function is unknown. a r is a subunit of rna polymerase. if the mutation we identified acts like some other reported rna polymerase mutations to decrease transcription elongation [ , ] , this mutation might result in less dsrna production and therefore less pkr activation. a r is a gene of unknown biochemical function that may be involved in evasion of the adaptive immune response [ , ] . all three passaged virus pools contained nucleotide deletions in a r at greater than % frequency. a r orthologs are conserved across most poxviruses but, intriguingly, variola virus contains a truncation in its a r consistent with previous studies of rhtrs in other agm cell types [ ] , pkr, but not eif a, was phosphorylated in pro cells infected with vv-a. thus, the improved replication of the passaged virus pools in pro cells was likely due to enhancement of the basic rhtrs -mediated inhibition of pkr, and not due to another mechanism, such as dsrna sequestration or reduction in the abundance of dsrna. vvdedk+rhtrs infected pro cells had phospho-eif a levels lower than that found in vvde l infected cells (fig. , lanes and ) , suggesting that even just a single copy of rhtrs is able to inhibit pkr function to a small degree, but amplification of the locus appears to be needed to express enough rhtrs to inhibit eif a phosphorylation potently and enable efficient viral replication. it is also possible that elevated expression of rhtrs is necessary to block another activity of pkr, such as autophagy or inflammasome responses [ ] [ ] [ ] that might aid in replication. adaptation of vvdedk+rhtrs to pro cells provided a substantial replication advantage in both human and rhesus monkey fibroblasts. although vv-a replicated to much higher titers and expressed more rhtrs than vvdedk+rhtrs in hff, s metabolic labeling revealed relatively low protein synthesis rates and eif a phosphorylation was still elevated after vv-a infection compared to vv-bg. a substantial proportion of the block to vv-a replication in hff is still mediated by pkr despite rhtrs overexpression (fig. b) , suggesting that further adaptation in hff may be necessary to block the pkr pathway in hff completely. these results indicate that rhtrs overexpression blocks human pkr incompletely. nonetheless, viruses that had adapted by initial passage in pro s replicated in hff at a level sufficient to enable sustained passage in hff (fig. b) . furthermore, these pro -adapted viruses acquired additional changes as a result of serial passage in hff, although the biological relevance of these changes is currently unclear. combined with the observation that serial passage of vvdedk+rhtrs in hff failed to generate any adapted viruses (fig. a) , these data suggest that adaptation of rhtrs in minimally permissive pro cells was a critical intermediate step in the generation of a virus with broadened species tropism. cross-species pathogen transmission is an important source of emerging infections worldwide. our study illustrates that gene amplification of a weak viral antagonist of pkr can broaden the host range of vaccinia virus. the presence of gene families in other large dna viruses, e.g. the cytomegalovirus us family, of which rhtrs is a member, provides indirect evidence that episodes of locus amplification have also occurred in other viruses. adaptation of a viral antagonist through non-synonymous mutations has the potential to confer a species-specific advantage for the virus in a specific host. in contrast, gene amplification and subsequent overexpression of the antagonist is more likely to increase protein activity through mass action effects in a variety of hosts. thus, gene amplification may be a common evolutionary strategy employed by large dna viruses, permitting modest replication in otherwise resistant host species and providing a molecular foothold that enables further adaptations necessary for more efficient replication and spread in the new host. viruses and cell culture agm fibroblasts (pro , coriell institute for medical research) human foreskin fibroblasts (hff), rhesus fibroblasts (rf), bsc and hela cells, and derivative cell lines were maintained in dulbecco's modified eagle's medium supplemented with % nuserum (bd biosciences) as previously described [ ] . at times, pro cell lines were also propagated in minimal essential medium with % fetal calf serum and anitbiotics to enable more rapid growth. pro cells were transduced with a nonsilencing control or pkr-targeting shrna lentiviral vectors (open biosystems, catalogue numbers rhs - and rhs , respectively) and selected in puromycin ( mg/ml) to generate pro -ctrl k/d and pro -pkr k/d cells. peq was constructed by moving the rhtrs gene, with a c-terminal biotinylation signal and x-his tag, as a hindiii/pmei fragment from peq [ ] into the hindiii/hpai sites of plhcx (clontech laboratories, inc). hff-lhcx and hff+rhtrs were produced by transducing hff with retroviral vectors made using lhcx and peq , respectively and selecting with hygromycin b ( mg/ml). vaccinia virus (vv) copenhagen strain (vc ) [ ] and vvde l [ ] , both obtained from bertram jacobs (arizona state university), and vv-bg (vc -lacz in [ ] were propagated and titered in bsc cells. vc-r (vvde ldk l) was constructed by replacing the e l gene in the k l-deleted vacv vp strain (dk l in vc background, provided by bertram jacobs) [ ] by homologous recombination. the bp arm was created by pcr amplification of vc dna with primers c ( -gattaaggg-tactagcggcaccg ) c ( -ttttagagagaacta-acacaaccagc- ). the bp arm was created by pcr amplification of vc dna with primers c ( -gtgtag-taagctagcgagctcggtaccttctagttatcaataa-cagttagtagtttag- ) c ( -ccaacaaactgttc-tcttatgaatcg- ). the reading frame of egfp including the pest sequence was amplified with primers c ( -gct-ggttgtgttagttctctctaaaacccgggatccaccg-gtcgcc- ) c ( -ggtaccgagctcgctagcttac-tacacattgatcctagcagaagc- ) using pd egfp-n (clonetech) as the template. pcr products were gel-purified and mixed together as template for fusion pcr using c c . pfuultra polymerase (agilent technologies) was used for these pcr reactions. pcr products were cloned into the pcr . topo vector to generate plasmid s . s was used as template for pcr amplification of marker + and arms using c c followed by gel-purification. bs-c- cells grown on well plates were infected with vp at moi = and transfected hours after infection with mg of the purified pcr product. cell lysates were collected after hours and plated in a dilutions series on rk +e l+k l cells [ ] . green plaques were picked after hours at the highest dilution possible and plaque purified an additional three times on rk +e l+k l cells. egfp expression in vc-r is under the control of the endogenous e l promoter. vvdedk was propagated and titered using hff+trs cells (hf-trs in [ ] ). vvdedk+rhtrs was constructed by homologous recombination of plasmid peq [ ] into the thymidine kinase (tk) locus of vvdedk. recombinant virus was plaque purified three times in bsc under g selection and subsequently propagated and titered on bsc cells. total rna from pro cells was amplified using previously reported pkr specific primers [ ] . the amplification product was gel purified and cloned using the strataclone pcr cloning kit (agilent). multiple plasmids were submitted for sanger sequencing using pkr specific sequencing primers (# : -atggctggt-gatcttgcac; # : -gtgaacaactcacttgcttc; # : -gaaactagacaaagttttggc; # : -ctaa-catgtatgtcgttcct; # : -aaggcacttagtctt-tgatc; # : -tctgatatctcaagcaatgc). contigs were assembled and curated in geneious pro v . . (genbank #kf - ). the predicted amino acid sequence was aligned to the predicted amino acid sequences of previously reported agm (genbank # eu ), rhesus macaque (genbank# eu ), and human pkr (genbank # nm ) using clustalw (http://www.ch.embnet.org/software/clustalw.html) [ ] . for every round of infection, triplicate confluent cm dishes ( figure ) or -well plates (figure ) of pro or hff were infected with vvdedk+rhtrs (moi = . ). two days postinfection, cells were collected, pelleted and resuspended in ml dmem+ % nuserum. after three freeze/thaw cycles, virus titers were determined on bsc by plaque assays and used for the next round of infection. if insufficient virus was produced to infect cells at moi = . , the entire volume of virus lysate was used in the subsequent round of infection. vaccinia virus dna from passage in pro ( figure ) or passage in hff (figure right) viruses was purified from infected cell cytoplasmic extracts for genetic analyses described below [ ] . the passage pools were further expanded (passage ) in pro for use in virologic assays. pro cells were co-infected with . moi of either vvdedk+rhtrs or vv-a, and . moi of vvde l+ rhtrs as a common competitor. two days post-infection cells were collected, pelleted and resuspended in ml dmem+ % nuserum. after three freeze/thaw cycles, virus titers were determined on bsc by plaque assays. vvde l+rhtrs plaques were detected with the b-gal substrate imagene red c rg following the manufacturer's directions (life technologies). plaques were imaged on a typhoon trio imager (egfp - nm excitation, bp filter; imagene red - nm excitation, bp filter) at mm/pixel resolution, and classified as gfp+, imagene red+, or double positive using imagej software (http:// rsb.info.nih.gov/ij/). externally directed pcr. externally directed oligonucleotides were designed that bind near the or end of rhtrs (# : -tgtgggaggatgcattgcag; # : -ggc-gactacaatccccattg). high-fidelity pcr was performed with ng of purified virus dna following the manufacturer's directions (phusion, thermo scientific). reactions were pulsed at uc s followed by cycles of uc s, uc s, uc min. amplification products were gel-purified and cloned using the strataclone pcr cloning kit (agilent) following the manufacturer's suggested protocol. recombination sites were identified by sanger sequencing of these products. library preparation and sequencing. from each viral pool, ng of viral dna was sheared and ligated to adaptors by transposition using the nextera kit (epicentre), following the manufacturer's direction with several modifications: transposition was performed in ul volume using . ml nextera transposase enzyme and ''hmw'' reaction buffer at final x. transposition was carried out for minutes at uc, after which sheared, adaptor-ligated templates were transferred directly to pcr reactions for amplification. in addition, pcr incorporated a sample-specific barcode tag on the reverse primer. amplified libraries were pooled and cleaned using ampure beads (beckman coulter) at a . : ratio of beads to input. the pooled libraries were sequenced on an illumina hiseq instrument (illumina) using -bp forward and reverse reads with a -bp index to read the per-sample barcode. sequence analysis. de novo assembly guided by the vaccinia virus copenhagen genome (genbank #m . ) was used to construct a reference assembly sequence for vvdedk+rhtrs (tables s and s ). shotgun reads from the parental virus were assembled to contigs using abyss . . [ ] ; parental virus reads were then realigned against the resulting assembly contigs using bwa . . [ ] . discrepancies between reads and contigs, reflecting de novo assembly errors, were corrected in the contigs following manual examination, and contigs were ordered and concatenated by comparison to the copenhagen sequence. reads from each evolved viral pool were aligned to the parental reference genome, and single-base and short-indel variants were called using the genome analysis toolkit [ ] . custom scripts were used to determine the allele frequency (fraction reads supporting variant allele out of all aligned reads at a given site) for all variants found in any sample. copy number was estimated by counting the depth of read coverage within sliding windows of bp, correcting for effects of differences in g+c composition, and dividing by the parental strain read depth. everted reads at duplication breakpoints were identified using bwasw [ ] and custom scripts, as previously described [ ] . cells were mock infected or infected with vaccinia viruses (moi = ). one day postinfection, the cells were lysed in % sodium dodecyl sulfate (sds). equivalent amounts of the lysates were separated on % sds-polyacrylamide gels, transferred to polyvinylidene difluoride (pvdf) membranes, and probed with one of the following antibodies: pkr (sc- ; santa cruz biotechnology, inc.), phospho-pkr (t ; - ; epitomics), eif a or phospho-eif a (ser ) antibody (both from cell signaling technology, catalog numbers and , respectively), trs a [ ] , or actin (a ; sigma). all purchased antibodies were used according to the manufacturer's recommendations. proteins were detected using the western star chemiluminescent detection system (applied biosystems) according to the manufacturer's recommendations. densitometry measurements were performed using imagej. total rna was extracted from pro , pro -ctrl kd, and pro -pkr kd cells with trizol reagent following the manufacturer's protocol (invitrogen), and ng of total rna was assayed per reaction. the standard curve was generated from fold serial dilutions of a pro pkr containing plasmid (described above in pro pkr sequence analysis, peq ) diluted in pg/ml salmon sperm dna. all samples were amplified in triplicate using the gotaq -step rt-qpcr kit following the manufacturer's protocol (promega) using pkr specific primers (# : -cacagaattgacggaaagac; # : -atcccaacagccattgtagt). rt-qpcr was performed on a rotor-gene q thermocycler (qiagen) with temperature holds at uc min and uc min followed by cycles of uc s, uc s. raw data was analyzed using the included rotor-gene q series software using the automatic cycle threshold (ct) setting for assigning baseline and threshold for ct determination. nonlinear regression analysis to determine pkr copy number in the experimental samples was performed in graphpad prism . sds. equivalent amounts of protein from each sample were separated on % sds-polyacrylamide gels, dried, and visualized by autoradiography. figure s rt-qpcr analysis of pkr copy number in pro -derived cell lines. rt-qpcr was performed on serial -fold dilutions of a pkr containing plasmid from to copies of pkr to generate a standard curve (top). data are represented as the mean +/ the % confidence interval. pkr copy number/ng total rna was interpolated from this standard curve for pro , pro -ctrl kd, and pro -pkr kd cells (bottom). relative to pro cells, pro -ctrl kd cells expressed slightly less pkr ( % decrease), while pro -pkr kd cells had a % decrease in pkr expression. similarly, pro -pkr kd cells expressed % less pkr than pkr-ctrl kd cells, consistent with fig. b. (tif) figure s amino acid alignment of african green monkey, human, and rhesus pkr. predicted pkr amino acid sequences from agm (pro t and eu ), rhesus macaque (genbank# eu ), and human (genbank # nm ) were aligned using clustal w ( . ) [ ] . we identified three non-synonymous amino acid differences (grey or green highlighted residues) between the two agm alleles. one of these differences is at a site that is evolving under positive selection in primates (green highlighted residue) [ ] . relative to pro pkr, rhesus and human pkr are . % and . % identical at the amino acid level, respectively. (tif) figure s increased fitness of vv-a compared to vvdedk+rhtrs assessed by indirect competition assay. pro cells were co-infected with either vvdedk+rhtrs or vv-a (moi = . ) and the same competitor virus, vvde l+rhtrs (moi = . ). two days post-infection viral progeny were collected and titered on bsc cells. (a) representative typhoon image of virus titer plates produced by infection with vvde+rhtrs in combination with either vvdedk+rhtrs (top panels) or vv-a (bottom panels). (b) plaques were scored for egfp expression (vvdedk+rhtrs or vv-a), b-gal expression (vvde l+ rhtrs ) or both (double +) as described in materials and methods. vv-a replicated , -fold better than vvdedk+ rhtrs relative to vvde l+rhtrs . data are represented as the mean + sd. (tif) figure s predominant recombination sites identified in rhtrs locus amplification. (a) schematic of externally directed pcr. externally directed oligonucleotides (grey arrows) were designed to bind to the ends of rhtrs (white). only tandem duplications will produce an amplification product. (b) externally directed pcr revealed enrichment of rhtrs amplification products in the passaged virus pools. (c) schematic of the initial rhtrs locus in vvdedk+rhtrs (top), and the predominant recombination sites identified in all three passaged viruses (middle), or in vv-a alone (bottom). vaccinia virus genes are colored green or blue, and exogenous sequences are colored dark red (rhtrs ) or brown (neor). pcr products amplified by externally directed primers (black bars), the duplicated sequence (grey boxes), and the recombination sites (red arrowheads), are indicated. recombination sites occur (middle) after nt (j r) and before nt (l r) relative to the reference vaccinia virus. copenhagen strain (genbank #m . ), or (bottom) after nt (j r) and before nt (neor -pcdna . , invitrogen). (tif) the global threat of emerging infectious diseases the role of wildlife in emerging and re-emerging zoonoses crossspecies virus transmission and the emergence of new epidemic diseases nipah virus outbreak in malaysia the emergence of nipah and hendra virus: pathogen dynamics across a wildlife-livestock-human continuum origins of hiv and the aids pandemic the cytoplasmic body component trim alpha restricts hiv- infection in old world monkeys structure and function of the protein kinase r protein synthesis and translational control during viral infection rules of engagement: molecular insights from host-virus arms races protein kinase r reveals an evolutionary model for defeating viral mimicry rapid evolution of protein kinase pkr alters sensitivity to viral inhibitors multiple duplications of yeast hexose transport genes in response to selection in a glucose-limited environment the influence of ccl l gene-containing segmental duplications on hiv- /aids susceptibility amplification-mutagenesis-how growth under selection contributes to the origin of genetic diversity and explains the phenomenon of adaptive mutation poxviruses deploy genomic accordions to adapt rapidly against host antiviral defenses vaccinia virus-encoded ribonucleotide reductase: sequence conservation of the gene for the small subunit and its amplification in hydroxyurea-resistant mutants gene duplication as a mechanism of genomic adaptation to a changing environment species specificity of protein kinase r antagonism by cytomegalovirus trs genes amplification of the ribonucleotide reductase small subunit gene: analysis of novel joints and the mechanism of gene duplication in vaccinia virus species specificity of protein kinase r antagonism by cytomegalovirus trs genes the dsrna protein kinase pkr: virus and cell control global trends in emerging infectious diseases temperature-sensitive mutants in the vaccinia virus a r gene increase double-stranded rna synthesis as a result of aberrant viral transcription mapping and phenotypic analysis of spontaneous isatin-beta-thiosemicarbazone resistant mutants of vaccinia virus vaccinia virus a r inhibits mhc class ii antigen presentation deletion of the a gene from modified vaccinia virus ankara increases immunogenicity and isotype switching regulation of starvation-and virus-induced autophagy by the eif alpha kinase signaling pathway novel role of pkr in inflammasome activation and hmgb release chemical genetics reveals a kinase-independent role for protein kinase r in pyroptosis evasion of cellular antiviral responses by human cytomegalovirus trs and irs nyvac: a highly attenuated strain of vaccinia virus distinct patterns of ifn sensitivity observed in cells infected with vaccinia k l-and e l-mutant viruses vaccinia virus-encoded eif- alpha homolog abrogates the antiviral effect of interferon myxoma virus protein m is a dual function immunomodulator that inhibits pkr and also conscripts rha/dhx to promote expanded host tropism and viral replication essential role for either trs or irs in human cytomegalovirus replication multiple sequence alignment with the clustal series of programs the preparation of orthopoxvirus dna abyss: a parallel assembler for short read sequence data fast and accurate short read alignment with burrows-wheeler transform the genome analysis toolkit: a mapreduce framework for analyzing nextgeneration dna sequencing data fast and accurate long-read alignment with burrows-wheeler transform versatile and open software for comparing large genomes we thank bertram jacobs (arizona state university) and michael axthelm (oregon health & science university) for reagents. we thank members of the geballe lab for helpful discussions and harmit malik (fhcrc), matt daugherty (fhcrc), and nels elde (university of utah) for advice and critical reading of the manuscript. we thank the fred hutchinson cancer research center genomic core and scientific imaging core for technical assistance. key: cord- -ao kenbx authors: lin, yao-tang; prendergast, james; grey, finn title: the host ubiquitin-dependent segregase vcp/p is required for the onset of human cytomegalovirus replication date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: ao kenbx the human cytomegalovirus major immediate early proteins ie and ie are critical drivers of virus replication and are considered pivotal in determining the balance between productive and latent infection. ie and ie are derived from the same primary transcript by alternative splicing and regulation of their expression likely involves a complex interplay between cellular and viral factors. here we show that knockdown of the host ubiquitin-dependent segregase vcp/p , results in loss of ie expression, subsequent suppression of early and late gene expression and, ultimately, failure in virus replication. rnaseq analysis showed increased levels of ie splicing, with a corresponding decrease in ie splicing following vcp knockdown. global analysis of viral transcription showed the expression of a subset of viral genes is not reduced despite the loss of ie expression, including ul / . furthermore, immunofluorescence studies demonstrated that vcp strongly colocalised with the viral replication compartments in the nucleus. finally, we show that nms- , a small molecule inhibitor of vcp, is a potent hcmv antiviral with potential as a novel host targeting therapeutic for hcmv infection. viruses are obligate intracellular pathogens, meaning that they are completely dependent on the host cellular machinery to replicate. identifying which host genes are necessary for virus replication extends our understanding of how viruses replicate, how cells function and provides potential targets for novel antivirals. here, we show that a cellular factor called valosin containing protein (vcp) is essential for human cytomegalovirus replication. we demonstrate that vcp is required for the expression of an essential virus gene called ie . finally we show that a chemical inhibitor of vcp is a potent antiviral against human cytomegalovirus, demonstrating the potential for vcp inhibitors as novel therapeutics against this virus. human cytomegalovirus (hcmv) is a highly prevalent herpesvirus, infecting to % of the global population depending on the socio-economic status. although normally asymptomatic in healthy individuals, hcmv infection is a significant cause of morbidity and mortality in immunocompromised populations, individuals with heart disease and recipients of solid organ and bone marrow transplant. hcmv is also the leading cause of infectious congenital birth defects [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . during infection, hcmv initiates a programmed cascade of gene expression, resulting in production of infectious virus. two of the first genes to be expressed are the major immediate early (mie) genes ie (ie ) and ie (ie ). the mie proteins have multiple roles during infection including transactivation of viral genes, which drives replication and virus production [ ] [ ] [ ] . because of this, they are thought to play a pivotal role in controlling the switch between latent and productive infection in hcmv [ , ] . while ie is required for efficient virus replication at low multiplicity of infection [ , ] , ie expression is essential, with deletion resulting in non-viable virus [ ] . ie and ie are generated from the same primary transcript by differential splicing and alternative polyadenylation [ , , ] . they share the first three exons, with splicing to the fourth or fifth exon determining expression of ie or ie transcript, respectively (fig ) . independent polyadenylation signals exist downstream of both exon four and exon five. such genomic arrangements, that require terminal exon skipping, are considered relatively unusual in the host cell, with specific factors and mechanisms involved in regulating the process not fully understood [ ] . valosin containing protein (vcp) belongs to the hexameric aaa atpase family and plays a pivotal role in ubiquitin mediated signaling through remodeling target proteins, often leading to proteosomal degradation [ ] . vcp contains two atpase domains, which hydrolyze atp to generate the energy required to remodel or unfold target proteins. through this action, vcp is able to segregate target proteins from associated cellular membranes or larger protein complexes. once segregated, the target protein is relocalised or degraded via the proteosomal complex. vcp can also affect which proteins are modified through its interaction with multiple ubiquitin regulatory co-factors, making vcp a central signalling hub for ubiquitin mediated regulation. in addition to ubiquitin, vcp has been linked to other post-translational modifiers such as sumo and nedd [ , ] . as such it is linked to a wide range of biological functions, including protein quality control, autophagy, chromatin remodeling, dna damage response and more recently rna processing [ ] . functionally, vcp plays a central role in protein homeostasis by facilitating proteosomal degradation of misfolded or damaged proteins as part of the endoplasmic reticulum associated degradation pathway (erad). in addition to its role in protein degradation, vcp has been linked to non-degradative functions involving removal and relocation of proteins from membranes and protein complexes. examples include removal of aurora b protein from mitotic chromosomes, removal of transcription factors from chromatin and disassembly of rnp complexes [ , ] . it has also been linked to a number of functional roles in virus replication including entry of sindbis virus and replication of poliovirus [ ] [ ] [ ] . while vcp plays a role in us -specific degradation of mhc class i protein during hcmv infection [ ] a direct role for vcp in the replication of hcmv has not previously been reported. using a focused sirna screen, we identified vcp as an essential factor for hcmv replication. we show that vcp is essential for the onset of virus replication and knockdown of vcp results in changes in alternative splicing of the mie transcripts, loss of ie expression and ultimately failure in virus replication. previously, we identified the host gene atp v c as a critical factor in hcmv virus production [ ] . atp v c is a component of the vacuolar atpase, and among other functions, is involved in membrane organization [ ] . to identify additional host membrane organization factors involved in hcmv replication we performed a focused sirna screen against host genes involved in membrane organization using pools of four sirnas against each target. primary human fibroblast cells were double transfected with each sirna pool in well format. two days post-transfection, the cells were infected with a low passage hcmv strain, tb e, expressing gfp from an sv promoter. gfp levels were monitored over a period of seven days by fluorometry to determine virus infection and replication levels (s table) . analysis of the screen indicated both increased and decreased virus replication ranging from % reduction in gfp levels to a % increase (fig a) . z-score analysis based on three biological repeats of the sirna screen identified five clear outliers that resulted in reproducible reductions in virus replication, as measured by gfp fluorescence intensity ( fig b) . of these, knockdown of vcp resulted in the largest negative z-score. to validate that the observed phenotype was due to knockdown of vcp, rather than potential off-target effects, the original sirna pool was deconvoluted to test the four individual sirnas against vcp. all four sirnas resulted in reduced vcp expression and reduced hcmv replication based on gfp fluorescence ( fig c) . the third sirna of the pool generated less efficient vcp knockdown, and corresponded with less inhibition of hcmv in the sirna assay, providing additional evidence that knockdown of vcp results in a direct reduction in virus replication. these results indicate that the initial observed phenotype is due to specific knockdown of vcp rather than off-target artefactual effects of the sirnas. although gfp expression serves as an effective read-out for virus replication, loss of gfp signal may not necessarily reflect reduced virus production. to determine the direct effect of vcp knockdown on hcmv virus production, one-step growth curves were generated. primary human fibroblast cells were transfected with sirna pools targeting vcp or a negative control non-targeting sirna. two days post transfection cells were infected at a multiplicity of infection (moi) of three with tb e-gfp. supernatant was collected at hour time points for seven days total. knockdown of vcp resulted in significant reduction in hcmv replication, with viral titers over four log lower in cells depleted of vcp compared to negative control cells at day seven ( fig d) . no amplification of infectious virus was observed in cells knocked down for vcp, suggesting a complete block in virus replication. cell viability assays showed a moderate decrease in cell viability at days post transfection with vcp sirna compared to negative control sirna, although visual inspection of cells did not indicate gross cytotoxicity and the reduction is not sufficient to account for the substantial reduction in virus replication (s fig). vcp is required for ie expression but not ie expression further characterization was performed to determine at what stage of the hcmv replication cycle vcp is required. analysis of gfp fluorescence at hours post infection (hpi) indicated that all cells were infected, demonstrating that vcp expression is not required for virus entry, translocation of genome to the nucleus or initial transcription of the viral genome ( fig a) . western blot analysis was performed to measure the levels of each of the major kinetic classes of hcmv genes, including immediate early (ie and ie ), early (pp ) and late (pp ), to define when disruption in virus replication occurs ( fig b) . in control cells, protein expression of each class of virus gene is clearly observed. however, in cells knocked down for vcp, neither ie nor downstream viral gene expression was detected. strikingly, despite being encoded by the same primary transcript, ie expression levels were higher relative to control cells, up until day four post infection. this data indicates that vcp expression is required for the onset of hcmv replication and suggests knockdown of vcp affects expression of the major immediate early genes. because knockdown of vcp resulted in specific loss of ie but not ie , we hypothesized that vcp may play a role in ie protein stability, transcript stability or regulation of alternative splicing and polyadenylation. to determine whether vcp was required for ie protein stability we inhibited the two main pathways of protein degradation using mg (proteasome inhibitorubiquitin dependent) and pepstatin a or e (protease inhibitors-ubiquitin independent) in the context of vcp knockdown (fig a) . if vcp regulation of ie was due to protein degradation, inhibition of these pathways should rescue ie protein levels. neither mg , pepstatin a, nor e rescued the loss of ie proteins levels following knockdown of vcp. instead, higher concentrations of mg phenocopied vcp knockdown in control cells, indicating that proteosomal function is required for ie but not ie gene expression. as vcp targets ubiquitinated proteins for proteosomal degradation, this result suggests that an intermediate protein, targeted by vcp, may inhibit ie expression and is consistent with previous publications showing inhibition of the proteasome reducing ie expression and hcmv replication [ ] . to determine whether regulation of the mie genes was occurring at the rna level, northern blot analysis was performed. fibroblast cells transfected with sirna against vcp or control sirna were infected h post transfection at high moi with tb e and total rna harvested at , and hpi. as shown in fig b, knockdown of vcp resulted in a substantial reduction of ie rna levels at and hpi, including the smaller ie and ie species. this result was confirmed by qrt-pcr, using primers specific for ie and ie transcript. supplemental fig a demonstrates the clear difference in the kinetics of ie and ie transcript accumulation in control cells, with ie transcript levels rapidly increasing following infection, while ie levels initially increase, then plateau between and hpi, before accumulating to substantially higher levels from hpi onwards. this accumulation fails to occur following knockdown of vcp, where levels of ie do not substantially increase after hpi, in contrast to control cells (s b and s d fig) . mie transcription is also affected at earlier time points with levels of both ie and ie reduced at hpi and increased at hpi (s c and s d fig) . an increase in ie and ie rna levels at hpi may be a result of increased mie promoter activity due to the associated loss of ie protein expression at early time points, as ie is a negative regulator of the mie promoter. ie transcript levels decrease rapidly to hpi, while protein levels remain high, suggesting that ie protein is stable and the increase in ie transcription at hours may contribute to the prolonged increase in ie protein levels. these results suggest that vcp may be affecting other aspects of mie transcription. however, northern, western and qrt-pcr data are all consistent with a substantial effect on ie expression from hpi onwards. to determine whether loss of vcp results in specific destabilization of ie transcript, rna levels were measured following treatment of cells with actinomycin d hpi. actinomycin d inhibits rna polymerase, blocking new transcription of ie and ie , allowing for monitoring of transcript stability over time. total rna was harvested at the indicated time points and ie and ie transcript levels determined by northern blot analysis. calculation of the half-life of ie and ie transcripts indicated that stability of both ie and ie modestly increased by . and . fold respectively, suggesting loss of vcp does not result in ie transcript destabilization ( fig ) . defining changes in alternative splicing of ie and ie is challenging as multiple factors, including promoter activity, rna stability and viral genome amplification can all contribute to an increase or decrease in total ie and ie mrna levels. to determine whether knockdown of vcp altered differential splicing of the mie region, rnaseq analysis was performed. this allows a direct comparison of read counts from the first three shared exons of the mie transcripts to the ie and ie specific exons four and five, respectively. this controls for changes in promoter activity and effects of genome amplification. therefore, based on our current understanding of rna processing, changes in the absolute ratio of exon four or five to the shared exons must be due to changes in splicing or changes in rna stability. cells were transfected with vcp sirna or a negative control sirna and infected with hcmv at an moi of three. total rna was harvested at , and hpi and subjected to strand-specific, pairedend, illumina sequencing. the annotated exons of the major immediate early region were clearly apparent from the mapped reads in all six samples and reads corresponding to the splice junctions were consistent with previous data for the region, indicating splicing between shared exons one to three and splicing between exon three and four, resulting in ie transcript, and three and five resulting in ie transcript (s fig) . consistent with the northern blot and qrt-pcr data, mie transcription increased at hpi but decreased at and hpi following knockdown of vcp (s fig) . furthermore, splicing of the mie primary transcript is heavily biased towards ie at hours, with ie splicing increasing as the infection progresses (s a fig). to determine whether knockdown of vcp alters the balance of splicing between ie and ie , the proportion of total reads mapping to the five exons of the ie region were calculated (s table) . fig a repre sents the absolute difference in exon frequencies from vcp knockdown samples compared to negative control samples. at hpi, knockdown of vcp has no effect on the relative proportion of reads mapping to exon four and five. however, by hpi there is a clear increase in the relative proportion of reads mapping to exon four in vcp knockdown samples, with a corresponding decrease in exon five read frequencies. in contrast knockdown of vcp has no effect on the relative proportion of reads mapping to the shared exons, indicating reduced splicing from exon three to five (ie ) and a corresponding increase in splicing from exon three to four (ie ). knockdown of vcp also resulted in lower frequencies of read pairs spanning exon three to five splice junction (ie ) and a corresponding increase in read pairs spanning exon three to four splice junction (ie ) (fig a and b, s b fig and s and s tables) . these results show that knockdown of vcp alters splicing of mie transcripts resulting in a failure to switch from ie splicing to ie splicing. to determine whether knockdown of vcp resulted in more generalized effects on virus transcript splicing, we analysed read counts for other well-characterized hcmv spliced transcripts (s fig). based on exon counts, only ul showed a similar pattern to mie transcripts in response to vcp knockdown where absolute exon frequencies for ul exon increased with a corresponding decrease in exon read frequencies. however, unlike the mie transcripts, analysis of paired-end reads did not support substantial splicing between ul exon to exons and (s fig). instead, exon and exons and of ul are likely to be predominantly independent transcriptional units with differing kinetics, with exon and requiring ie expression for transactivation. to determine the effect of vcp knockdown on global viral gene expression, read counts were mapped to the entire viral genome. as shown in supplemental fig total read counts mapping to the viral genome were relatively similar at hpi. however, while viral gene expression increased in control cells by approximately . fold between and hpi, expression in vcp knockdown cells only increased by -fold, indicating a general reduction in viral gene expression following vcp knockdown. this is unsurprising given the vital role ie protein plays in transactivation of viral gene expression. to determine whether knockdown of vcp results in a similar effect on all viral genes or differential effects, total reads were mapped to individual viral open reading frames and viral gene expression compared between control cells and vcp knockdown cells (fig and s table) . at hpi moderate reductions in viral gene expression can be observed, with a subset of transcripts expressed at relatively higher levels following vcp knockdown. in particular, the proportion of total reads mapping to each of the five exons was calculated, with the absolute difference in these values between vcp knockdown and corresponding negative control shown (numbers within exons). knockdown of vcp has no effect on the proportion of reads originating from exons , or , but is associated with a greater proportion of reads derived from exon (ie ) and decreased numbers from exon (ie ). (b) log ratios of the proportions of reads mapping to exon whose matching pair maps to either exons , or are shown. the proportion of read pairs spanning the shared splice junction between exon two and three is unaffected by vcp knockdown, whereas exon to frequency increases and to decreases. https://doi.org/ . /journal.ppat. .g expression levels of ul and ul , ul / and the mie transcripts ul (ie ) and ul (ie ) were relatively higher at hpi in vcp knockdown cells compared to control cells. however, at and hpi there is more profound global suppression of virus gene expression in vcp knockdown cells. this is particularly apparent for the non-coding genes rna . and . and other genes that are highly up regulated at later stages of infection, including ul , ul , ul and ul . despite the loss of ie expression, the expression of a subset of viral genes remained equivalent to control levels following knockdown of vcp (fig and s fig) . these include ul a, ul and ul , ul a and ul / , ul (ie ) and ul . this is particularly surprising for ul / which has previously been identified as a target for ie transactivation [ , ] . vcp is essential for hcmv replication altered mie splicing is not due to changes in cell cycle or delay in replication progression following vcp knockdown previous studies have shown that the cell cycle impacts on the regulation of mie alternative splicing with ie splicing and virus replication blocked at the g /m phase [ ] . furthermore, cyclin dependent kinases are required for efficient ie expression and cyclin a overexpression inhibits ie splicing and virus replication [ ] . to determine whether knockdown of vcp results in alterations in cell cycle, which in turn regulates mie splicing, cells were stained with propidium iodide following vcp knockdown. the results clearly show that knockdown of vcp has no gross effect on cell cycle control (fig ) . consistent with the majority of cells being in g /g phase, cyclin a levels remained undetectable by western blot in control and vcp knockdown cells (s fig). these results show that regulation of mie splicing by vcp is not a consequence of alterations in cell cycle control and instead represents a novel regulatory pathway. to determine whether the effect of vcp on mie expression could be the result of a simple delay or inhibition of progression in virus replication, we compared mie protein and transcript levels following treatment of cells with ganciclovir, a well characterized drug, which inhibits viral dna synthesis. while both ganciclovir treatment and vcp knockdown result in substantial reductions in ie gene expression, only knockdown of vcp results in a corresponding increase in ie gene expression and relative changes in ie and ie splicing (s fig) . furthermore, in contrast to vcp knockdown, ie and pp expression could be detected four days post infection following ganciclovir treatment. these data suggest alterations in mie levels, caused by vcp knockdown are not an artifact of delayed virus replication. previous studies have demonstrated that vcp plays an important role in the cytoplasm of hcmv infected cells, mediating us -dependent degradation of mhc class i, by stripping the protein from the er membrane in a ubiquitin dependent manner [ ] . to determine the cellular localisation of vcp during hcmv infection, primary fibroblast cells were infected at high moi with hcmv, fixed at -hour time points and stained for vcp. as shown in fig , vcp displayed dynamic temporal cellular localisation during hcmv infection. in uninfected cells, vcp staining resulted in diffuse signal throughout the cell. however following infection with hcmv, distinct puncta can be observed in the nucleus of infected cells. by hpi vcp was consistently found within two large puncta within the nucleus, which increased in size by hours. such staining is characteristic of the viral replication compartments. following infection with hcmv the viral genomes are deposited adjacent to nd domains before forming two distinct replication compartments within the nucleus, characterized by colocalisation of viral dna, dna replication proteins and ie protein [ ] . therefore, vcp localisation to the virus replication compartments in the nucleus coincides with increased ie expression. given that vcp is essential for hcmv replication, we investigated whether small molecule inhibitors against vcp are viable antiviral candidates. because vcp is a potential anti-cancer target, a number of effective small molecule inhibitors of vcp have been developed. nms- is a highly potent and selective inhibitor of vcp [ ] . to determine whether treatment of infected cells with nms- results in the same phenotype as vcp sirna knockdown, cells were pretreated with nms- or dmso and infected at high moi with hcmv. total protein was harvested every hours for a total of five days and subjected to western blot analysis. as shown in fig a, treatment with μm of nms- recapitulated the phenotypic effects of vcp sirna, with loss of ie transcript and protein expression. in contrast to sirna knockdown of vcp, treatment with nms- did not result in increased ie expression. this may be due to additional off target effects of the drug compared to sirna knockdown or a consequence of inhibiting the activity of vcp compared to directly knocking down the protein. next, we compared the antiviral activity of nms- to ganciclovir, currently the most commonly used hcmv antiviral. primary human fibroblast cells were pretreated with different concentrations of ganciclovir, nms- or dmso control, with cells and supernatant harvested seven days post infection for plaque assay. as shown in fig b, nms- and ganciclovir clearly inhibited the production of infectious virus. however, nms- displayed a higher level of potency at ten-fold lower concentrations, with an ic of . μm compared to . μm for ganciclovir. co-treating cells with nms- at the same time as infection also resulted in reduced ie levels and a similar reduction in infectious virus (fig c and s a fig). in contrast, treating cells hpi did not reduce ie expression or virus production to the same extent (fig c and s b fig) . while immediate early and early viral gene expression was not substantially affected by treatment of cells with nms- at hour post infection, expression of the late gene pp was substantially reduced, with infectious virus reduced fold, suggesting vcp may also be involved in late processes of virus replication. this is consistent with the pleiotropic nature of vcp and its potential to affect hcmv replication in a multitude of ways. treatment with nms- also showed little toxicity at effective concentrations (fig d) , suggesting nms- and small molecule inhibitors of vcp show significant potential as hcmv antiviral therapies. vcp plays a pivotal role in ubiquitin-dependent signaling through remodeling target proteins, often leading to proteosomal degradation. as such it is linked to a wide range of biological functions, including protein quality control, autophagy, chromatin remodeling and more recently rna processing [ ] . following systematic screening of host genes involved in membrane organization, we identified vcp as a critical host factor for hcmv replication. further characterization demonstrated that knockdown of vcp resulted in an initial increase in mie transcription, followed by a substantial reduction in the expression of the major immediate early transcript ie . strikingly, despite being expressed from the same primary transcript, ie levels increased following vcp knockdown. our data also suggests that vcp plays a role during late stages of virus replication as inhibition of vcp hpi did not substantially affect ie expression but still resulted in a two-log reduction in infectious virus. given the functionally pleotropic nature of vcp, it is unsurprising that knockdown results in multifactorial effects on the virus and potentially reflects the diverse roles of ubiquitin modulation and signaling during virus infection. viral gene expression during hcmv replication occurs through a tightly regulated cascade mechanism with expression of the mie genes ie and, in particular, ie , required for transactivation of early viral genes and subsequent late viral gene expression. due to the central role of the mie proteins in driving replication of the virus they have been suggested to play a pivotal role in regulating the balance between productive and latent infection. ie and ie are generated from the same primary transcript by alternative splicing, sharing the first three exons, while splicing to independent final exons through the relatively unusual process of terminal exon skipping [ , ] . consistent with previous publications [ , ] , our data shows that ie expression levels increase more rapidly than ie , due to preferential splicing to the ie terminal exon , resulting in lower levels of ie transcription, which plateaus between and hpi (s , s and s figs). as infection progresses, splicing to ie terminal exon increases, with a corresponding decrease in ie splicing. regulation of alternative splicing of the mie transcripts is therefore crucial for determining whether replication of the virus progresses, as expression of ie is dependent on this switch in splicing. although the timing and precise splicing pattern of the mie region has been well documented, how the splicing is regulated and the downstream consequences for virus replication are poorly understood [ ] . however, it is likely to depend on a complex interplay of cellular and viral factors and the architecture of the mie transcriptional region [ ] . expression of the mie transcripts and their splicing are intimately linked to multiple factors including cell cycle, cell type and differentiation status [ , , ] . suppression of mie transcripts during hcmv infection of myeloid cultures is thought to be key for establishing latent infection. conversely, activation and differentiation of latently infected cells results in up-regulation of mie gene expression and subsequent reactivation. during infection of fibroblast cells, expression of mie transcripts and replication are closely linked to cell cycle status, with mie expression suppressed during s/g phase [ , ] . this suppression is due in part to cyclin a , which alters splicing of the mie transcripts in a manner similar to knockdown of vcp, resulting in loss of ie expression and failure in virus replication [ ] . while expression of cyclin a results in a similar phenotype, our data shows that vcp knockdown does not cause gross changes in the cell cycle or increased cyclin a expression, indicating an alternative mechanism of action. although the majority of research on vcp has focused on its cytoplasmic activity, recent studies have identified equally important and diverse roles for vcp in the nucleus. the association of vcp with the viral replication compartments early in infection suggests that the nuclear functions of vcp may be playing a critical role in hcmv replication. studies in yeast and mammalian cell lines have demonstrated that vcp modulates the association of factors with chromatin, leading to regulation of cell cycle, transcription, dna replication and dna damage response, all of which could potentially affect virus replication and mie expression [ ] . previous reports have shown that hcmv induces the host dna damage response, with response factors including atm, γh ax and e f required for efficient hcmv replication and ie expression [ ] . γh ax also colocalises with the viral replication compartments in the nucleus. vcp acts downstream of atm and is required for the removal of ubiquitinated factors associated with dsdna break regions, allowing repair to proceed [ ] . if knockdown of vcp disrupts the dna damage response during hcmv infection, this could contribute to the observed loss of ie expression and inhibition of virus replication. reports are also emerging that vcp plays a role in transcript stability and regulation of splicing. hur is a rna binding protein that stabilizes transcripts by binding to canonical au rich regions within 'utrs. following ubiquitination of hur in response to stress signals, vcp removes hur from associated transcripts resulting in transcript destabilization [ ] . in drosophila, vcp has been linked to dendrite pruning in neuronal cells [ ] . expression of mutant forms of vcp results in incorrect splicing of a gene important to dendritic pruning, mical, possibly through mis-localisation of the rna binding protein tdp- . given the known mechanism of action of vcp it is likely that any effects on mie splicing are indirect, occurring through targeting of a ubiquitinated intermediate protein. one possible model is that a negative regulator of ie splicing exists at early stages during hcmv infection, which promotes ie expression at the expense of ie expression. based on the transcriptional architecture of the major immediate region, such a factor could regulate mie splicing by promoting polyadenylation of the ie transcript, thereby inhibiting read-through to the ie final exon. the factor would be ubiquitinated and removed by vcp between and hpi, allowing read-through of the first polyadenylation site and splicing of the ie transcript. this would be consistent with the initial plateau in ie transcription and localisation of vcp to the replication compartments at to hpi, where spliceosome and proteasome components have also been reported to colocalise [ , ] . the nms- data also supports this model, with inhibition of ie expression only effective with either pre-treatment or addition of the drug at the same time as infection. treatment at hpi was ineffective at blocking ie expression, suggesting a window in which vcp activity is required for regulation of mie splicing. interestingly, blocking de novo protein translation by cycloheximide treatment, effectively rescues ie transcription following nms- treatment, suggesting the inhibitory factor is induced by virus infection (s fig) . although not common, there are a number of examples of terminal exon skipping in the human genome, which would be analogous to the proposed model. maturation of b cells to differentiated plasma cells results in a switch from predominantly membrane bound igm to secreted igm. studies have shown that increased expression of the polyadenylation factor cstf- promotes polyadenylation upstream of the exons required for membrane bound igm resulting in increased expression of the secreted form [ , ] . tissue specific regulation of calcitonin peptide is also regulated through differential polyadenylation of an embedded terminal exon [ ] . here, the splicing factor srsf (srp ) binds to a polya enhancer sequence and directs polyadenylation of the upstream polya site through recruitment of polya factors. restricting srsf activity results in removal of the upstream terminal exon by splicing and enhanced expression of the downstream terminal exon. in these examples splicing is directly linked to cellular differentiation, in the case of igm, and tissue specificity, in the case of calcitonin. these factors are fundamental to the regulation of hcmv latency with cell type specificity determining the site of latency and differentiation of latently infected cells linked to reactivation [ , ] . given the central role of ie in determining virus replication, linking regulation of mie splicing to cellular differentiation and tissue specificity would be a potent mechanism of regulating the establishment, maintenance and reactivation of latency in hcmv. experiments are currently underway to determine whether regulation of mie splicing by vcp follows a similar paradigm to cellular examples and whether such regulation is involved in hcmv latency. finally, as vcp is clearly essential for virus replication, small molecule inhibitors of vcp are potentially attractive antiviral candidates. hcmv-related illness accounts for more than % of diseases associated with solid organ transplant patients. prolonged treatment, especially in patients with severely suppressed immune systems, greatly increases the risk of antiviral resistance [ ] [ ] [ ] . very few antivirals have been developed for use against hcmv since the licensing of ganciclovir, and of these, the same viral genes are targeted, reducing the likely usefulness of these drugs against resistant strains [ ] . an alternative strategy for the development of novel antivirals involves targeting of host genes required by the virus for successful replication. vcp has been identified as a potential anti-cancer target and as such a number of small molecule inhibitors have been developed, the most potent of which is nms- [ ]. we show that nms- is times more potent than ganciclovir at equivalent concentrations, with little sign of toxicity at active levels. whether the drug is toxic in vivo remains to be determined. deletion of vcp in transgenic mice is non-viable and naturally occurring mutations in humans are linked to severe developmental defects [ , ] . however, transient inhibition of vcp by small molecule inhibitors may be a viable treatment option, especially in patients where resistance to current antivirals poses a significant risk to health. normal human dermal fibroblasts (nhdf; gibco) were maintained in dulbecco's modified high glucose media (dmem; sigma) supplemented with % fetal bovine serum (fbs; gibco) and % penicillin-streptomycin (invitrogen). a low passage hcmv strain tb e, which expresses gfp from an sv promoter was used for sirna library screening, western blot analysis, and northern blot analysis. laboratory adapted hcmv strain ad was used for immunofluorescence experiments. the human sigenome sirna library that targets membrane trafficking genes ( sir-nas per gene; dharmacon, inc.) and other selected genes were included in the primary screen. in brief, nhdfs were seeded in -well plate a day before sirna transfection. next day, cells reached - % confluency and were transfected with sirna twice ( hours apart between first and second transfections) using lipofectamine rnaimax (invitrogen) according to the manufacturer's protocol. transfected nhdfs were incubated for hours and then infected with gfp expressing tb e virus at an moi of three. gfp intensity was monitored every hours with synergy ht microplate reader (biotek). the entire screen was performed in duplicate and repeated three times. vcp gene identified from the primary screen was subsequently silenced with four individual sirnas to eliminate the possibility that the phenotype was associated with off target effects. cells were lysed with ripa buffer ( . % sds, % triton x- , % deoxycholate, mm edta, mm nacl, and mm tris at ph . ) containing protease inhibitor cocktail (roche). ten micrograms of the total lysate was separated in % sds-polyacrylamide gels and transferred to pvdf membranes (millipore). primary antibodies used in this paper are mouse anti-cmv ie / monoclonal antibody (mab , millipore), mouse anti-cmv pp monoclonal antibody (ch , santa cruz biotechnology), mouse anti-cmv pp monoclonal antibody (ch , santa cruz biotechnology), rabbit anti-vcp polyclonal antibody (h- , santa cruz biotechnology), and mouse anti-β-actin monoclonal antibody (abcam). blots were probed with primary antibody ( : - : ) diluted in % dehydrated milk in tris buffered saline (tbs) and subsequently the hrp-conjugated secondary antibodies (pierce) at : . blots were washed in tbs three times, incubated with chemiluminescent substrate (supersignal west pico; thermo scientific) according to the manufacturer's protocol, and exposed in g:box (syngene) for visualization of bands. total rna was isolated by using trizol solution according to the manufacturer's protocol. northern blot analysis for ie and ie mrnas was conducted using total rna that was separated on a . % agarose formaldehyde gel and then transferred using whatman turboblotter rapid downward transfer systems. ie and ie probes were generated by pcr using cdna from tb e infected nhdfs (as template) and labelled with amersham rediprime ii dna labelling system (ge healthcare) with the following primers (ie : tcaaacagattaaggt tcgagtgg, and atccacatctcccgcttatcctcg; ie : tcatggtgcgcatcttc tccacc, and ttactgagacttgttcctcaggtcc). after hybridization and wash, the membranes were exposed to autoradiography film with an intensifying screen (biomax transcreen he, kodak) for visualization of bands. cells were transfected and infected as described above. total rna was harvested using trizol according to manufacturers guidelines. total rna was submitted to edinburgh genomics for generation of truseq stranded libraries and subjected to hiseq high output v pe sequencing. the strand-specific rna-seq reads were mapped to a combined version of the human (hg ) and human herpesvirus (kf ) genomes using the hisat spliced read mapper (pmid: ). only valid read pairs mapped together in the correct orientation were retained (i.e. carrying a sam flag of one of , , or ) and a custom perl script was used to count the number of these reads mapping to each exon as well as the number of read pairs spanning different combinations of exons. drug inhibitor studies and cytotoxicity assay nms- (xcess biosciences) and ganciclovir (cambridge bioscience) were dissolved in dmso and added to the cell culture at a working concentration hours before hcmv infection. viability for nms- treated cells was assessed using the celltiter-glo luminescent cell viability assay kit (promega) and celltiter-blue (promega) hours after nms- addition. mg (cambridge bioscience), pepstatin a (sigma), and e (sigma) were dissolved in dmso and added to the cell culture hours after hcmv infection. μg/ml cycloheximide (in dmso, sigma) was added to the cell culture minutes before hcmv infection to block protein biosynthesis. laboratory adapted hcmv strain ad infected cells were fixed and permeabilized in methanol:acetone solution ( : ) for minutes, and then blocked with % human serum in pbs for minutes. primary and secondary antibodies were diluted with % human serum in pbs. cells were washed with pbs after primary and after secondary antibody incubations. primary antibodies used in this paper are mouse anti-cmv ie monoclonal antibody ( e , santa cruz biotechnology), rabbit anti-vcp polyclonal antibody (h- , santa cruz biotechnology) at : . alexa-fluor- conjugated goat anti-mouse or alexa-fluor- conjugated goatanti-rabbit igg secondary antibodies were diluted : . all images were acquired with zeiss lsm confocal microscope fitted with x/ . oil-immersion objective lens. total rna was isolated by using trizol solution according to the manufacturer's protocol followed by dnase (turbo dna-free kit, ambion) treatment, and then reverse transcribed with poly t primers using high capacity cdna reverse transcription kit (invitrogen). real-time pcr was carried out using by taqman assays with pre-designed gene-specific primer/probe set (applied biosystems) on rotor gene (corbet research). custom primer/probe set are cgtcaaacagattaaggttcgagtgg, ccacatctcccgcttatcctcg, and -fam/catgctctg/zen/catagttagcccaatacacttcatctc-ctcg/ iabkfq for ie , and atggtgcgcatcttctccacc, ttactgagacttgttcctcaggtcctg, and -fam/caggctcag/zen/ggtgtccaggtcttcgggagg/ iabkfq for ie . cells were harvested with trypsin, washed with pbs, and followed by fix with % cold ethanol for minutes at ˚c. then the fixed cells were washed twice with pbs, treated with rnasea, and labelled with μl of propidium iodide ( mg/ml stock solution, sigma). cell cycle analysis was first carried out using bd lsrfortessa x , and then further analyzed using flowjo software. quantification of difference in ie protein levels between knockdown of vcp versus ganciclovir treatment. quantification is compared to negative control for each time point. (d) relative ie and ie transcript levels normalised to mie shared exons. levels were determined by qrt-pcr using primer probes specific to exon to , exon or exon . exon and levels were then normalised to exon - for vcp knockdown cells (d) or cells treated with ganciclovir ( μm). cells were treated μg/ml cycloheximide, minutes prior to infection to block de novo protein synthesis and total rna harvested at indicated times. ie and ie transcript levels were determined by northern blot analysis. (docx) s table. sirna screen data. raw data from the three repeated sirna screens including technical repeats. average over the three experiments as well as z scores and standard deviations are shown. (xlsx) s table. exon read count ratios. total read counts for each of the five exons for each condition are shown along with ratio to total calculations and differential between negative control and vcp knockdown samples. (xlsx) s table. splice junction counts. total read counts across each of the mie junctions are shown along with ratio calculations defining the differential between exon to , to and to in log . (xlsx) s table. relative expression levels of exons and are significantly dependent on the combination of timepoint and vcp status. linear model results of testing associations between the relative expression levels of each exon and both time and sivcp treatment. relative expression levels being measured as the proportion of the transcript's reads that mapped to the corresponding exon in that sample. coefficients, standard errors (in brackets) and p values are shown. associations between relative expression levels of exons and and timepoint are dependent on vcp status as demonstrated by the significant interaction term (time: sivcp). regulation and tissue-specific expression of human cytomegalovirus cytomegalovirus disease of late onset following renal transplantation: a potentially fatal entity acquired cytomegalovirus infection in preterm infants transfusion-associated cytomegalovirus infections cytomegalovirus infection of human blood cells death in the aids patient: role of cytomegalovirus interstitial pneumonia and cytomegalovirus infection as complications of human marrow transplantation the use of cytomegalovirus-screened blood in neonates progress on human cytomegalovirus vaccines for prevention of congenital infection and disease analysis of splice variants of the immediate-early region of human cytomegalovirus the human cytomegalovirus ie -kilodalton protein interacts with an early gene promoter via site-specific dna binding and protein-protein associations structural analysis of the major immediate early gene of human cytomegalovirus human cytomegalovirus persistence aspects of human cytomegalovirus latency and reactivation human cytomegalovirus with ie- (ul ) deleted fails to express early lytic genes multiple spliced and unspliced transcripts from human cytomegalovirus immediate-early region and evidence for a common initiation site within immediate-early region widespread mrna polyadenylation events in introns indicate dynamic interplay between polyadenylation and splicing emerging functions of the vcp/p aaa-atpase in the ubiquitin system role of cdc /p as a sumo-targeted segregase curbing rad -rad interaction p complexes as signal integration hubs the vcp/p system at a glance: connecting cellular function to disease pathogenesis should i stay or should i go: vcp/p -mediated chromatin extraction in the dna damage response cdc /p promotes reformation of the nucleus by extracting the kinase aurora b from chromatin valosin-containing protein (vcp/p ) is required for poliovirus replication and is involved in cellular protein secretion pathway in poliovirus infection genome-wide rnai screen identifies sec a and vcp as conserved regulators of sindbis virus entry genome-wide screen reveals vcp requirement for coronavirus exit from endosomes the aaa atpase cdc /p and its partners transport proteins from the er into the cytosol systematic microrna analysis identifies atp v c as an essential host factor for human cytomegalovirus replication v for victory-a v -atpase structure revealed proteasome subunits relocalize during human cytomegalovirus infection, and proteasome activity is necessary for efficient viral gene transcription identification of binding sites for the -kilodalton ie protein of human cytomegalovirus within an ie -responsive viral early promoter separate dna elements containing atf/creb and ie binding sites differentially regulate the human cytomegalovirus ul - promoter at early and late times in the infection inhibition of human cytomegalovirus immediate-early gene expression by cyclin a -dependent kinase activity cyclin-dependent kinase activity is required at early times for accurate processing and accumulation of the human cytomegalovirus ul - and ul immediate-early transcripts and at later times for virus production we would like to acknowledge paul digard, jay nelson and christopher snyder for critical review of the manuscript and steve west and greg kudla for helpful discussion. rnaseq was performed by edinburgh genomics, the university of edinburgh. ahn jh, jang wj, hayward gs ( ) the human cytomegalovirus ie and ul - proteins accumulate in viral dna replication compartments that initiate from the periphery of promyelocytic leukemia protein-associated nuclear bodies (pods or nd key: cord- - aqt cc authors: tan, jinzhi; vonrhein, clemens; smart, oliver s.; bricogne, gerard; bollati, michela; kusov, yuri; hansen, guido; mesters, jeroen r.; schmidt, christian l.; hilgenfeld, rolf title: the sars-unique domain (sud) of sars coronavirus contains two macrodomains that bind g-quadruplexes date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: aqt cc since the outbreak of severe acute respiratory syndrome (sars) in , the three-dimensional structures of several of the replicase/transcriptase components of sars coronavirus (sars-cov), the non-structural proteins (nsps), have been determined. however, within the large nsp ( amino-acid residues), the structure and function of the so-called sars-unique domain (sud) have remained elusive. sud occurs only in sars-cov and the highly related viruses found in certain bats, but is absent from all other coronaviruses. therefore, it has been speculated that it may be involved in the extreme pathogenicity of sars-cov, compared to other coronaviruses, most of which cause only mild infections in humans. in order to help elucidate the function of the sud, we have determined crystal structures of fragment – (“sud(core)”) of nsp , which comprises of the residues of the domain. both the monoclinic and triclinic crystal forms ( . and . Å resolution, respectively) revealed that sud(core) forms a homodimer. each monomer consists of two subdomains, sud-n and sud-m, with a macrodomain fold similar to the sars-cov x-domain. however, in contrast to the latter, sud fails to bind adp-ribose, as determined by zone-interference gel electrophoresis. instead, the entire sud(core) as well as its individual subdomains interact with oligonucleotides known to form g-quadruplexes. this includes oligodeoxy- as well as oligoribonucleotides. mutations of selected lysine residues on the surface of the sud-n subdomain lead to reduction of g-quadruplex binding, whereas mutations in the sud-m subdomain abolish it. as there is no evidence for nsp entering the nucleus of the host cell, the sars-cov genomic rna or host-cell mrna containing long g-stretches may be targets of sud. the sars-cov genome is devoid of g-stretches longer than – nucleotides, but more extended g-stretches are found in the ′-nontranslated regions of mrnas coding for certain host-cell proteins involved in apoptosis or signal transduction, and have been shown to bind to sud in vitro. therefore, sud may be involved in controlling the host cell's response to the viral infection. possible interference with poly(adp-ribose) polymerase-like domains is also discussed. the sars coronavirus (sars-cov) is much more pathogenic for humans than any other coronavirus. therefore, protein domains encoded by the sars-cov genome that are absent in other coronaviruses are of particular interest, because they may be responsible for the extraordinary virulence. the most prominent such domain has been identified by bioinformatics as part of nonstructural protein (nsp ) of the virus and appropriately named the ''sars-unique domain'' (sud) [ ] . with a molecular mass of kda, nsp is the largest of the non-structural proteins of sars coronavirus (see figure ). comprising amino-acid residues (polyprotein a/ ab residues ala to gly ), sars-cov nsp is larger than the entire replicase of picornaviridae. it contains at least seven subdomains [ ] : an n-terminal acidic domain (ac, also called nsp a); an x-domain (also designated as adrp, or nsp b); the sud (nsp c); a papain-like proteinase, pl pro (also called nsp d); and additional domains (nsp e-g) that include a transmembrane (tm) region. at present, it is completely unclear whether and how the individual domains of nsp interact with one another or with other components of the coronaviral replicase complex. also, some of them possibly recognize proteins of the infected host cell [ ] . in the absence of functional data on these domains, attempts have been made to derive their possible biological role from their three-dimensional structures (see [ ] for a review). the nmr structure of an n-terminal fragment of the acidic domain (nsp a) has revealed a ubiquitin-like fold complemented by two additional short a-helices ( [ ] , pdb code idy). nmr chemical-shift analysis suggested that these non-canonical structural elements might bind single-stranded rna with some specificity for auacontaining sequences, although the k d values observed are relatively high (, mm). interestingly, a second ubiquitin-like domain occurs in nsp , as part of the papain-like proteinase (pl pro , nsp d, [ ] ; pdb code fe ). the pl pro cleaves the viral polyprotein after two consecutive glycine residues to release nsp , nsp , and nsp , respectively (the remaining cleavage reactions are performed by the coronaviral main proteinase (m pro ; [ - ]) ). in addition to its proteolytic activities on the n-terminal third of the polyproteins, the sars-cov pl pro has also been shown to be a deubiquitinating enzyme [ ] [ ] [ ] [ ] . lindner et al. [ ] have shown that in addition to its proteolytic and deubiquitinating activity, the sars-cov pl pro acts as a de-isgylating enzyme. induction of isg and its subsequent conjugation to proteins protects cells from the effects of viral infection [ , ] . since the isg gene is induced by interferon as part of the antiviral response of the innate immune system, the de-isgylation activity of nsp d could explain the suppression of the interferon response by the papain-like protease, in addition to a possible direct interaction between the pl pro and irf [ ] . among the subdomains of the nsp multidomain protein, there is also the so-called ''x-domain'' (nsp b), which shows structural homology to macrodomains. the latter name refers to the nonhistone-like domain of the histone macro a [ ] [ ] [ ] . in animal cells, such domains are occasionally physically associated with enzymes involved in adp-ribosylation or adp-ribose metabolism. because of this linkage and on the basis of sequence similarity to poa p, a yeast protein involved in the removal of the -phosphate group from adp-ribose -phosphate (a late step in trna splicing; [ ] ), it has been proposed that the coronaviral x-domains may have the function of adp-ribose- -phosphatases (adrps; [ ] ). the crystal structures of x-domains of sars-cov [ , ] as well as of hcov e and infectious bronchitis virus (ibv) [ ] show that the protein has the three-layer a/b/a fold characteristic of the macrodomains. embedded between the x-domain (nsp b) and the pl pro (nsp d), the sars-unique domain (sud; nsp c) fails to show sequence relationship to any other protein in the databases [ ] . we have produced full-length sud (residues to of nsp ), and a more stable, shortened -residue version (residues to ; henceforth called sud core ), by expression in escherichia coli. this definition of the boundaries of the sud is based on the structural results described here. we report crystallization of sud core and its x-ray structure in two crystal forms, at . and . Å resolution, respectively. the structure turns out to consist of two further copies of the macrodomain, in spite of the complete absence of sequence similarity. in addition, we demonstrate that each of the subdomains binds g-quadruplexes, both in dna and rna fragments, and that selected mutations of lysine residues in the first subdomain, sud-n, lead to reduced nucleic-acid binding, whereas those in the second subdomain, sud-m, abolish it. out of the many sud constructs designed and tested by us, sud core (nsp residues - ) turned out to be relatively stable and could be crystallized (table ) . two crystal forms were observed under identical crystallization conditions: form- crystals the genome of the sars coronavirus codes for nonstructural proteins that are involved in replicating this huge rna (approximately kilobases). the roles of many of these in replication (and/or transcription) are unknown. we attempt to derive conclusions concerning the possible functions of these proteins from their three-dimensional structures, which we determine by x-ray crystallography. non-structural protein contains at least seven different functional modules within its -amino-acid polypeptide chain. one of these is the so-called sars-unique domain, a stretch of about residues that is completely absent from any other coronavirus. it may thus be responsible for the extraordinarily high pathogenicity of the sars coronavirus, compared to other viruses of this family. we describe here the three-dimensional structure of the sars-unique domain and show that it consists of two modules with a known fold, the so-called macrodomain. furthermore, we demonstrate that these domains bind unusual nucleic-acid structures formed by consecutive guanosine nucleotides, where four strands of nucleic acid are forming a superhelix (so-called g-quadruplexes). sud may be involved in binding to viral or host-cell rna bearing this peculiar structure and thereby regulate viral replication or fight the immune response of the infected host cell. were monoclinic (space group p , two sud core molecules per asymmetric unit) and diffracted x-rays to . Å resolution; form- crystals were triclinic (space group p , four sud core molecules per asymmetric unit) and diffracted to . Å . both structures were determined by molecular replacement (see materials and methods). the r.m.s. deviations (on ca atoms) between the models derived from the two different crystal structures are around . Å . the models have good stereochemistry (table ) . . % of the amino-acid residues are in the favoured regions of the ramachandran plot and . % are in allowed regions. . % are outliers. in all six independent copies of the sud core monomer, residue val adopts forbidden conformational angles. this residue is located in a turn described by the polypeptide chain where it leaves the subdomain interface (see below) and reaches the surface of the molecule. the side chain makes a hydrophobic contact across the subdomain interface and is also contacting the side chain of phe of a symmetry-related sud core dimer in the crystal lattice in the monoclinic crystal form (this also applies to two of the four monomers in the triclinic form). overall structure sud core exhibits a two-domain architecture (figure a ). the n-terminal subdomain (sud-n) comprises nsp residues - , and the c-terminal subdomain of sud core contains residues - . we call the latter the ''middle sud subdomain'', or sud-m, because full-length sud has a c-terminal extension of residues compared to sud core . the sud-n and sud-m subdomains have a similar fold and can be superimposed with an r.m.s.d. of . - . Å (based on ca positions); they share % sequence identity (see figure c for a structural alignment). of the amino-acid residues identical between the two subdomains, four form a conserved leu-glu-glu-ala motif at the n-terminus of helix a . the linker between the two subdomains (residues - ) has no visible electron density. this is due to elevated mobility of the linker, rather than proteolytic cleavage, since we showed by sds-page of dissolved crystals that the sud core polypeptide (in the presence of b-mercaptoethanol) has the apparent molecular mass to be expected (, kda; not shown). in addition to the linker, sud-n and sud-m are connected by a disulfide bond between cysteines and ( figure b ). disulfide bonds are rare in cytosolic proteins, but in coronaviral nsps, examples of such bonds have been reported [ , ] . the fold of each sud subdomain is that of a macrodomain ( figure a) . macrodomains consist of a largely parallel central bsheet surrounded by - a-helices. the order of regular secondary-structure elements in sud-n is bn -an -bn -an -bn -bn -an -bn -an -bn , and in sud-m bm -am -bm -am -bm -bm -am -bm -am -bm -am . the topology of the b-strands is b -b -b -b -b -b , all of which are parallel except b (figure a ). between the two subdomains, most of the secondary-structure elements are conserved with respect to their position in the three-dimensional structure, although they often differ in length. this is particularly obvious for a-helix , which comprises just four residues in the n-terminal subdomain but eleven in the m subdomain. similarly, a-helix has vs. amino-acid residues in the two subdomains. in general, the strands of the central b-sheet appear to align better between the two subdomains than do the a-helices. each of the sud core subdomains is related to the macrodomain of the histone macro a ( [ ] ; pdb code zr , molecule c; for sud-n: z-score . , r.m.s.d. . Å for out of ca atoms, % sequence identity; for sud-m: z-score . , r.m.s.d. . Å for out of ca atoms, % sequence identity). called ''xdomains'', single macrodomains are also found in alphaviruses, in hepatitis e virus, and in rubella virus, in addition to coronaviruses [ , ] . the sars-cov x-domain (nsp b), the domain immediately preceding the sud in nsp , shares no recognizable sequence identity with sud-n ( %) or sud-m ( %) ( figure c ), but its three-dimensional structure [ , ] (pdb code acf, chain a) can be superimposed onto each of the two sud subdomains with an r.m.s.d. (based on ca atoms) of . Å and . Å , respectively ( figure d ). thus, within nsp , sars-cov has three macrodomains aligned one after the other. in both crystal forms, sud core displays the same head-to-tail dimer, with the sud-n subdomain of monomer a interacting with the sud-m subdomain of monomer b, and vice versa. approximately Å of solvent-accessible surface per monomer is buried upon dimerization ( figure ). due to the two-domain architecture of each monomer, the resulting four lobes give the dimer a quasi-tetrahedral shape ( figure a ). involving , hydrogen bonds and four well-defined salt-bridges (as-pb …arga , argb …glua , argb …aspa , and glub …arga ), interactions between the monomers are largely hydrophilic. as to be expected, the structures of the monomers are very similar to one another, with r.m.s.d. values (for ca atoms) of . Å between monomers a and b of the monoclinic crystal form, and . - . Å between monomers a-d of the triclinic form. the structure of sud-m alone is even better conserved between the individual copies of sud core . also, the fold of the sud-m subdomain is similar to the model of the sud fragment - derived from nmr measurements, which was published very recently (r.m.s.d. , . Å ) [ ] . the sud core macrodomains fail to bind adp-ribose the function of the coronaviral x-domain is still unclear; for some coronaviruses such as hcov e and sars-cov, it has been shown to exhibit a low adp-ribose- -phosphate phosphatase (appr- -pase, occasionally also called ''adrp'') activity and to bind the product of the reaction, adp-ribose [ ] [ ] [ ] ] . however, the two subdomains of sud core do not bind adpribose, as we have demonstrated by zone-interference gel electrophoresis ( figure s ). when we investigated possible interactions between sud and nucleic acids by zone-interference gel electrophoresis, we found that the domain binds oligo(g) and oligo(dg) stretches with a k d of , mm, but not oligo(da), (dc), or (dt) [ ] . single-stranded nucleotides of random sequence are only bound if they are longer than , nucleotides. here we demonstrate that each of the two individual sud subdomains also binds oligo(dg) ( figure a ). with oligo(dh), where h stands for a, c, or t, but not g, only very small gel shifts, if at all, were observed. as oligo(g) stretches are known to form g-quadruplexes, i.e. four-stranded nucleic-acid structures formed by contiguous guanines [ ] , we also examined the binding to the oligodeoxynucleotide -gggcgcgggag-gaattgggcggg- , a g-rich sequence present in the bcl- promoter region. this oligonucleotide has been shown by nmr spectroscopy to form a g-quadruplex ( [ ] ; pdb code f u). we found that both full-length sud and sud core do indeed bind this oligodeoxynucleotide and that this process is enhanced by the addition of k + ions, which are known to stabilize g-quadruplex structures ( figure b ). in agreement with the ability of sud to non-specifically bind to oligonucleotides of . bases [ ] , both sud and sud core were found to bind the reverse-complementary sequence, but with low affinity and, more importantly, independent of k + ions. as there is no evidence for sars-cov nsp entering the nucleus and binding to dna, we examined whether sud would bind to an rna known to form a quadruplex structure. indeed, zone-interference gel shift experiments revealed major shifts for both sud and sud core in the presence of the oligoribonucleotide -uggggggagggagggaggga- , which is a proteinbinding element in the -nontranslated region of chicken elastin mrna [ ] and forms g-quadruplexes [ ] ( figure c ). furthermore, we observed a significant gel shift for sud core when we added the short oligonucleotide uggggu, which has also been shown to form a g-quadruplex ( [ ] ; pdb code j g). this shift was also enhanced by the addition of k + ( figure d ). thus, sud binds rna (rg)-quadruplexes and dna (dg)-quadruplexes with comparable affinity. inspection of the structure of the sud dimer reveals a central narrow cleft running across the dimer surface, but distinct from the monomer-monomer interface ( figure c ), which could be a binding site for another protein. in addition, there are several positively charged patches in the center of the dimer ( figure b ), and on its backside ( figure c ), which could be involved in binding to g-quadruplexes. we have prepared four sets of mutations by replacing lysine residues (and one glutamate) in these patches by alanines. the first two pairs of mutations, k a+k a (m , at the end of helix an ) and k a+k a (m , in the loop between an and bn ), are located on the surface of the sud-n subdomain and lead to reduced shifts with g-quadruplexes in the zone-interference gel electrophoresis experiment, both with the g-quadruplex from the the narrow cleft running across the dimer surface (with a , u orientation relative to the monomer-monomer interface, which runs horizontal in this illustration) could be a potential protein-binding site. the monomer-monomer interface is largely hydrophilic and buries , Å of exposed surface per monomer. doi: . /journal.ppat. .g , or (dt) shows that the binding is specific for (dg) . ''h'' stands for a, c, or t. (b) binding of increasing concentrations (indicated above the lanes) of the quadruplex-forming oligodeoxynucleotide -gggcgcgggag-gaattgggcggg- (labeled ''bcl- '') as occurring within the bcl- promoter region, in the presence and absence of mm kcl, which is known to promote quadruplex formation. left panel, full-length sud; right panel, sud core . the reverse-complementary oligodeoxynucleotide (labeled ''rc''), which fails to form a quadruplex but exceeds the minimum length of , nucleotides for non-quadruplex interaction with sud, is also bound, but with reduced affinity and independently of kcl. (dg) when the sars-unique domain was first predicted [ ] , the boundaries of the domain were set approximately at nsp residues and . we made major efforts to produce this protein in a stable form, but with little success. only when we used in-vitro protein synthesis, were we able to obtain small amounts of a relatively stable preparation comprising nsp residues - [ ] . at the n-terminus of this construct, up to eleven residues actually correspond to the c-terminus of the preceding x-domain (nsp b). when we expressed a gene construct coding for sud ( - ) in e. coli, we observed rapid proteolytic degradation of the n-terminal segment. the relatively stable intermediate obtained had its n-terminus at nsp residue . the n-terminal segment , - is predicted to be intrinsically unfolded by several prediction programs (not shown). therefore, we assume segment - to be merely a linker between nsp b and sud, and to be the first residue of the latter. this assignment is justified by the observation that in our crystal structures reported here, the sud-n subdomain is a complete macrodomain without any residues lacking at the n-terminus. therefore, the protein corresponding to nsp residues - is called ''full-length sud'' here. in this communication, we describe the crystal structures at . Å and . Å resolution (monoclinic and triclinic form, respectively) of the core of the sars-unique domain (sud core , nsp residues - ). sud core turns out to consist of two subdomains, sud-n (nsp residues - ) and sud-m ( - ), each exhibiting the fold of a macrodomain. the two subdomains are connected by a flexible linker (residues - ) and a disulfide bond. even though coronavirus replication occurs in the cytosol, where the environment is reductive, it is unlikely that the formation of this disulfide is an artifact owing to handling of the protein: as the linker between the sud-n and sud-m subdomains is very short (seven residues), and the mutual orientation of the subdomains is fixed due to the tight dimerization, cysteine residues no. and will be very close to one another irrespective of the exact conformation of the linker. in fact, disulfide bonds are not uncommon in coronaviral nonstructural proteins (nsps) involved in rna replication or transcription. among others, they have been observed in hcov- e nsp [ ] and turkey coronavirus nsp [ ] , but in these cases, the disulfide bond connects two subunits of the homo-oligomeric proteins, whereas the occurrence in sud core is the first case of an intramolecular disulfide bond described in a coronavirus nsp. coronavirus replication in the perinuclear region of the cell is localized to double-membrane vesicles that have been hijacked from the endoplasmic reticulum or late endosomes [ ] [ ] [ ] [ ] . these vesicles are around - nm in diameter and present alone or as clusters in the cytosol [ ] . the milieu inside or at the surface of these vesicles is unknown, but it is well possible that it is partially oxidative. it has also been speculated [ ] that formation of disulfide bonds may be a way for the coronaviral nsps to function in the presence of the oxidative stress that is the consequence of the viral infection [ ] [ ] [ ] . our identification of two macrodomains in sud core brings the number of these domains in sars-cov nsp to three. what are the functions of these modules? the original sars-cov ''xdomain'' (nsp b) has been shown to have low adp-ribose- phosphate phosphatase (appr- -pase or ''adrp'') activity [ ] [ ] [ ] . however, this assignment is controversial. a nuclear appr- pase (poa p in yeast, [ ] ) is an enzyme of a trna metabolic pathway, but there is no evidence for coronavirus nsp ever being translocated to the nucleus, and the other enzymes involved in this pathway are missing in coronaviruses (with the exception of the cyclic , -phosphodiesterase (cpdase) in group a viruses such as mouse hepatitis virus, bovine coronavirus, and human coronavirus oc ). therefore, it has been proposed that the xdomain may be involved in binding poly(adp-ribose), a metabolic product of nad + synthesized by the enzyme poly(adp-ribose) polymerase (parp; [ ] ). however, we have recently demonstrated that the x-domain of infectious bronchitis virus (ibv) strain beaudette, a group- coronavirus, does not have significant affinity to adp-ribose [ ] . this can be explained on the basis of crystal structures: in the x-domain (nsp b) of sars-cov [ ] , and in that of hcov e [ ] , a stretch of three conserved glycine residues is involved in binding the pyrophosphate unit of adp-ribose, whereas in the corresponding domain of ibv strain beaudette (but not in all ibv strains, see [ ] ), the second glycine is replaced by serine, leading to steric interference with adpribose binding [ ] . in the two sud subdomains, the tripleglycine sequence is not conserved (see figure c ), and hence, they do not bind adp-ribose either. neuman et al. [ ] reported that full-length sud binds cobalt ions, whereas a domain called sud-c by these authors, which is however almost identical (residues - ) to our sud-m ( - ), does not. from this, they concluded that the metal-binding activity is associated with the cysteine residues in the n-terminal subdomain. we were also able to observe binding of cobalt ions to sud core by following the occurrence of a peak at nm in the uv spectrum, which, in contrast to the data presented by neuman et al. [ ] , could be reverted by addition of zinc ions. however, when we removed the n-terminal his-tag, this phenomenon could no longer be observed. furthermore, we note that of the four cysteine residues in the sud-n subdomain (residues , , , and ), and are non-accessible in the interior of the subdomain, and is involved in the buried disulfide bond to cys ; therefore, cys and perhaps the solvent-exposed his would remain the only potential ligands for cobalt ions in sud-n. however, these residues are . Å apart and thus unlikely to chelate cobalt ions. for sud-m, a recent publication [ ] reported binding to oligo(a). however, we fail to observe this ( figure a, lane labeled ''a'') . instead, we have demonstrated that full-length sud and sud core bind oligodeoxynucleotides and oligoribonucleotides that form g-quadruplexes. for full-length sud and sud core , we had previously shown binding to oligo(dg) and oligo(g) stretches [ ] , but the demonstration here of oligo(dg) binding to the individual sud core subdomains, sud-n and sud-m, is unexpected because their overall electrostatic properties are very different from one another: sud-n is acidic (pi = . ), whereas sud-m is basic (pi = . ). however, even sud-n shows surface patches with positive electrostatics that could bind nucleic acid ( figure b) . we have used automatic docking procedures to place the gquadruplex found in the bcl- promoter region ( [ ] ; pdb code f u) into our crystal structures. one potential binding site identified is in the cleft between the sud-m and the sud-n subdomains within the sud core dimer ( figure s a ); this binding site is spatially close to the mutations m and m , consistent with the observation that these mutations abolish binding completely. however, we have previously shown by dynamic light-scattering that g-quadruplex binding leads to oligomerization of sud core [ ] . consequently, we have also constructed models based on the packing modes of sud core dimers observed in our crystal structures. one potential binding site for g-quadruplexes might be in a cleft between two consecutive sud core dimers as they occur in both the monoclinic and triclinic crystal forms ( figure s b ), but for confirmation, any of these models will have to await crystallographic determination of the complex. in summary, our mutation experiments demonstrate an involvement of several of the many lysine residues of sud in binding g-quadruplexes, but as it is probably extended surfaces of sud core oligomers that participate in this process, it is not possible to pinpoint any single amino-acid residue. the target of sud binding could be g-quadruplexes in rna of viral or/and cellular origin. the sars-cov genome contains three g -stretches (one on the plus-strand and two on the minusstrand) and an additional two g -sequences, which could perhaps form local g-quadruplexes. however, the g-stretch binding capabilities of sud and sud core seem to have been optimized for recognition of longer g-rich sequences. by systematic variation of the length of oligo(dg), we found that sud core exhibits strongest affinity (k d , . mm) for (dg) to (dg) [ ] . the nontranslated regions of several host-cell mrnas coding for proteins involved in the regulation of apoptosis and in signaling pathways contain long g-stretches and could also be targets of sud. examples of such mrnas are those coding for the proapoptotic protein bbc [ ] , rab b (a member of the ras oncogene family, [ ] ), map kinase [ ] , and tab , a component of the nf-kb signaling pathway [ ] . it is conceivable that these proteins might be targets for the virus when interfering with cellular signaling. changes in the stability and/or translation efficiency of these mrnas due to the binding of a viral regulatory factor could result in an altered reaction of the infected cell to apoptotic signals, or it could silence the antiviral response. the idea that coronaviral x-domains might function as modules binding poly(adp-ribose) [ ] received support from the observation that some macrodomains are connected with domains showing poly(adp-ribose) polymerase (parp) activity, i.e. in the so-called macroparps (parp- and parp- ) [ ] . there are human genes for members of the parp family; the prototype enzyme, parp- , catalyzes the post-translational modification of many substrate proteins, including itself, in a multitude of cellular processes (dna repair, transcriptional regulation, energy metabolism, and apoptosis) [ ] [ ] [ ] . interestingly, sud-m and the c-terminal -residue subdomain (sud-c) that is missing in our sud core construct together show a , % sequence identity ( % similarity) to the catalytic domain of parp- . however, the three-dimensional structures of sud-m (this work) and the c-terminal domain of parp- [ ] are different and cannot be superimposed. another feature common between sars-cov sud and parp- is that the latter has recently been shown to bind to g-quadruplexes [ ] , although it is generally assumed that this occurs through the dna-binding domain rather than the catalytic domain of parp- . parp- and most of its family members are located to the nucleus, while parp- and others predominantly act in the cytoplasm [ ] [ ] [ ] . parp- is incorporated into vaults, rnacontaining subcellular particles in the cytoplasm [ ] . furthermore, zap, a human antiviral protein comprising a c-terminal parp-like domain devoid of catalytic activity, has been shown to exhibit antiviral activity on alphaviruses [ ] , which contain an xdomain similar to that of coronaviruses [ , , ] . in addition, zap contains an n-terminal zinc-finger domain, a central tiparp ( , , , -tetrachlorodibenzo-p-dioxin (tcdd)-inducible parp) domain, and a wwe domain (a protein-protein interaction module in ubiquitin and adp-ribose conjugation proteins). in fact, zap appears to be part of the human innate immune system and to play a role comparable to apobec g in hiv infection [ ] . it is possible that this group of viruses has evolved macrodomains to counteract the antiviral activity of zap. indeed, macrodomains can inhibit parps, as has been shown for the macrodomain of the histone mh a . , which downregulates the catalytic activity of parp- [ ] . having three macrodomains at its disposal, sars-cov may be much more efficient in knocking down the antiviral response of the host cell than other coronaviruses. whether this involves a direct interaction between sud and zap or another member of the parp family, or competition for g-quadruplexes in viral or host-cell rna, remains to be shown. full-length sud (nsp residues - ) and the fragment sud core (nsp residues - , previously called ''sudc b'') of sars-cov strain tor (acc. no. ay ) were produced recombinantly in e. coli as described [ ] . the coding regions for the sud-n subdomain (nsp residues - ) and the sud-m subdomain (nsp residues - ) were constructed by introducing an appropriate deletion into the previously described plasmid pqe -xa-c b [ ] using site-directed mutagenesis. plasmids encoding sud-n and sud-m were prepared using primers listed in table s . the coding regions for four sets of mutations of sud core , m (k a+k a), m (k a+k a), m (k a+k a+k a), and m (k a+k a+e a), were constructed by introducing appropriate mutations into plasmid pqe -xa-c b [ ] using site-directed mutagenesis. plasmids encoding these mutants were prepared using primers also listed in table s . all plasmids provided an n-terminal his-tag and a short linker sequence encoding a factor-xa cleavage site. the coding regions of the expression plasmids were verified by dna sequencing. e. coli m (prep ) was used as expression host for these constructs. sud-n, sud-m, and the mutated proteins were purified according to the same protocol as for sud core [ ] . crystallization sud core displayed . % purity in sds-page, and monodispersity in dynamic light-scattering. initial crystallization screening was performed using the sitting-drop vapor-diffusion method in -well intelli-plates (dunn laboratories). several commercial kits (sigma, jena bioscience) were used for the screening. the protein concentration was mg/ml. using a phoenix robotic system (art robbins), drops were made of nl protein and nl precipitant solution. the optimized crystallization condition consisted of % polyethylene glycol monomethyl ether and . m ammonium sulfate in . m morpholinoethane sulfonic acid (ph . ). plate-like crystals grew in - days, to maximum dimensions of . . . mm . many sud core crystals had to be tested for diffraction until one yielding data to . Å resolution was found. the best diffracting crystals belonged to space group p . under the same crystallization conditions, a second crystal form belonging to space group p was observed, diffracting to lower resolution of about . Å . crystals were cryoprotected in reservoir solution that included % glycerol, and were harvested into a loop prior to flash-cooling in liquid nitrogen. all data were collected at k from a single crystal each at beamline bl . , bessy (berlin, germany), using an mx ccd detector (rayonics), or at beamline i - at max-lab (lund, sweden), using a mar ccd detector (marresearch). data were processed with mosflm [ ] , and reduced and scaled using the scala [ ] program from the ccp suite [ ] . crystals belonging to space group p had unitcell parameters a = . we attempted to solve the structure by molecular replacement into the p form using the nmr coordinates of a subdomain comprising sars-cov nsp residues - ; pdb code jwj [ , ] ), which is almost identical to the sud-m subdomain of sars-cov nsp . using the program phaser [ , ] , we could find two solutions, and the c-terminal part of sud core was well defined in the electron-density maps. however, for the n-terminal half, only a few segments of poly(ala) chain could be built into the maps. this starting model was then refined in buster-tnt [ ] using local structure similarity restraints (lssr) [ ] as non-crystallographic symmetry (ncs) restraints to give r and r free values of . and . , respectively. the resulting mf o -df c electron density was subjected to density modification using solvent flattening, histogram matching, and -fold ncs-averaging using dm [ ] . the averaging masks were calculated and updated using the auto-correlation procedure [ ] as implemented in dm. using the automatic building program buccaneer [ ] together with refmac [ ] (as implemented in the ccp i [ ] interface for ccp ) in an iterative procedure for cycles resulted in a model for residues in chains (the longest having residues), in which residues were assigned both a chemical identity and a sequential residue number, while the remaining residues were modeled as poly(ala) in shorter chains. the r and r free values resulting from refmac were . and . , respectively. this model was refined in buster-tnt, again using lssr as ncs restraints for the common parts in the already sequenced residues of the dimer, to r and r free values of . and . . the improved electron density was again subjected to density modification using dm as detailed above, but using a lower solvent content of % as well as anisotropically scaled observed amplitudes as output by buster-tnt. the resulting density-modified and ncs-averaged map was then used for automatic model building using the iterative buccaneer/refmac procedure described above. this produced a model with residues in chains with residues sequenced. the r and r free values from refmac for this model were . and . , respectively. since the refinements in buster-tnt at that point showed some problematic low correlations between f o and f c at low resolution, the original images collected from the p crystal were reprocessed using xds [ ] and scala, applying different highresolution cutoffs for different segments of the collected images. details for this dataset are given in table . subsequent refinement of the p form with refmac, under application of weak ncs restraints, yielded a model with r = . , r free = . . the advanced handling of ncs restraints through lssr in buster-tnt gave a final model r = . and r free = . . the final model in the p form comprises residues (a -a ; a -a ; b -b ; b -b ). chain a of the p form was used for molecular replacement with the program molrep [ ] into the p form. there was an unambiguous solution for four molecules in the asymmetric unit. this model was refined with buster-tnt (using lssr for ncs restraints) and rebuilt in coot [ ] to final values of r = . and r free = . . the final model of the p form comprises residues. the figures were made with pymol [ ] . the zone-interference gel electrophoresis (zige) device was adapted from abrahams et al. [ ] . zige assays were performed using a horizontal % agarose gel system in tbe buffer ( mm tris, mm boric acid, . mm ethylenediaminetetraacetic acid (edta), ph . ). the protein was incubated at room temperature for min with different concentrations of oligodeoxynucleotides, such as (dg) and bcl- promoter region ( -gggcgcgggag-gaattgggcggg- ), or oligoribonucleotides ( -uggggg-gagggagggaggga- and -uggggu- ). the samples were mixed with dimethylsulfoxide (dmso; final concentration % (v/v)) and a trace of bromophenolblue (bpb). these proteinoligonucleotide samples were applied to the small slots. oligonucleotide with the same concentration as in the small slots was also mixed with dmso and bpb in xtbe buffer and applied to the long slots of the gel (total volume ml). electrophoresis was performed at uc for h with a constant current of ma. staining was performed as outlined in [ ] . protein data bank: coordinates and structure factors have been deposited with accession code w g (p crystal form) and wct (p crystal form). figure s zone-interference gel electrophoresis experiment showing that sud core fails to bind nad+ and adp-ribose. sud core alone (label ) and decreasing concentrations ( , . , . , . and . mm) of nad + , or decreasing concentrations ( , . , . , . and . mm) of adp-ribose. found at: doi: . /journal.ppat. .s ( . mb doc) figure s alternative models of g-quadruplex binding to sud core , obtained by automated docking into the crystal structures. the sud-n and sud-m subdomains are in violet and cyan, respectively, the g-quadruplex as found in the bcl- promoter region (pdb code: f u) is in orange. the pairs of mutations in sud-n are indicated by green (m , k a+k a) and blue (m , k a+k a) spheres. the m set of mutations in sud-m is indicated by olive (k a) and orange (k a+k a) spheres. the m set of mutations, also in sud-m, is indicated by orange (k a+k a) and yellow (e a) spheres. (a) a possible binding site is in a cleft between monomers in the sud core dimer. the binding site is close to the lysine residues replaced by the m and m mutations, compatible with the inability of these mutants to bind g-quadruplexes. (b) a second potential binding site is a cleft between two neighboring sud core dimers as found in both crystal packing arrangements (space groups p and p ). this binding mode is compatible with the observation of sud core oligomerization upon g-quadruplex binding. unique and conserved features of genome and proteome of sars-coronavirus, an early split-off from the coronavirus group lineage proteomics analysis unravels the functional repertoire of coronavirus nonstructural protein structural proteomics of emerging viruses: the examples of sars-cov and other coronaviruses nuclear magnetic resonance structure of the n-terminal domain of nonstructural protein from the severe acute respiratory syndrome coronavirus severe acute respiratory syndrome coronavirus papain-like protease: structure of a viral deubiquitinating enzyme coronavirus main proteinase ( cl pro ) structure: basis for design of anti-sars drugs the crystal structures of severe acute respiratory syndrome virus main protease and its complex with an inhibitor phdependent conformational flexibility of the sars-cov main proteinase (m pro ) dimer: molecular dynamics simulations and multiple x-ray structure analyses deubiquitination, a new function of the severe acute respiratory syndrome coronavirus papain-like protease? the papain-like protease of severe acute respiratory syndrome coronavirus has deubiquitinating activity the papain-like protease from the severe acute respiratory syndrome coronavirus is a deubiquitinating enzyme deubiquitinating activity of the sars-cov papain-like protease selectivity in isg and ubiquitin recognition by the sars coronavirus papain-like protease isg : the immunological kin of ubiquitin regulation of irf- -dependent innate immunity by the papain-like protease domain of the severe acute respiratory syndrome coronavirus macroh a, a core histone containing a large nonhistone region splicing regulates nad metabolite binding to histone macroh a structural characterization of the histone variant macroh a a highly specific phosphatase that acts on adp-ribose -phosphate, a metabolite of trna splicing in saccharomyces cerevisiae adp-ribose- -monophosphatases: a conserved coronavirus enzyme that is dispensable for viral replication in tissue culture structural basis of severe acute respiratory syndrome coronavirus adp-ribose- -phosphate dephosphorylation by a conserved domain of nsp structural and functional basis for adp-ribose and poly(adp-ribose) binding by viral macro domains crystal structures of the x-domains of a group- and a group- coronavirus reveal that adp-ribose-binding may not be a conserved property variable oligomerization modes in coronavirus non-structural protein turkey coronavirus non-structure protein nsp -an endoribonuclease computer-assisted assignment of functional domains in the nonstructural polyprotein of hepatitis e virus: delineation of an additional group of positivestrand rna plant and animal viruses differential activities of cellular and viral macro domain proteins in binding of adp-ribose metabolites nuclear magnetic resonance structure shows that the sars-unique domain contains a macrodomain fold adp-ribose- -phosphatase activities of the human coronavirus e and sars coronavirus x domains the ''sarsunique'' domain (sud) of sars coronavirus is an oligo(g)-binding protein quadruplex structures in nucleic acids nmr solution structure of the major g-quadruplex structure formed in the human bcl promoter region identification of a ga-rich sequence as a protein-binding site in the -untranslated region of chicken elastin mrna with a potential role in the developmental regulation of elastin mrna stability crystal structure of an rna purine-rich tetraplex containing adenine tetrads: implications for specific binding in rna tetraplexes x-ray analysis of an rna tetraplex (uggggu) with divalent sr + ions at subatomic resolution ( . Å ) coronavirus replication complex formation utilizes components of cellular autophagy ultrastructure and origin of membrane vesicles associated with the severe acute respiratory syndrome coronavirus replication complex sars-coronavirus replication/transcription complexes are membrane-protected and need a host factor for activity in vitro sars-coronavirus replication is supported by a reticulovesicular network of modified endoplasmic reticulum transmissible gastroenteritis coronavirus induces programmed cell death in infected cells through a caspase-dependent pathway glucose- -phosphate dehydrogenase deficiency enhances human coronavirus e infection identification of oxidative stress and toll-like receptor signaling as a key pathway of acute lung injury crystal structures of two coronavirus adp-ribose- -monophosphatases and their complexes with adpribose: a systematic structural analysis of the viral adrp domain bbc mediates fenretinide-induced cell death in neuroblastoma the structure of human neuronal rab b in the active and inactive form signal transduction in sars-cov-infected cells identification of a human nf-kb-activating protein, tab the macroparp genes parp- and parp- are developmentally and differentially regulated in mouse tissues poly(adp-ribose): the most elaborate metabolite of nad + poly(adp-ribose): novel functions for an old molecule the diverse biological roles of mammalian parps, a small but powerful family of poly-adp-ribose polymerases structure of the catalytic fragment of poly(adp-ribose) polymerase from chicken first evidence of a functional interaction between dna quadruplexes and poly(adp-ribose) polymerase- the formation of vault-tubes: a dynamic interaction between vaults and vault parp positive selection and increased antiviral activity associated with the parp-containing isoform of human zincfinger antiviral protein hiv- vif the histone variant mh a . interferes with transcription by down-regulating parp- enzymatic activity recent changes to the mosflm package for processing film and image plate data data reduction the ccp suite: programs for protein crystallography solvent content of protein crystals nmr assignment of the domain - from the sars-cov nonstructural protein nsp likelihoodenhanced fast translation functions phaser crystallographic software buster-tnt, version . . . cambridge annual meeting of the american crystallographic association dm: an automated procedure for phase improvement by density modification demon/angel: a suite of programs to carry out density modification the buccaneer software for automated model building refinement of macromolecular structures by the maximum-likelihood method a graphical user interface to the ccp program suite automatic processing of rotation diffraction data from crystals of initially unknown symmetry and cell constants molrep: an automated program for molecular replacement coot: model-building tools for molecular graphics delano wl the pymol molecular graphics system zone-interference gel electrophoresis: a new method for studying weak protein-nucleic acid complexes under native equilibrium conditions on the use of the merging r-factor as a quality indicator for x-ray data we thank d. mutschall for expert technical assistance, and u. müller (bessy, berlin, germany) as well as t. ursby (max-lab, lund, sweden) and k.h.g. verschueren for assistance with synchrotron data collection. this publication is dedicated to professor wolfram saenger on the occasion of his th birthday. key: cord- - a au c authors: gastaldello, stefano; chen, xinsong; callegari, simone; masucci, maria g. title: caspase- promotes epstein-barr virus replication by targeting the large tegument protein deneddylase to the nucleus of productively infected cells date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: a au c the large tegument proteins of herpesviruses contain n-terminal cysteine proteases with potent ubiquitin and nedd -specific deconjugase activities, but the function of the enzymes during virus replication remains largely unknown. using as model bplf , the homologue encoded by epstein-barr virus (ebv), we found that induction of the productive virus cycle does not affect the total level of ubiquitin-conjugation but is accompanied by a bplf -dependent decrease of nedd -adducts and accumulation of free nedd . expression of bplf promotes cullin degradation and the stabilization of cullin-ring ligases (crls) substrates in the nucleus, while cytoplasmic crls and their substrates are not affected. the inactivation of nuclear crls is reversed by the n-terminus of cand , which inhibits the binding of bplf to cullins and prevents efficient viral dna replication. targeting of the deneddylase activity to the nucleus is dependent on processing of the catalytic n-terminus by caspase- . inhibition of caspase- severely impairs viral dna synthesis and the release of infectious virus, pointing a previously unrecognized role of the cellular response to danger signals triggered by ebv reactivation in promoting virus replication. post-translational modification of proteins by covalent linkage of ubiquitin (ub) or ubiquitin-like proteins (ubls), such as sumo, nedd , isg , regulates diverse cellular processes, including cell cycle progression, dna repair, transcription, signal transduction and immune responses [ , ] . cytosolic and nuclear proteins tagged with multiple lys -linked ub moieties are targeted to the proteasome for degradation, whereas the attachment of single or multiple ub or ubls regulates a variety of non-proteolytic events, including protein-protein interactions and intracellular traffic [ ] . conjugation of the modifiers is accomplished by an enzymatic cascade composed of activating enzymes (e ), conjugating enzymes (e ) and substrate-specific ligases (e ), and is reversed by substrate-specific cysteine or metallo-protease that control the turnover of the modification and play thereby a key role in determining the functional outcome. although each modifier is involved in unique cellular functions, important cross-talk has been highlighted by the demonstration that nedd and sumo regulate the activity of certain ubiquitin ligases. thus, the best characterized substrates of nedd conjugation are cullins that function as scaffolds for the assembly of cullin-ring ubiquitin ligases (crls) [ , ] , while sumoylation is required for substrate recognition and subsequent ubiquitination by a family of sumotargeted ubiquitin ligases (stubls) [ ] . furthermore, several deconjugases, including usp [ ] , ataxin- [ ] pfuch [ ] , uch-l and uch-l [ ] , exhibit dual specificity for ub and nedd conjugates, while senp is both a nedd and sumo deconjugase [ ] . the significance of these multiple specificities in the context of biological processes remains, however, undefined. viruses rely on the host cell machinery for replication. given the key role of ub and ubl modifications in the regulation of cellular and immunological functions, modulation of these signaling pathways is essential for viral dna synthesis and for the survival of the virus during acute, chronic and latent infections [ ] . two viral interference strategies have been documented in the infected cells. viral proteins were shown to regulate the expression or capture the activity of cellular components of the ub and ubl signaling networks and to redirect their function towards preferred cellular or viral substrates [ ] . in addition, there are numerous examples of virus-encoded homologs of cellular ligases and deconjugases [ ] . these viral enzymes are often multifunctional proteins that share little homology with their cellular counterparts and are therefore attractive targets for selective inhibition. adenovirus [ ] , severe acute respiratory syndrome coronavirus [ ] , and all members of the herpesvirus family [ , ] , encode their own deconjugase. the homologs encoded in the n-terminus of the large tegument proteins of herpesviruses show very little sequence similarity but the residues implicated in the formation of the catalytic site are strictly conserved. all the tested homologs have potent ubiquitin-specific protease activity and their overexpression in transfected cells is associated with a dramatic decrease of polyubiquitinated substrates [ , ] . expression of the active enzymes was confirmed during infection by human cmv (hcmv) [ , ] , murine gamma-herpesvirus (mhv- ) [ ] , marek's disease virus (mdv) [ ] , and pseudorabies virus (prv) [ ] . although not essential for viral dna synthesis, disruption of the catalytic function correlated with severe impairment of viral replication in in vivo models of infection [ , , ] . the epstein-barr virus (ebv) encoded homolog, bplf , has very potent ubiquitin deconjugase activity in various experimental models. anchoring the enzymatic domain to the er membrane [ ] or injection of the purified protein in semi-intact cells [ ] promoted the dislocation of ubiquitinated erad substrates, resulting in their stabilization in the cytosol. overexpression of the n-terminus was associated with deubiquitination of the viral ribonucleotide reductase (rr) [ ] and the cellular dna polymerase processivity factor pcna [ ] , resulting in downregulation of the viral rr activity and attenuation of polg at dna damage sites. furthermore, expression of the catalytically active bplf was shown to correlate with deubiquitination of traf and inhibition of nf-kb signaling during productive ebv infection [ ] . we have previously reported that, in addition to their deubiquitinating activity, bplf and the homologs encoded by hsv , kshv and mhv- exhibit strong activity against nedd conjugates [ ] . bplf hydrolyzes nedd conjugates in vitro and stabilizes several crl substrates in transfected cells, including the cellular dna-replication licensing factor cdt . expression of bplf alone or in the context of the productive virus cycle induced the accumulation of cdt and arrest of the cells in s-phase, while re-expression of cdt was sufficient to revert the inhibition of virus replication induced by bplf knockdown. this phenotype is dependent on direct binding of bplf to crls via interaction of the conserved helix- of bplf with the c-terminal domain (ctd) of cullins, at a site that is also engaged by the crl regulator cand [ ] . while the double specificity of this family of viral enzymes is experimentally documented, the importance of the ubiquitin and nedd -specific deconjugase activities in infected cells is not easily understood since deubiquitination or inactivation of the specific ubiquitin ligase may have similar effects on individual substrates. thus, it remains unclear whether both the ubiquitin-and nedd -specific deconjugase activities operate during virus replication and, if so, how the different functions are regulated or compartmentalized in the infected cells. here we report that induction of the productive virus cycle has no appreciable effects on the global levels of protein ubiquitination but is accompanied by a bplf -dependent decrease of cullin neddylation and stabilization of nuclear crl substrates. targeting of catalytic nterminus of bplf to the nucleus is dependent on cleavage by caspase- , and inhibition of the caspase halts virus replication and the release of infectious virus. the akata-bx cell line was used to study the contribution of the ub-and nedd -specific deconjugase activities of the ebv large tegument protein bplf to the productive virus cycle. treatment of akata-bx with anti-igg antibodies promotes upregulation of the lytic cycle transactivator bzlf , followed in temporal succession by the expression of immediate early, early and late viral gene products (supplementary, figure s ). conjugated and free ub and nedd were quantified in western blots probed with specific antibodies using cells lysates made in the presence of cysteine protease inhibitors ( figure ). the levels of conjugated and free ub remained virtually unchanged over time ( figure a and b), whereas, consistent with the occurrence of deneddylation, the conjugated nedd progressively decreased in parallel with increase of free nedd ( figure c and d). in order to assess the involvement of bplf , a previously characterized specific shrna [ ] was transfected in akata-bx before induction of the productive cycle. as shown in figures a and b , the effect was abrogated in cells expressing the bplf specific shrna, confirming that the phenotype is dependent on bplf expression and supporting the conclusion that endogenous enzyme acts as a deneddylase during virus replication. transfection of the catalytically active bplf in hela cells promotes cullin deneddylation and their proteasomal degradation [ ] . we asked therefore whether this phenotype is reproduced when the endogenous enzyme is expressed during virus replication. as illustrated by the representative western blot shown in figure a , induction of the productive virus cycle was accompanied by a gradual decrease of the cul , cul , cul a and cul specific bands in akata-bx . this was not due to a global impairment of protein synthesis since neither cul , nor the crl subunit rbx were affected. furthermore, the decrease was rescued by treatment with mg confirming that cullins are degraded by the proteasome ( figure b ). this finding is consistent with a scenario where the inactivation of crls by bplf mediated deneddylation of cullins, and their subsequent proteasomal degradation, are key requirements for efficient virus replication. however, the failure to degrade cul is surprising since the bplf binding site on cullins is highly conserved [ ] . to explore the possible cause of this observation, the abundance of cul , cul , cul , cul a and cul was monitored over time in the nucleus and cytoplasm of the induced cells. the fractionation procedure was validated by probing of western blots with antibodies to parp, histone h and b-actin. in line with their known subcellular localization, parp and h were exclusively detected in the nuclear fractions, whereas b-actin was enriched in the cytoplasmic fraction ( figure c ). variable amounts of nuclear and cytoplasmic cullins were detected in untreated akata-bx , with prevalent nuclear localization of cul , cul , cul a and viruses rely on the host cell for replication and have evolved sophisticated strategies to manipulate and harness the cellular metabolic pathways and defense responses. a better knowledge of these viral strategies will provide new targets for antiviral therapies. the nterminus of the large tegument proteins of herpesviruses encodes an ubiquitin and nedd -specific deconjugase, but the function of the enzyme during virus replication is largely unknown. here we report that, endogenously expressed bplf , the homolog encoded by epstein-barr virus (ebv), promotes a dramatic decrease of nedd conjugates and the accumulation of free nedd in cells entering the productive virus cycle. bplf exerts its deneddylase activity in the nucleus, which promotes the accumulation of cullin-ring ligase (crl) substrates that are required for efficient virus replication. targeting of the viral enzyme to the nucleus is dependent on processing of the catalytic n-terminus by caspase- . inhibition of caspase- severely impairs viral dna synthesis and the release of infectious virus, pointing to an unexpected role of the cellular response to danger signals triggered by ebv reactivation in promoting virus replication. cul , whereas cul was detected almost exclusively in the cytoplasmic fraction ( figure c ). induction of the productive virus cycle was accompanied by a progressive decrease of nuclear pool of cul , cul , cul a and cul , whereas the amount of proteins detected in the cytoplasm remained unchanged throughout the observation period ( figure c and d ). there was no detectable change in the expression of cytoplasmic cul , further supporting the conclusion that only nuclear cullins are affected. we then monitored the abundance of known nuclear and cytoplasmic crl substrates. in agreement with the destabilization of the ligases, induction of the productive cycle was accompanied by the accumulation of several nuclear substrates of crl and crl a, including p , p , cdt and cdc a, whereas two cytosolic substrates of crl , the rho gtp exchange factor vav [ ] , and the hypoxia induced factor hif a that is continuously degraded in normoxic conditions [ ] , were not affected ( figure a and b) . ikba, a cytosolic substrate of crl -btrcp, was progressively degraded, confirming that the ligase is inactivated only in the nucleus. the involvement of bplf in the degradation of nuclear cullins and stabilization of nuclear crl substrates was confirmed by shrna knockdown ( figure c ). thus, expression of the bplf specific shrna rescued the downregulation of cul , cul , cul a and cul and promoted destabilization of their nuclear substrates p , p , cdt and cdc a, whereas the levels of cul , vav and hif a (not shown) remained unchanged. interestingly, the levels of ikba were significantly increased in cells expressing the bplf specific shrna suggesting that the viral protein may indirectly regulate the activity of nfkb. we have previously shown that the capacity of bplf to promote the deneddylation and degradation of cullins is dependent on binding to cullins at a site overlapping with the binding site of the regulator cand . the interaction is inhibited by overexpression of the n-terminus of cand , which rescues cullin deneddylation and degradation [ ] . in order to assess whether this regulatory interaction may operate during virus replication, the productive cycle was induced in akata-bx cells transiently transfected with plasmids expressing myc-tagged cand or the cand nterminus that compete for bplf binding to cullins, or, as controls, the cand c-terminus that binds to the opposite end of the cullin scaffold, and the empty vector ( figure a ). expression of comparable amounts of the transfected proteins was confirmed in western blots probed with a myc-specific antibody ( figure a , upper panels). in line with the above documented effects, low levels of cul , cu a and cul and high levels of the crl substrate cdt were detected upon induction of the productive cycle in cells transfected with the empty vector. the degradation of cullins and stabilization of cdt were reversed in cells expressing the full length or the n-terminus of cand that compete for binding of bplf , whereas the c-terminus of cand had no effect ( figure a ). to assess the functional significance of this finding, the efficiency of viral dna replication was measured by q-pcrs in akata-bx transfected with cand or the deletion mutants using primers specific for unique sequences in the bzlf and ebna coding genes ( figure b ). induction of the productive cycle was associated with more than -fold increase of viral dna content in cell transfected with the empty vector or the cand cterminus while viral dna replication was strongly impaired in cells expressing the full-length cand or the cand nterminus. it is noteworthy that only the full-length cand that blocks both the n-terminal and c-terminal domains of cullins regulates the neddylation cycle in non-infected cells. thus, the capacity of the n-terminal domain to fully reverse the inactivation of crls in infected cells is consistent with a mechanism of action based on inhibition of the binding of bplf to cullins, and identifies cand as a potent and specific cellular inhibitor of ebv replication. the large tegument proteins of herpesviruses are huge proteins predominantly localized in the cytoplasm of the infected cells where they play a key role in the delivery of viral dna to the nuclear pore during primary infection and in the secondary envelopment and egress of mature virions [ ] . however, the preferential effect on nuclear cullins and their substrates implies that the enzymatic activity of endogenous bplf is compartmentalized to the nucleus. to find a possible cause of this puzzling observation, the subcellular localization of bplf was investigated using a polyclonal rabbit serum raised against the catalytic nterminus (amino acids - ). to confirm recognition of the active enzyme, lysates of untreated and induced akata-bx were labeled with the ha-ub-vs and flag-nedd -vs functional probes that covalently bind to the catalytic cysteine. western blots of anti-ha and anti-flag immunoprecipitates were then probed with antibodies to ha, flag and bplf . as illustrated by the representative blots shown in figure a , the anti-ha and anti-flag antibodies detected two de-novo expressed enzymatic activities associated with polypeptides of . kd and approximately kd in the lysates of induced cells. probing of parallel blots with affinity purified antibodies to bplf confirmed that the high molecular weight species corresponds to the full-length bplf , while the kd species is likely to represent an approximately kd n-terminal catalytic domain cross-linked to the kd probe. prediction algorithms were then used to screen the amino acid sequence of bplf for the presence of cleavage sites for known cytosolic endo-peptidases. several putative caspase-cleavage sites were identified in the first amino acids of bplf (supplementary, figure s ). in particular, a high-score caspase- cleavage site in position asp may generate a catalytically active fragment of the observed size. to test whether bplf is cleaved by capsase- , the productive cycle was induced in akata-bx treated with the pan-caspase inhibitor zvad-fmk, and the specific caspase- inhibitors yvad-cho and small molecule vx- . cell lysates collected after h were labeled with ha-ub-vs (not shown) and flag-nedd -vs. as shown in figure b , both the full length and the kd species were readily detected by the anti-flag antibody, although the kd species was significantly stronger, which may be due to more efficient cross-linking of the probe or to poorer transfer of the high molecular weight full length enzyme. the intensity of the kd fragment was strongly decreased when the induction was carried out in the presence of caspase inhibitors while the intensity of the full length species was slightly but consistently increased, confirming that the catalytic nterminus of bplf is cleaved by caspase- . having established the specificity of the antibody, we then turned to investigate the subcellular localization of bplf . staining of akata-bx with the affinity purified rabbit serum revealed a diffuse cytoplasmic and nuclear fluorescence h after induction ( figure c ), with essentially no background in cells stained with tritc-conjugated secondary antibody alone. the specificity of the staining was confirmed by its virtual abrogation in induced cells expressing a bplf -specific shrna. the nuclear fluorescence was virtually abolished when the induction was performed in the presence of the pan-caspase inhibitor (not shown) or the caspase- inhibitors yvad-cho ( figure c , d) and vx- (not shown). thus, accumulation of the catalytic n-terminus of bplf in the nucleus is dependent on cleavage of the cytosolic protein by caspase- . since the enzymatic activity of bplf is required for efficient ebv dna replication [ , ] , we tested whether the latter is affected by inhibition of caspase- . in line with the constitutive il- production of b-lymphoma cell lines [ , ] , two bands of approximately kd and kd, corresponding to the pro-caspase and active caspase- , were detected in western blots of untreated akata-bx probed with a caspase- specific antibody ( figure a ). the intensity of the kd band increased upon induction of the productive cycle, which was prevented by addition of the caspase- inhibitors yvad-cho ( figure a ) and vx- (not shown). inhibition of caspase- did not affect the expression of the viral transactivator bzlf , nor the subsequent expression of the early antigen borf and late antigen gp / ( figure a ). nevertheless, the treatment abrogated the bplf -dependent degradation of cul and cul a and consequent stabilization of the crl substrates cdt and cdc a ( figure b ). in line with the requirement of cdt stabilization for efficient viral dna replication [ ] , the yield of viral dna was significantly impaired ( figure c ). similar levels of inhibition were achieved in cells treated with the pan-caspase inhibitor zvad-fmk and the caspase- specific inhibitors yvad-cho and vx- . thus, caspase- appears to be the sole responsible for bplf processing. in the final set of experiments we asked whether the effect of caspase- is restricted to akata-bx cell line. to this end, the productive virus cycle was induced by culturing the prototype ebv producer cell line b . in medium supplemented with % fcs, ng/ml tpa in the presence or absence of increasing concentrations of vx- . as shown in figure s , the marmoset cell line expresses a conserved caspase- species that is detected by the cross-reactive antibody used in our experiments, and the intensity of a polypeptide of approximately kd, corresponding to the active caspase- , increased upon induction of the productive cycle. inhibition of caspase- activity by addition of vx- resulted in a dose-dependent inhibition of viral dna synthesis, with maximal inhibition observed in the presence of mm vx- ( figure d ). this was paralleled by a corresponding dosedependent inhibition of the release of infectious virus assessed by the capacity of spent supernatants to induce the expression of ebna in the ebv negative bjab cell line ( figure e ). our present study addresses an ongoing debate on the contribution of the deneddylase encoded in the large tegument protein of herpesviruses to virus replication, and provides a clear example of how, under physiologic conditions of expression, several lines of evidence support the notion that endogenously expressed bplf promotes ebv replication by acting as a deneddylase. the recombinant enzyme has potent ubiquitin deconjugase activity in vitro, and overexpression of the catalytic n-terminus induces a global decrease of ubiquitin conjugates in transfected cells [ , ] . however, induction of the productive cycle was not accompanied by significant changes in the amount of ubiquitinated proteins or free ubiquitin in akata-bx , whereas neddylated proteins decreased and free nedd increased in a bplf -dependent manner (figures and ). while inconsistent with the potent deconjugase activity of bplf in experimental settings, our failure to detect appreciable change in the global levels of ubiquitination during productive infection does not formally exclude that the viral enzyme might selectively target few ubiquitinated substrates. yet, it should be stressed that evidence for the capacity of bplf to deubiquitinate specific substrates, such as the ebv ribonucleotide reductase [ ] , pcna [ ] and traf [ ] , was in all cases obtained by overexpressing the catalytic n-terminus in transfected cells. in the experiments of saito et al. reconstitution of the viral enzyme in cells infected with a mutant ebv strain that lacks bplf was associated with deubiquitination of traf [ ] . however, induction of the productive virus cycle was associated with strong traf deubiquitination also in the absence of bplf , suggesting that other factors are primarily responsible for this effect and for the subsequent downregulation of nf-kb target genes. this interpretation is also supported by our findings that the nfkb inhibitor ikba was stabilized upon silencing of the endogenous bplf in induced akata-bx ( figure c ), which may be explained by failure to phosphorylate ikba due to a bplf -independent inhibition of traf signaling associated with replicative cycle. similar to the effect of bplf in transfected cells [ , ] , we found that endogenously expressed bplf is required for the selective degradation of cullins in productively infected cells and for the stabilization of several crl substrates that regulate the cell cycle and facilitate ebv dna replication (figures and ) . the reversion of this phenotype by overexpression of the crl regulator cand ( figure ) strengthens the notion that bplf plays a key role in cullin deneddylation and degradation under physiological conditions of expression. we have previously reported that the conserved n-terminal domains of bplf and cand share a binding site on the c-terminus of cullins [ ] . cand regulates the activity of crls by sequestering cullins that are deneddylated after substrate ubiquitination, which promotes the exchange of substrate-adaptor modules, broadening the substrate repertoire and allowing rapid adaptation to a variety of metabolic conditions [ ] . cullins are the only known binding partners of cand and it is therefore reasonable to assume that the reduced ebv dna replication in cells overexpressing cand , and in particular the truncated cand n-terminus that lacks the protein exchange function of the intact protein, is due to inhibition of the binding of bplf of cullins. collectively these findings support the notion that binding to the neddylated substrate determines the activity of this potentially promiscuous enzyme under physiological conditions of expression. this has two important implications. first, it underscores the possibility of experimental artifacts due to altered stoichiometry of the interacting partners in transfected cells. more importantly, it emphasizes the likelihood that interference with binding may have substantial downstream effects. in the case of ebv infection this could offer a new target for specific inhibition of virus replication. we have shown that endogenously expressed bplf acts on nuclear cullins and stabilizes nuclear crl substrates while cytosolic crls and their substrates are not affected. this nuclear compartmentalization is dependent on cleavage of the catalytic nterminus by caspase- ( figure ). the processed fragment does not contain a putative nuclear localization signal but the size is sufficiently small for free diffusion through the nuclear pore that accommodates particles of up to kd. processing of bplf is likely to be a key regulatory event in virus replication. it is noteworthy that a catalytically active n-terminal fragments of the bplf homolog ul has been detected in cells infected with hsv [ ] , and preliminary results suggest that blockade of caspase- has a comparable inhibitory effect on hsv replication (gastaldello et al. unpublished) . although processing of the tegument protein was not detected in cells infected with hcmv [ , ] , kshv [ ] or mhv- [ ] , and the full length enzymes encoded by these viruses are active dubs, we have previously shown that the catalytic n-terminus of kshv and mhv- share the cullin-binding capacity of bplf and inactivate crls in transfected cells [ , ] . it remains to be seen whether the failure to detect processing of some tegument proteins is explained by different experimental procedures or whether it might reflect true differences in the interaction of these viruses with the infected cells. in the case of bplf , it is tempting to speculate that, in addition to facilitating nuclear accumulation, cleavage of the catalytic nterminus may also activate the viral enzyme. this possibility is supported by the observation that the band corresponding to the processed bplf bound to the ub-vs and nedd -vs functional probes was significantly stronger than the full length protein in lysates of induced cells ( figure a and b) . furthermore, cullins were not degraded in the cytosol, suggesting that the cytosolic enzyme is either inactive or cannot reach its targets. it is also possible that, while acting as a deneddylase in the nucleus, bplf , or perhaps the unprocessed form of the enzyme, may act as an ubiquitin-specific deconjugase in the cytoplasm of the infected cells. experimental testing of this possibility remains an interesting focus for future work, pending the identification of substrates that are affected under physiologic conditions of expression. in this context it is noteworthy that cytosolic tegument proteins associated with the virion play important roles in the early and late phases of infection by contributing to the delivery of viral dna to the nuclear pore and to the secondary envelopment and egress of mature virions [ ] . conceivably, a different set of cellular and viral substrates may be affected during these phases of the infection. an unforeseen outcome of our study is the demonstration that the infected cell may regulate the efficiency of virus replication via caspase- mediated processing of bplf . caspase- is well known for its role as the converging target of danger signals such as physical stress, extracellular atp, bacterial and viral products, figure . cleavage by caspase- releases the catalytic n-terminus of bplf and promotes its accumulation in the nucleus. a. a catalytically active n-terminal fragment of bplf is produced during ebv replication. lysates of control and induced akata-bx were labeled with the ha-ub-vs and flag-nedd -vs functional probes and immunoprecipitated with ha-or flag-specific antibodies. western blots were probed with antibodies to ha or flag and with an affinity purified rabbit serum raised against the n-terminus of bplf . cellular dubs labeled by the ha-ub-vs functional probe are indicated by asterisks. a non-specific band of approximately kd detected by the anti-ha antibody in both untreated and induced akata-bx is indicated by an empty circle. one representative experiment out of four performed with different batches of affinity-purified antibodies is shown. b. inhibition of caspase- prevents the production of the catalytic n-terminal fragment. lysates of akata-bx induced in the presence of the indicated caspase inhibitors were labeled with the flag-nedd -vs functional probe and flag immunoprecipitates were probed with a flag-specific antibody. one representative experiment out of three is shown. c. inhibition of caspase- prevents the accumulation of bplf in the nucleus. fluorescence micrographs of akata-bx induced in the presence of absence of caspase- inhibitors stained with the affinity purified antibody to bplf . as controls induction was performed in akata-bx expressing a bplf specific shrna. scale bar mm. representative cells are enlarged. d. quantification of nuclear and cytoplasmic fluorescence in cells from independent experiments. the images were analyzed using the imagej software. doi: . /journal.ppat. .g that are detected in the cytosol by sensing molecules and adaptors that promote the assembly of a multisubunit complex known as the inflammasome [ ] . the inflammasome triggers the self-activation of caspase- , which in turn mediates the maturation of proinflammatory cytokines like interleukin (il)- b and il- , and executes a rapid program of cell death known as pyroptosis [ ] . additional cellular substrates of caspase- include the sterol regulatory element binding proteins (srebps) that are activated following changes in intracellular ions [ ] . thus, caspase- plays pleyotropic roles in the activation of innate and adaptive immune responses as well as in processes that link changes of the intracellular environment with lipid metabolism, membrane biogenesis and cell survival. many viruses are known to inhibit the inflammasome or directly block the activity of caspase- in order to counteract antiviral responses [ ] . our findings highlight a previously unrecognized role of the cellular response to danger signals triggered by ebv reactivation in promoting rather than inhibiting virus replication. this further illustrates the complexity and multilayer regulation of the interaction of ebv with its host where the capacity of the virus to adapt to and exploit physiologic cellular responses underlies the establishment of life-long persistent infections. . inhibition of caspase- prevents the inactivation of nuclear crls and hampers ebv dna replication. a. caspase- is constitutively active in akata-bx and is further activated during virus replication but is not required for expression of the lytic cycle genes. akata-bx cells were induced in the presence or absence of caspase- inhibitors and western blots were probed as indicated. the asterisks in the caspase- blot indicate residual igg heavy chains detected by the secondary antibody. one representative experiment out of three is shown. b. inhibition of caspase- prevents the degradation of cullins and stabilization of crl substrates. western blots of cell lysates produced as described in figure a were probed with the indicated antibodies. one representative experiment out of three is shown. plasmids encoding a bplf specific shrna [ ] , and myctagged full-length human cand , the cand c-terminus (cand -c) and n-terminus (cand -n) [ ] were described previously. recombinant lentiviruses were produced in hek t cells transfected with the recombinant plko. expressing a control scrambled or bplf -specific shrna and the packaging plasmids, pspax and pmd .g [ ] (addgene, dr. didier trono, epfl lousanne, switzerland) by calcium phosphate precipitation and supernatant containing viral particles was collected after h. virus titers were assessed by quicktiter lentivirus titer kit, a hiv p specific elisa (cell biolabs inc., san diego, ca). akata-bx cells that carries a recombinant ebv where the thymidine kinase gene was replaced by a cmv immediate early promoter driven gfp [ ] the productive virus cycle was induced in akata-bx by incubating cell pellets for h at uc with polyclonal rabbit antihuman igg ( : , dako, denmark) and monitored by the increased of gfp fluorescence or by probing western blots with antibodies specific for the ebv transactivator bzlf . for induction of the productive cycle in the b . cell line the cells were placed in a six well plate at the density of cells/ml in ml medium supplemented with % fcs and ng/ml tpa and the spent supernatant was harvested after weeks [ ] . where indicated, the caspase- inhibitor vx- was added to the culture medium. quantitative-pcr was performed on dna extracted from the cell pellets and culture supernatant. total dna was purified with the dnazol reagent (invitrogen) and ebv dna content was assayed by qpcr with the kapa sybr fast qpcr kit (kk , kapa biosystems, cape town, south africa) and an abiprism sequence detection system using ng of dna and the probes: ebna : ggacgtggagaacagt-catc / cactcctgcccttcctcacc , product bp; bzlf : cactaccaggtgccttttgt / gagactgg-gaacagc tgagg , product bp; gapdh: aaggtcg-gagtcaacggatt / ctcctggaagatggtgatgg , product bp. the cycling conditions were: initial step uc min, denaturation uc min, followed by cycles of uc for seconds, uc for min, uc for seconds, uc for seconds and uc for seconds. a final extension for min at uc and melting curve between uc to uc, uc/second transition were incorporated. optical raw data were exported to microsoft excel for analysis. all qpcr reactions were performed in triplicate and ct values were averaged. the fold change in the target gene relative to the gapdh endogenous housekeeping control gene is determined by: fold change = d (dct) where dct = ct target -ct gapdh the subcellular localization of bplf was investigated by immunofluorescence in akata-bx cells h after induction in the presence or absence of caspase inhibitors. eight cells were deposited on glass slides by cytospin centrifugation, fixed with % paraformaldehyde and permeabilized with . % v/v tritonx- in pbs for min. the slides were then treated with blocking solution ( . % bsa in pbs) for h at rt and incubated with the anti-bplf antibody o/n at uc followed by incubation for h with tritc conjugate anti-rabbit ig ( : , r , dako) . the slides were mounted with vectashield mounting medium containing dapi (h- , vector laboratories) and images were captured with a zeiss lsm meta confocal microscope and analyzed with the imagej . q software (wayne rasband, national institutes of health, usa). akata-bx cells were washed in cold pbs containing . m freshly added iodoacetamide and resuspended in hypotonic lysis buffer ( mm hepes, ph . , . mm mgcl , mm kcl, . % np- , mm dtt, mm iodoacetamide, mm orthophenantroline, mm nem and protease inhibitors cocktail. after incubation on ice for min, the membranes were broken by passage through a - g needle and the nuclei were pelleted by centrifugation for min at rpm. the supernatant was used as cytoplasmic fraction. the nuclei were washed three times with hypotonic buffer and lysed in buffer containing mm tris-hcl, ph . , mm nacl, % np , . % doc and protease inhibitors cocktail. protein concentration was measured with a protein assay kit (bio-rad laboratories, ca). total cell lysates were prepared in lysis buffer ( mm tris-hcl ph . , mm nacl, mm dtt, mm edta ph . , . % np , . % sds, mm nem, mm ortho-phenantroline, protease inhibitors cocktail) and protein concentration was measured with a protein assay kit (bio-rad laboratories, solna, sweden). twenty mg of cell lysate were denatured for min at uc in loading buffer and fractionated in acrylamide bis-tris - % gradient gel (invitrogen, carlsbad, ca). after transfer to pvdf membranes (millipore, bedford, ma), the filters were blocked in pbs containing . % tween- and % non-fat milk or % bsa and incubated with the primary antibodies for either h at room temperature or overnight at uc followed by incubation for h with the appropriate horseradish peroxidaseconjugated secondary antibodies. the complexes were visualized by chemiluminescence (ecl, ge healthcare, uppsala, sweden). deconjugase activity was assayed by labeling with the ha-ub-vs and flag-nedd -vs functional probes (boston biochem, boston, ma) as described [ ] . ten cells were lysed in ml of buffer containing mm tris-cl ph . , mm nacl, mm dtt, mm pmsf, . % np , mm glucose, mm mgcl (lysis and labeling buffer, llb) followed by strokes through a g needle. functional labeling was performed by addition of . mg of ha-ubiquitin-vs or flag-nedd -vs followed incubation for min at uc. the cross-linked proteins were immunoprecipitated with anti-ha-agarose or anti-flagagarose beads for h at uc with rotation and eluted by competition with mg/ml of the ha or flag peptides in llb. enzymatically active proteins were detected in western blots probed with anti-ha or anti-flag antibodies. putative caspase cleavage sites were searched in the bplf amino acid sequence using the grabcas software [ ] . sites with the highest probability of cleavage were identified by setting the cut-off scores to . . statistical analysis was performed using student's t-test. p-values , . were considered as significant. figure s kinetics of expression of immediate early, early and late antigens in induced akata-bx . akata-bx cells were treated for h with anti igg antibodies and western blots of cell collected at the indicated times were probed with antibodies to the immediate early antigen bzlf , early antigen borf and late antigen gp / . western blots from one representative experiment are shown. figure s effect of caspase- inhibition on the nuclear localization of bplf . representative localization profile of dapi and tritc fluorescence in induced akata-bx and cells treated with the caspase- inhibitor yvad-cho or bplf specific shrna. the bplf specific fluorescence was homogeneously distributed in the nucleus and cytoplasm of untreated cells but was excluded from the nucleus of caspase- inhibitor treated cells. background levels of bplf fluorescence were observed in cells expressing a bplf specific shrna. (tif) figure s induction of the productive cycle promotes the activation of caspase- in b . cells. the productive cycle was induced in b . cells by treatment with the indicated amounts of tpa or tpa and nabut in medium containing % fcs. induction of the ebv productive cycle was confirmed after one week by probing western blots of total cell lysates with antibodies specific for immediate early (bzlf ) early (borf ) and late (gp / ) antigens. human caspase- specific antibodies detected a band of approximately kd corresponding to the active caspase- in untreated cells and a stronger band was observed in the induced cells. the high levels of the active caspase- species detected in untreated cells is in line with the constitutive expression of the active enzyme in ebv transformed lcls and may be partly explained by spontaneous entry into the productive cycle. (tif) role of ubiquitin-and ubl-binding proteins in cell signaling ubiquitin-like proteins the ubiquitin-mediated proteolytic pathway: mode of action and clinical implications structural insights into nedd activation of cullin-ring ligases: conformational control of conjugation cullin-based ubiquitin ligase and its control by nedd -conjugating system rnf is a poly-sumo-specific e ubiquitin ligase required for arsenic-induced pml degradation identification of a novel isopeptidase with dual specificity for ubiquitin-and nedd -conjugated proteins nedd : a new ataxin- interactor identification by functional proteomics of a deubiquitinating/deneddylating enzyme in plasmodium falciparum specific and covalent targeting of conjugating and deconjugating enzymes of ubiquitin-like proteins desumoylating enzymes-senps a gammaherpesvirus ubiquitin-specific protease is involved in the establishment of murine gammaherpesvirus infection targeting of host-cell ubiquitin pathways by viruses viral avoidance and exploitation of the ubiquitin system deubiquitinating function of adenovirus proteinase the papain-like protease of severe acute respiratory syndrome coronavirus has deubiquitinating activity a deubiquitinating enzyme encoded by hsv- belongs to a family of cysteine proteases that is conserved across the family herpesviridae epstein-barr virus encodes three bona fide ubiquitin-specific proteases cleavage specificity of the ul deubiquitinating protease activity of human cytomegalovirus and the growth of an active-site mutant virus in cultured cells highmolecular-weight protein (pul ) of human cytomegalovirus is a competent deubiquitinating protease: mutant viruses altered in its active-site cysteine or histidine are viable a herpesvirus ubiquitin-specific protease is critical for efficient t cell lymphoma formation mutagenesis of the active-site cysteine in the ubiquitin-specific protease contained in large tegument protein pul of pseudorabies virus impairs viral replication in vitro and neuroinvasion in vivo enzymatic blockade of the ubiquitin-proteasome pathway a viral deubiquitylating enzyme restores dislocation of substrates from the endoplasmic reticulum (er) in semiintact cells positive reciprocal regulation of ubiquitin c-terminal hydrolase l and beta-catenin/ tcf signaling epstein-barr virus bplf deubiquitinates pcna and attenuates polymerase eta recruitment to dna damage sites epstein-barr virus deubiquitinase down-regulates traf -mediated nf-kappab signaling during productive replication a deneddylase encoded by epstein-barr virus promotes viral dna replication by regulating the activity of cullin-ring ligases herpes virus deneddylases interrupt the cullin-ring ligase neddylation cycle by inhibiting the binding of cand suppressor of cytokine signaling- inhibits vav function through protein degradation hifalpha targeted for vhl-mediated destruction by proline hydroxylation: implications for o sensing the c terminus of the large tegument protein pul contains multiple capsid binding sites that function differently during assembly and cell entry of herpes simplex virus b-cellderived interleukin (il- )-like factor. i. relationship of production of il- -like factor to accessory cell function of epstein-barr virus-transformed human blymphoblast lines b-cell-derived interleukin- (il- )-like factor. ii. sources, effects, and biochemical properties the epstein-barr virus (ebv) deubiquitinating enzyme bplf reduces ebv ribonucleotide reductase activity cand promotes assembly of new scf complexes through dynamic exchange of f box proteins kaposi's sarcoma-associated herpesvirus encodes a viral deubiquitinase a functional ubiquitin-specific protease embedded in the large tegument protein (orf ) of murine gammaherpesvirus is active during the course of infection inflammasomes and their roles in health and disease inflammasomes: caspase- -activating platforms with critical roles in host defense caspase- activation of lipid metabolic pathways in response to bacterial pore-forming toxins promotes cell survival inflammasomes and viruses: cellular defence versus viral offence a lentiviral rnai library for human and mouse genes applied to an arrayed viral high-content screen inhibition of heavy chain and beta -microglobulin synthesis as a mechanism of major histocompatibility complex class i downregulation during epstein-barr virus replication epstein-barr virus: transformation, cytopathic changes, and viral antigens in squirrel monkey and marmoset leukocytes a novel active site-directed probe specific for deubiquitylating enzymes reveals proteasome association of usp grabcas: a bioinformatics tool for score-based prediction of caspase-and granzyme bcleavage sites in protein sequences we are grateful to drs kazuhiro iwai (department of biophysics and biochemistry, graduate school of medicine, osaka university, osaka, japan), ron t. hay (college of life sciences, university of dundee, dundee, uk) and jaap middeldorp (cca-vumc, amsterdam, the netherlands) for providing plasmids, antibodies and technical advice, and to teresa frisan for useful comments and critical reading of the manuscript. the contribution of the undergraduate students sandra zlodej, gianluca spaltro and jim baggen is gratefully acknowledged. key: cord- - lnj w authors: de vries, erik; tscherne, donna m.; wienholts, marleen j.; cobos-jiménez, viviana; scholte, florine; garcía-sastre, adolfo; rottier, peter j. m.; de haan, cornelis a. m. title: dissection of the influenza a virus endocytic routes reveals macropinocytosis as an alternative entry pathway date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: lnj w influenza a virus (iav) enters host cells upon binding of its hemagglutinin glycoprotein to sialylated host cell receptors. whereas dynamin-dependent, clathrin-mediated endocytosis (cme) is generally considered as the iav infection pathway, some observations suggest the occurrence of an as yet uncharacterized alternative entry route. by manipulating entry parameters we established experimental conditions that allow the separate analysis of dynamin-dependent and -independent entry of iav. whereas entry of iav in phosphate-buffered saline could be completely inhibited by dynasore, a specific inhibitor of dynamin, a dynasore-insensitive entry pathway became functional in the presence of fetal calf serum. this finding was confirmed with the use of small interfering rnas targeting dynamin- . in the presence of serum, both iav entry pathways were operational. under these conditions entry could be fully blocked by combined treatment with dynasore and the amiloride derivative eipa, the hallmark inhibitor of macropinocytosis, whereas either drug alone had no effect. the sensitivity of the dynamin-independent entry pathway to inhibitors or dominant-negative mutants affecting actomyosin dynamics as well as to a number of specific inhibitors of growth factor receptor tyrosine kinases and downstream effectors thereof all point to the involvement of macropinocytosis in iav entry. consistently, iav particles and soluble fitc-dextran were shown to co-localize in cells in the same vesicles. thus, in addition to the classical dynamin-dependent, clathrin-mediated endocytosis pathway, iav enters host cells by a dynamin-independent route that has all the characteristics of macropinocytosis. influenza a virus (iav) is an enveloped, segmented negativestrand rna virus infecting a wide variety of birds and mammals. as its first step in infection iav attaches to host cells by the binding of its major surface protein, the hemagglutinin (ha), to sialic acids, which are omnipresent on the glycolipids and glycoproteins exposed on the surfaces of cells. where the structural requirements for this interaction have been studied in great detail, much less is known about whether and how the attachment to specific sialylated receptors (e.g. to n-linked glycoproteins, olinked glycoproteins or gangliosides or even to specific receptors within these groups) affects the subsequent endocytic steps. obviously, knowledge about the repertoire of endocytic pathways that can successfully be used by iav will increase our insights into cell and species tropism of iav. in turn, this will contribute to our understanding of the requirements for the generation of novel viruses with pandemic potential that can arise by exchange of rna segments between currently circulating human serotypes and an animal virus during occasional co-infection in a human or an animal host. clathrin mediated endocytosis (cme) has for long been identified and studied as the major route of iav cell entry [ , ] and is, by far, the best characterized endocytic pathway. evidence obtained from live cell imaging has revealed the de novo formation of clathrin-coated pits at the site of virus attachment [ ] and the requirement for the adapter protein epsin , but not eps , in this process [ ] . still, specific transmembrane receptors linking viral entry to epsin or to other adapters have not been identified although a recent study performed in cho cells indicated the specific requirement for n-linked glycoproteins in iav entry [ ] . some recent papers provided indications for the utilization of alternative entry pathways by iav. studies in which cme was obstructed by pharmacological or genetic intervention indicated the ability of iav to enter host cells via alternative endocytic routes [ , , ] . also live cell imaging revealed the simultaneous availability of entry routes involving non-coated as well as clathrin-coated pits [ ] . however, this alternative iav entry route has not been characterized in any detail and requirements for any specificity in receptor usage apart from the need for the proper sialic acid moiety have not been established. during the past decades quite a variety of endocytic pathways have been identified in eukaryotic cells [ , , ] . their occurrence, abundance and mechanistic details appear to vary between cell types, tissues and species and their utilization by viruses as a route of entry makes them an important factor in host and cell-type permissiveness for infection [ , ] . besides by cme, different viruses have been shown to enter cells via caveolae, macropinocytosis or other, less well described, routes [ , ] . most often, the selection of a specific endocytic route is linked to the utilization of a specific receptor that facilitates traveling via that particular route. nevertheless, many receptors allow flexibility by their capacity to enter through multiple pathways. for iav, an additional level of complexity to the dissection of potential entry routes is added by the apparent lack of an iav-specific protein receptor. a full experimental characterization of the iav entry pathways will benefit from separation of the iav entry pathways into routes that can be studied independently. whereas co-localization with clathrin is an established marker for endocytosis via this route, the complete lack of unique markers for macropinosomes or most other endocytic compartments [ , ] complicates such studies. furthermore, crucial to any study concerning endocytic pathways is the abundantly documented fact that such pathways are highly dependent on experimental cell culture conditions [ ] [ ] [ ] [ ] [ ] . pathways that are constitutive in one cell type may be absent or inducible by specific experimental conditions in other cell types. moreover, the manipulation of specific endocytic pathways may result in up or down regulation of other specific pathways. here we have established entry assay conditions that allow dissecting cell entry of iav into a dynamin-dependent (dyna-dep) and a dynamin-independent (dyna-ind) component. dynamin is a large gtpase forming multimeric assemblies around the neck of newly formed endocytic vesicles. gtp hydrolysis is required for pinching off of the vesicles [ ] . whereas cme is completely dependent on dynamin, several other endocytic routes do not require dynamin [ ] . we performed an extensive characterization of the dynamin-independent iav entry route using pharmacological inhibitors as well as by expressing dominant-negative mutants and applying sirna induced gene silencing as tools. taken together the results identify a pathway that closely resembles macropinocytosis as a novel entry pathway for iav. to identify and characterize potential non-cme entry routes taken by iav, we adapted a luciferase reporter assay [ ] to enable the quantitative determination of infection or entry by measuring the activity of secreted gaussia luciferase. twentyfour hours prior to infection hela cells were transfected with a plasmid (phh-gluc) allowing constitutive synthesis (driven by the human poli promoter) of a negative strand viral rna (vrna) encoding a gaussia luciferase under control of the untranslated regions (utrs) of the np segment of influenza a/wsn/ (h n ) (hereafter called iav-wsn) np segment. upon iav infection, the combined expression of the viral polymerase subunits and np will drive transcription of luciferase mrna from the negative strand vrna and subsequent synthesis of gaussia luciferase. a dose-response curve demonstrating the applicability of the assay to inhibitor screening (fig. a) was obtained for bafilomycin a (bafa ), a known inhibitor of iav entry [ ] . bafa acts upon the vacuolar-type h(+)-atpase, thus preventing endosomal acidification and thereby trapping iav in peri-nuclear immature endosomes with a lumenal ph that does not permit viral membrane fusion. remarkably, dynasore, a small molecule inhibitor of the gtpase dynamin that is crucial for endocytic vesicle formation in clathrin-and caveolin-mediated endocytosis [ ] as well as in a poorly described clathrin-and caveolin-independent endocytic pathway [ , ] , did not give significant inhibition (fig. b) . bafa specifically inhibits iav during the entry phase as demonstrated in fig. c . the continuous presence of nm bafa (added to the cells hr prior to infection) for hrs completely prevents infection. in contrast the addition of bafa at hr or hrs post infection resulted in high levels of luciferase activity (again measured at hrs p.i.) that were % or % respectively of the control to which no bafa was added, indicating that entry was essentially completed within hrs. the last bar of fig. c shows that the inhibition by bafa is reversible as withdrawal of the inhibitor after hrs resulted in high levels of infection. the specific effect of bafa on iav entry was confirmed by confocal microscopy demonstrating that bafa , as expected, traps iav particles in a peri-nuclear location, presumably in nonacidified endosomes (fig. d) . bafa was subsequently exploited to establish a specific iav entry assay (hereafter further referred to as the gluc-entry assay). hela cells transfected with phh-gluc were inoculated with iav at a range of mois and incubated for hrs after which the entry medium was replaced by complete growth medium containing % fcs and nm bafa to prevent any further entry of virus. entry was indirectly quantified by determination of luciferase activity after further incubation for hrs demonstrating a quantitative correlation between infection dose and luciferase activity across a wide range of mois (fig. e) . the indirect gluc-entry assay was next tested for its capacity to examine the effects of inhibitors on iav entry. dynasore or bafa (fig. f) were included in the medium (dmem containing % attachment to and entry into a host cell are the first crucial steps in establishing a successful virus infection and critical factors in determining host cell and species tropism. influenza a virus (iav) attaches to host cells by binding of its major surface protein, hemagglutinin, to sialic acids that are omnipresent on the glycolipids and glycoproteins exposed on the surfaces of cells. iav subsequently enters cells of birds and a wide variety of mammals via receptormediated endocytosis using clathrin as well as via (an) alternative uncharacterized route(s). the elucidation of the endocytic pathways taken by iav has been hampered by their apparent redundancy in establishing a productive infection. by manipulating the entry conditions we have established experimental settings that allow the separate analysis of dynamin-dependent (including clathrin-mediated endocytosis) and independent entry of iav. collectively, our results indicate macropinocytosis, the main route for the non-selective uptake of extracellular fluid by cells, as an alternative iav entry route. as the dynamindependent and -independent iav entry routes are redundant and independent, their separate manipulation was crucial for the identification and characterization of the alternative iav entry route. a similar strategy might be applicable to the study of endocytic pathways taken by other viruses. fcs) during entry (the first h of infection) and were removed when the inoculum was replaced by growth medium containing bafa . concentrations up to mm dynasore did not inhibit entry which is in agreement with the result shown in fig. b . in contrast, . nm bafa already inhibited entry for more than % (fig. f) . as a control, dynasore was also added at hrs post infection to analyze whether the drug affected iav replication during the post entry phase. as expected, mm dynasore did not significantly inhibit iav replication when present from to hrs p.i. (fig. f ). thus, with the gluc-entry assay we can study the effect of specific inhibitors on iav entry in a quantitative manner, at least as long as the inhibitors do not irreversibly affect iav replication during the post entry phase. furthermore, the lack of inhibition of iav entry by dynasore demonstrates that under these experimental conditions iav is able to enter cells via a pathway that is fully redundant to any dynamindependent (dyna-dep) entry route, including the classical cme pathway. also when iav travels via this novel dynamin-independent (dyna-ind) route, iav apparently enters via low ph compartments as entry is fully sensitive to bafa . as factors present in serum are known for their potential to induce specific endocytic pathways, we further explored the conditions required for the novel dyna-ind iav entry pathway (using the gluc-entry assay) by inoculating cells in pbs in the presence of increasing concentrations of fetal calf serum (fcs). whereas dynasore completely inhibited entry in pbs, inclusion of % and % fcs resulted in increasing levels of dynasore resistant entry ( fig. a) , suggesting the existence of a serum-inducible dyna-ind iav entry pathway. this effect was not caused by inactivation of dynasore during the experiment as vesicular stomatitis virus (vsv), which enters cells by cme [ , ] , was still sensitive to mm dynasore in the presence of % fcs (fig. b) . in agreement herewith, the uptake of transferin, known to occur via cme, was inhibited by dynasore regardless of the hela cells were grown on glass cover slips and infected with iav (strain wsn; moi of ) and fixated after min, hrs or hrs (column , or respectively). infection was performed in . % dmso (upper row panels) or in the presence of nm bafa (lower row panels). the nucleus was visualized by dna staining with topro- (red). iav infection was visualized by staining with monoclonal antiserum directed against np (green). in the absence of inhibitor, iav localized to the nucleus after hrs, while new virus particles spread to the cytoplasm after hrs. bafa (lower row panels) caused accumulation of incoming virus particles at a peri-nuclear location. (e) quantitative determination of iav entry by a single-cycle gluc-entry assay. hela cells ( , cells/well in dmem supplemented with % fcs) were transfected with phh-gluc hrs prior to infection with a serial dilution of infectious iav particles (plotted on the x-axis). two hours after infection nm bafa was added to block any further entry. cells were incubated for a further hrs to allow expression of luciferase activity (y-axis; relative light units, rlu). (f) effect of dynasore and bafa on iav entry in the gluc-entry assay. dynasore (dy, dark grey bars; , or mm) or bafa (light grey bars; . , . or nm) were present from hr prior to infection (strain wsn; moi . ) to hrs p.i. after which the inhibitor-containing medium was replaced with medium containing nm bafa to block any further entry. cells were incubated for a further hrs to allow the quantitative expression of luciferase activity (y-axes; rlu relative to the control infection without inhibitor). whereas bafa displayed dose-dependent inhibition of iav entry, dynasore did not significantly inhibit iav entry. presence of fcs (fig. s , panel a) . as expected, both dyna-dep entry in pbs and dyna-ind entry in the presence of % fcs and mm dynasore required sialic acid receptors for efficient entry as pre-treatment of hela cells with neuraminidases almost completely abolished entry via either pathway (fig. c ). the kinetics of the dyna-dep and dyna-ind entry pathways were compared by performing a time-course experiment in which iav entry was terminated by the addition of nm bafa at different time points (fig. d) . in comparison to entry via the dyna-dep pathway (the only pathway available in pbs) entry in the presence of fcs (when presumably both the dyna-dep and dyna-ind entry pathways are available) showed similar kinetics. in contrast, entry via the dyna-ind pathway (which is the only pathway that is active in the presence of % fcs and mm dynasore) was slower. the difference was most prominent after min, while after hrs similar levels of entry were reached. to validate and extend these results we visualized the reduction of the number of infected cells by immunoperoxidase staining using an antibody against np (fig. ) . a number of different cells of mammalian and avian origin were infected for hours at an moi of in pbs with or without serum. after hours the inoculum was replaced by growth medium containing % fcs and nm bafa and the expression of np was examined after hours later. after incubation in pbs, staining was completely prohibited by the presence of mm dynasore whereas in the presence of serum dynasore had no effect. a serum-inducible, dyna-ind route of entry was thus functional in all five cell lines, including the human epithelial airway carcinoma cell line a . to confirm our results and to obtain further proof for the utilization of dyna-dep and dyna-ind entry routes by iav, we additionally used an iav virus-like particle (vlp) direct entry assay [ ] . these vlps contain iav ha and na in their envelope and harbor a beta-lactamase reporter protein fused to the influenza matrix protein- (blam ), which allows the rapid and direct detection of entry, independent of virus replication. upon fusion of viral and endosomal membrane, blam gains access to the cytoplasmically retained fluorigenic substrate ccf- that, after cleavage by blam , shifts to a shorter fluorescent emission wavelength that can be detected by flow cytometry. entry into hela cells was performed in the absence or presence of % fcs using vlps containing ha and na either from iav-wsn (having a strict alpha - linked sialic acid binding specificity) or from the pandemic iav (ha from a/ newyork/ / , binding to alpha - and alpha - linked sialic acids; na from a/brevigmission/ / ). entry of vlps of both iav strains was severely inhibited by dynasore when no serum was added to the inoculum (fig. a, d) , whereas the presence of % fcs rendered entry completely dynasore resistant. (fig. b, e ). quantification of vlp entry is shown in fig. c and f. importantly, to confirm the existence of the serum-inducible entry pathway by a method that is independent of dynasore, we used sirna induced silencing of dynamin . fig. g shows that two different sirnas had a significant inhibitory effect ( hrs after sirna transfection) on entry of the renilla luciferaseencoding pseudovirus wsn-ren [ ] in hela cells in the absence of fcs, whereas the presence of % serum no reduction in entry levels was observed, confirming the results obtained with dynasore. knockdown of dynamin protein levels ( hrs after sirna transfection) was analyzed by western we conclude that a dyna-ind entry pathway can be induced by serum in different cell types from several species. the evidence was obtained using both replication-dependent (gluc-entry assay and immunodetection of infected cells) and replication-independent assays (entry of vlps), the latter allowing immediate detection of the fusion-mediated delivery of viral m protein into the cytoplasm. cascade blue). in the histograms entry is displayed by a shift to higher fluorescence (the grey area represents background fluorescence of noninfected cells). (c and f) quantification of facs results. background fluorescence was subtracted from each measurement (geometric mean) and data were normalized to vlp entry in optimem without dynasore (dy) (red curve of panel a and and d). vlp entry was not inhibited by dynasore in presence of % fcs whereas the access of blam to its ccf substrate in the cytoplasm was blocked by dynasore in pbs. vlp entry was more efficient in the presence of serum. (g) effect of downregulation of dynamin by sirna silencing. serum-inducible dyna-ind entry was analyzed in hela cells that were transfected hrs prior to infection with two different sirnas targeting dynamin- (dyna). sirna treated cells were infected with the pseudovirus wsn-ren in pbs (grey bars) or in pbs containing % fcs (black bars) and luciferase activity was determined after hrs post infection (y-axis; rlu relative to infection of cells transfected with a scrambled sirna). entry of pseudovirus wsn-ren (moi . ) was reduced by % to % when entry was performed in pbs (grey bars) whereas entry was not significantly affected in the presence of % serum (black bars). (h) western blot showing the knockdown of dynamin (in comparison to tubulin) at hrs after transfection with sirnas. (i) quantification of the residual levels of dynamin (dyna) mrna (determined by quantitative rt-pcr) and protein (determined by densitometric scanning of the western blot) hrs after sirna transfection. data were normalized to s rna (rt-pcr) or tubulin protein levels and calculated relative to the levels obtained after transfection with a scrambled sirna that served as a control. doi: . /journal.ppat. .g inhibitors of growth factor receptor tyrosine kinases and actomyosin network dynamics reduce dyna-ind entry of iav the dyna-ind entry pathway was further characterized by inhibitor profiling using an -compound kinase inhibitor library. serum-induced dyna-ind entry was examined in % fcs using the gluc-entry assay. mm dynasore was added in order to block cme and any other potential dyna-dep entry pathways. this allowed the independent inhibitor profiling of the novel pathway by avoiding the potentially masking effect of the presence of redundant entry pathways. cells were preincubated with the kinase inhibitors ( mm) for h at uc and then inoculated with virus (moi . ) in the presence of % fcs and mm dynasore for h at uc (dyna-ind entry). in parallel, inoculations were also done in pbs to compare the effects of the inhibitors on dyna-dep entry. after hr the medium and inhibitor were replaced by full growth medium containing % fcs and nm bafa to allow the subsequent expression of gluc activity under identical conditions for the dyna-ind and -dependent entry assay. six kinase inhibitors appeared to act non-discriminatively, inhibiting both dyna-dep and dyna-ind entry (fig. a ): the protein kinase c (pkc) inhibitors ro - , rottlerin (both displaying moderate cytotoxicity, result not shown) and hypericin, which have all three been previously identified as iav inhibitors [ , ] ; the highly cytotoxic pan-specific serine/threonine protease inhibitor staurosporine; the irreversible pi- kinase inhibitor wortmannin and the receptor tyrosine kinase inhibitor tyr . in order to investigate whether some of these inhibitors affect iav replication during the post-entry phase, we performed the same experiments but now adding the kinase inhibitors after viral entry. four of the inhibitors thus appeared to induce significant inhibition of post-entry processes (fig. a ). although unlikely, we cannot formally exclude that post-entry processes specific for only one of the two entry pathways are affected. interestingly, whereas no specific dyna-dep entry inhibitors were identified, inhibitors (none displaying cytotoxic effects, data not shown) caused significant (p, . ) inhibition (. -fold) of dyna-ind entry (fig. b ). this included inhibitors of the calmodulin dependent kinases myosin light chain kinase (mlck) and camkii and seven inhibitors of different growth factor receptor tyrosine kinases. in contrast to the three non-specific pkc inhibitors mentioned above, the pkc inhibitors bim- and hbdde appeared to have a specific inhibitory effect on dyna-ind entry. the specific effect of these drugs on dyna-ind entry is not only shown by the lack of inhibition of dyna-dep entry in pbs, but also by the observation that none of the fifteen compounds induced . -fold inhibition when added post-entry (at t = hr post infection). the kinase library screen was repeated on a human epithelial lung carcinoma cells in order to confirm the results in a potentially more natural host cell line. the inhibition profiles obtained were very similar to those found for hela cells with the exception of the strong effect of ag ( % inhibition) and moderate effects of ag ( % inhibition) and tyr ( % inhibition) on dyna-dep entry. (fig. c) . mlck inhibitors ml- and ml- have been reported to be highly specific for their target kinase [ ] . phosphorylation by mlck activates non-muscle myosin ii light chain, indicating that a functional actomyosin network might be essential for dyna-ind entry of iav. this was further examined by testing the effect of blebbistatin, an inhibitor of myosin ii heavy chain activity, and of several inhibitors that affect actin dynamics by disrupting actin microfilaments (cytochalasin b and d), by enhancing actin polymerization (jasplakinolide) or by inhibiting actin polymeriza-tion (latrunculin a). actin inhibitors were used at the minimal concentration required to induce clearly visible changes in the actin cytoskeleton as pre-determined by staining with fitcphalloidin (results not shown). whereas the inhibitors did not affect dyna-dep entry (fig. a) using gluc-entry assay, all inhibitors as well as ml- and ml- significantly inhibited dyna-ind entry (fig. b) . next, hela cells were transfected with plasmids encoding dominant negative or wildtype rab fused to green fluorescent protein (rab dn and rab wt in fig. ) h prior to infection with iav. rab is a small gtpase found in association with several endosomal compartments and crucial for the function and maturation of early endosomes. it is required for the trafficking of a wide range of endocytic cargo following different routes, including dyna-dep as well as dyna-ind routes [ ] . entry of iav has been shown to require rab [ ] . consistently, we found that hela cells expressing rab dn (as identified by gfp fluorescence, fig. c ) were much less susceptible to productive iav infection (as judged by indirect immunofluorescence using alexa- labeled np antibodies) than cells transfected with several dynamin-independent endocytic pathways have been described [ , ] . of these, macropinocytosis has been demonstrated to be stimulated by growth factors present in serum and to depend on actin dynamics [ ] [ ] [ ] . yet, studies on macropinocytosis are hampered by a lack of specific inhibitors, cargo, membrane markers and characteristic morphology. amiloride and the more potent derivative eipa are inhibitors of epithelial sodium channels (enac) as well as of several other na+/h+ antiporters. eipa has often been used as a hallmark inhibitor that specifically inhibits endocytosis via the macropinocytic pathway [ ] . whereas dyna-dep entry of iav was not inhibited by eipa (fig. a) , dyna-ind entry was fully blocked eipa (fig. b) . the existence of redundant entry pathways in the presence of % fcs is clearly demonstrated by the marginal inhibition by either eipa or dynasore whereas the combination of eipa and dynasore resulted in strong inhibition both in the gluc-entry assay (fig. c ) and in the direct vlp entry assay ( fig. d and e) . supplementary fig. s shows that other cell lines, including the human lung epithelial cell line a , display similar iav inhibition patterns for eipa and dynasore. consistently, virus production displayed a similar inhibitor sensitivity profile (fig. f and g) as virus entry indicating that the entry pathways we characterized lead to a productive infection. clearly, vlps and viral particles follow similar redundant entry pathways, distinguishable in a dyna-dep and a dyna-ind pathway, the latter being sensitive to eipa and dependent on actomyosin function. one characteristic of macropinocytosis is the nonselective uptake of large amounts of extracellular solutes [ ] . therefore, the uptake of soluble fitc labeled dextran (fdx) into relatively large vesicles ( . to mm) has often been applied as a morphological marker for macropinosomes. using this marker we found that the addition of % fcs to the culture medium slightly increased the uptake of fdx into hela cells (fig. a) . notably, the distribution of fdx changes in response to serum from a random distribution into a more granular pattern. at high magnification and at color settings adjusted to higher intensity it could be seen that these fdx granules were free of actin staining (by phalloidin) indicating that they were in the lumen of vesicles (result not shown). interestingly, in the presence of iav (moi of ) the uptake of fdx into vesicles was clearly enhanced. at a higher magnification viral particles could be found to co-localize in fdx loaded vesicles as well as outside these vesicles (fig. b) . phalloidin staining of actin was used to demonstrate that many virus particles localized to actin-rich protrusions at the periphery of the cell. the uptake of fdx was studied in a quantitative manner by flow cytometry (fig. c) . a moderate, but reproducible shift to higher fdx fluorescence was observed at uc when virus was added in presence of % fcs whereas such a shift was absent when no serum or virus was added. this result confirms the observations by confocal microscopy (fig. a) which showed that the combined presence of fcs and iav increases the uptake of fdx as compared to fcs alone. in a control experiment the uptake of fdx in % fcs in presence of iav was shown to be specifically inhibited by eipa, but not by dynasore (fig. s , panel b) . in contrast, transferrin uptake, which serves as a specific marker for cme, was affected by dynasore, but not by eipa (fig. s , panel a) . in conclusion, serum induces the uptake of fdx into large vesicles, which can be further enhanced by the addition of iav particles that, after entry, co-localize in part with these vesicles. these results indicate the utilization of a macropinocytic pathway for entry of iav, which is consistent with the observed sensitivity of the seruminducible dyna-ind entry of iav and vlps to eipa. macropinocytosis has been implicated in the entry of several viruses [ , ] . however, differences in susceptibility to inhibitors suggest that distinct forms of macropinocytosis might be used by different viruses [ , ] . by screening specific inhibitors in the gluc-entry assay using dyna-ind entry conditions we evaluated the possible involvement of a few signaling cascades that have been implicated in the induction of macropinocytosis. serum-inducible macropinocytosis has been shown to be activated via a myriad of signaling cascades initiated by growth factors binding to transmembrane tyrosine kinase receptors [ , , , ] , consistent with the results shown in fig. . a prominent downstream effect of these signaling cascades is the activation of p associated kinase (pak ) which in turn can activate a number of different pathways leading to actin network rearrangements that can ultimately lead to the induction of macropinocytosis [ ] . fig. a -b shows that mm ipa , an inhibitor of pak [ ] , specifically inhibits background fluorescence from fdx binding to the outside of cells was determined by performing the same experiment at uc (at which no endocytosis takes place) and was subtracted from the mean fluorescence intensity obtained at uc to determine the amount of fluorescent fitc-dextran that was internalized at uc. data were plotted relative to fitcdextran uptake in pbs in absence of iav. doi: . /journal.ppat. .g dyna-ind entry of iav. activation of pak in response to growth factor stimulation often involves upstream signal transduction by members of the rho sub-family of small gtpases like cdc and/or rac [ , , ] . alternatively, activated cdc and rac can induce actin rearrangements independently of pak [ , [ ] [ ] [ ] [ ] by direct interaction with wasp or wave family proteins, respectively [ , ] . however, inhibitors of cdc (pirl [ ] ), rac (nsc [ ] ) or n-wasp (wiskostatin [ ] ) did not display inhibitory effects on dyna-ind or dyna-dep entry of iav (fig. c-d) . instead, pirl and wiskostatin induced a significant, concentration dependent increase of entry. this stimulatory effect was not observed for the control vaccinia virus strain wr, which enters cells via a rac dependent, macropinocytotic pathway [ ] (fig. e) , indicating that this effect is specific for iav. the results suggest a requirement for pak in dyna-ind entry of iav that does not require activation by either cdc or rac . growth factor inducible activation of the tyrosine kinase src has also been linked to the induction of macropinocytosis [ ] [ ] [ ] ; consistent with this observation the src inhibitor pp [ ] specifically inhibited dyna-ind entry of iav (fig. a-b) . remarkably, -aageldanamycin, a specific inhibitor of the chaperone protein hsp [ ] , also caused specific inhibition of dyna-ind entry (fig. a-b) . hsp affects the folding and activity of many proteins but the recent demonstration of direct activation of the catalytic activity of src by hsp [ ] provides another indication of the involvement of src in dyna-ind endocytosis of iav. in conclusion, like for other viruses utilizing a macropinocytic entry pathway, pak seems to play a crucial role in dyna-ind entry by iav. however, this pathway is independent of rac or cdc but may require src, either upstream and/or downstream of pak . the data presented in this study demonstrate for the first time that iav can enter cells via dyna-ind macropinocytosis in addition to the previously described dyna-dep classical cme pathway [ , ] . several lines of evidence indicate that the dyna-ind entry route of iav that we identified corresponds with macropinocytosis. first of all, the entry pathway is dependent on the presence of serum, a well-known inducer of macropinocytosis. second, iav colocalized in vesicles with soluble fitc-dextran, a marker for macropinocytosis. third, dyna-ind iav entry was sensitive to the amiloride-derivative eipa, the hallmark inhibitor of macropinocytosis [ , [ ] [ ] [ ] [ ] . fourth, this iav entry pathway is sensitive to inhibitors or dominant-negative mutants affecting actomyosin dynamics. fifth, the specific inhibition of dyna-ind iav entry by a number of inhibitors of growth factor receptor tyrosine kinases as well as downstream effectors thereof also points at the involvement of macropinocytosis. finally, macropinocytosis is independent of dynamin [ , , ] . despite this extensive list of arguments, viral entry by macropinocytosis needs to be considered with caution. the characteristics of the dyna-ind route of cell entry by iav are similar, but not identical to the macropinocytic entry routes taken by other viruses, like two different strains of vaccinia virus and by coxsackie virus b [ , ] . as is shown in table and discussed in more detail below, the macropinocytic pathways used by each of these viruses have a few unique characteristics. this may very well reflect the growing notion that macropinocytosis represents a number of differentially induced and regulated processes, rather than being a single endocytic pathway [ , ] . macropinocytosis has collectively been described as an inducible form of endocytosis by which fluid-phase cargo travels via non-coated, relatively large and heterogeneous organelles that have emanated from extensive protrusions (e.g lamellar ruffles, circular ruffles or retracting blebs) of the plasma membrane [ ] . in the case of dyna-ind iav entry more extensive studies using electron microscopy will be required to study the morphology of membrane protrusions with which iav may associate. in addition, live cell imaging microscopy will be required to characterize the exact itinerary that is taken by iav virions traveling via a macropinocytic process. this is especially important as different routes of iav entry are likely to converge at some point in the endocytic pathway. although unlikely, co-localization of iav particles with fluid-phase dextran as shown in fig. b may thus represent a situation occurring after convergence of several different routes. the use of microscopy to study macropinocytosis is however complicated by the lack of specific membrane-associated markers for any early step of this endocytic process. a model (fig. ) based on our results explains the key steps involved in the macropinocytic entry pathway of iav, which are described in more detail below. by manipulating the inoculation conditions we were able to experimentally dissect iav entry into a dyna-dep and dyna-ind route. the dyna-ind route required the presence of % fcs in the entry assay medium. previously, a strict dependency on a dyna-dep entry route for iav was concluded from experiments with a cell line expressing an inducible dominantnegative mutant of dynamin [ ] . in that study, as well as in other entry studies of iav, entry was performed in dmem containing % serum or bsa. also in our hands . % serum ( fig. a) or . % bsa (result not shown) was not sufficient to allow dyna-ind entry. we are currently investigating which serum component is responsible for the observed effects on iav entry. dialysis of fcs (mw cut off . kda) did not affect its capacity to induce dyna-ind endocytosis (result not shown), indicating that low molecular weight solutes are not responsible for the observed effect. our evidence for a dyna-dep and a serum inducible dyna-ind entry route is based on the use of pharmacological (dynasore, a highly specific inhibitor of dynamin) as well as genetic (sirna directed against dynamin ) tools, ruling out the possibility that the inhibitory effect of dynasore was due for instance to absorption of the inhibitor by serum components. whereas dynasore resulted in near % inhibition of dyna-dep entry, only % inhibition was observed upon sirna induced silencing of dynamin indicating that the residual levels of dynamin that remain after hrs of silencing still support a low level of dyna-dep entry (fig. h) . reversible inhibitors like dynasore [ ] offer a major advantage for characterization of iav entry pathways. they can be applied for a limited period thus preventing the secondary adaptive effects of cells that may occur in response to long-term down regulation of a gene product by genetic methods like sirna interference. both entry routes were consistently identified by a viral entry assay quantified by virus induced expression of a luciferase reporter as well as by a vlp entry assay allowing direct analysis of the membrane fusion mediated entry step. the consistent performance of an ha with a strict preference for binding to a - linked sialic acids (from iav-wsn; our unpublished data) and an ha also binding to a - linked sialic acids (from iav [ ] ) in the vlp entry assay indicates that both pathways can be utilized by has of different specificity and may therefore be relevant to avian as well as human iav infections. consistently, serum-inducible dyna-ind entry was observed both in avian df cells and in a human lung epithelial carcinoma cell line a (fig. ) . the dyna-dep and dyna-ind iav entry pathways were found by our quantitative assays to be fully redundant. in the presence of serum, the combination of dynasore (inhibiting dyna-dep entry) and eipa (inhibiting dyna-ind entry) completely abolished entry whereas either drug alone had no effect. eipa, an inhibitor of plasma membrane na+/h+ exchangers, has been shown to invariably inhibit macropinocytosis [ , [ ] [ ] [ ] [ ] . as other routes of endocytosis are generally not affected, eipa is considered as a hallmark inhibitor of macropinocytosis [ ] , although results obtained with eipa should be considered with care as long as a mechanistic explanation for its effect on macropinocytosis is not yet fully clear [ ] . occasionally, a moderate two-to three-fold inhibition by dynasore alone was observed (result not shown) indicating that the capacity of the serum-inducible entry pathway is somewhat variable, possibly depending on slight variations in serum quality and factors like cell distribution in the wells that have been reported to influence viral infection [ ] . a redundancy in the utilization of cme as well as a clathrin-independent route for entry of iav has been visualized previously by quantitative live cell imaging [ ] . both routes were operative simultaneously in the same sample and the specific down-regulation of cme did not affect the total number of entry events. in response to specific extra-cellular signals (e.g. serum induction), changes in the actomyosin network occur that give rise to membrane protrusions required for macropinosome formation [ ] . compounds inhibiting actin polymerization (cytochalasin b and d), depolymerization (jasplakinolide) or sequestering soluble actin (latrunculin a) all specifically inhibited dyna-ind iav entry. in addition, the requirement for myosinii activity was established by a specific inhibitor (blebbistatin) of myosin ii atpase activity and by the expression of a dominant negative mutant of myosiniia heavy chain. also, the regulation of myosinii activity by phosphorylation of myosin light chain through the action of mlck is suggested by the inhibitory effect of mlck inhibitors ml- and ml- as well as by the similar effect of an expressed mlck dominant negative mutant. recently, a function for the actin cytoskeleton in iav entry was reported to be required for the entry into polarized epithelial cells but not for entry into non-polarized cells [ ] . when using the low-serum conditions used in that paper ( % fcs), we only observed dyna-dep entry that was not affected by actin dynamics inhibitors. perhaps, the polarized cells permit dyna-ind entry at lower serum concentrations. the changes in actin network dynamics that can lead to the formation of macropinosomes can be triggered by a number of signaling cascades. actin dynamics are induced by the activation of growth factor receptor tyrosine kinases by their respective figure . a model for iav entry by macropinocytosis. the model summarizes the inhibitory (red boxes) or stimulatory (blue boxes) effects of compounds on dynamin-independent iav entry. the effect of over-expression of dominant-negative mutants is indicated by red-lined boxes. the pathway requires the presence of serum factors in the entry medium and results in the enhanced uptake of dextran and its co-localization with iav in large vesicles (green boxes). we hypothesize that the interaction of serum factors and/or iav with receptor tyrosine kinases (rtks) is the primary signal for the induction of macropinocytosis. a number of rtks have been shown to be involved in this process in different cell lines. remarkably, a recently published genome-wide sirna screen of iav infection identified the fgf receptor as a host factor required for influenza virus replication [ ] . activation of rho family gtpases cdc and/or rac has been shown to be essential for signal transduction leading to macropinocytosis in many cases [ , ] but inhibitors are without effect or are stimulatory in the case of iav entry. downstream effectors of rho family gtpases include scaffold proteins like n-wasp and wave and protein kinases like pak . macropinocytic entry of iav however seems to require a rho family gtpaseindependent pak activation mechanism. in addition, src family kinases, which can be directly activated by rtks, play a role. pak and src have previously been linked to the activation of macropinocytosis via their effect on changes in actomyosin dynamics, a process which is crucial to any form of macropinosome formation [ , ] . apart from n-wasp-or wave-containing macromolecular assemblies other actin binding proteins can induce such changes (e.g. cortactin, which can be activated by src [ ] ) and thereby induce the formation of one of the different plasma membrane protrusions that can result in the formation of macropinosomes. in addition to an effect on the formation of plasma membrane protrusions and subsequent macropinosome formation, inhibitors can also affect downstream trafficking and maturation of macropinosomes which might be actindependent, but this is not depicted in the scheme. doi: . /journal.ppat. .g growth factor ligands that are normally present in serum [ ] [ ] [ ] , , ] the signal transduction cascades that link activation of growth factor receptor tyrosine kinases to actin remodeling and macropinocytosis are only beginning to be revealed. the specific inhibition of dyna-ind entry of iav by ipa , an inhibitor of pak , provides proof for the involvement of these cascades. pak is a key serine/threonine kinase regulating actin network dynamics but its crucial function in several pathways of endocytosis as well as numerous other cellular processes does not make it a very specific marker [ ] . even so, macropinocytosis has consistently been demonstrated to require pak activation, both in the induction of the process and/or in further downstream trafficking events of macropinosomes [ , ] . growth factor dependent activation of pak has most often been demonstrated to depend on upstream activation of small gtpases rac or cdc [ , , ] . different strains of vaccinia virus were recently shown to induce their uptake by macropinocytosis via activation of either rac or cdc [ ] . activation of rac has been linked to the induction of macropinocytosis via actin network-mediated formation of lamellipodia and/or circular ruffles whereas cdc has most often been implied in the formation of filopodia [ ] . an inhibitory effect of the rac inhibitor nsc or the cdc inhibitor pirl on iav entry, however, could not be demonstrated. remarkably, cdc inhibitor pirl enhanced iav entry and a similar effect was observed by wiskostatin, an inhibitor of n-wasp which functions directly downstream of cdc as a scaffolding complex required for the activation of actin polymerization leading to filopodia formation. similarly, the macropinocytosis-like entry pathway taken by coxsackie b virus was also shown to require pak activity that was independent of rac activation [ ] . direct examination of the magnitude and timing of the activation of pak will be required to obtain more insight in the involvement of this complex pathway. the induction of macropinocytosis by a pak dependent mechanism has been associated with ruffling at the cell membrane [ , , , ] . the identification of sub-membranous regions with increased actin staining by phalloidin has been interpreted as evidence for ruffling. this was not unambiguously identified by confocal microscopy in the experiments presented in fig. and fig. s and needs to be investigated in depth by life cell imaging techniques. in agreement with our observation that the dyna-ind entry of iav was inhibited by pp , an inhibitor of src family kinases, the non-receptor tyrosine kinase c-src has been shown to function as a key signaling intermediate in the induction of macropinocytosis via a mechanism independent of rac or cdc [ ] [ ] [ ] . downstream effects of c-src on actin networks proceed, amongst others, via phosphorylation of cortactin by c-src resulting in accelerated macropinosome formation [ ] . c-src has been shown to associate with macropinosomes [ , ] , both during their formation and their trafficking, while c-src kinase activity is required for macropinocytosis following egf stimulation of hela cells [ ] . interaction of hsp with c-src was recently shown to induce c-src kinase activity [ ] . also hsp has been demonstrated to associate with macropinosomes, while its specific inhibitor geldanamycin reduced the membrane ruffling that preceded macropinocytosis [ ] . thus, the inhibition of iav entry via macropinocytosis by aa-geldanamcyin may very well involve the effects of hsp on c-src. as detailed above, the dyna-ind entry pathway of iav shares many characteristics with the endocytic pathway macropinocytosis. this is corroborated by the observation that iav particles and dextran colocalize in large vesicles in the presence of fcs. several viruses have recently been reported to enter cells via macropinocytosis [ , ] . apart from common factors like the requirement for pak activation, actin dynamics and independence of dynamin, virus specific details have been described [ , ] (table ). in part these might be contributed to differences in experimental conditions (e.g. cell types tested) but diversity in the molecular mechanisms by which macropinocytosis can be induced and executed is likely to exist and to be exploited by viruses. whereas vaccinia virus is able to trigger its own macropinocytic uptake [ , ] , we have described a macropinocytosis pathway that is operational under conditions that are activated by components in serum. still, this does not exclude signaling induced by virus-host cell interactions, which are for instance suggested by the significant increase of fitc-dextran uptake in the presence of iav. the possible requirement for costimulatory signals from serum components and virus imposes an additional layer of complexity on the analysis of iav entry via dyna-ind pathways. influenza viruses cause respiratory infections by targeting the epithelial cells lining the respiratory tract. these surfaces are covered by a mucous layer composed of a variety of small solutes and glycoproteins derived among others from goblet cells [ ] . this semi-fluid layer in turn conditions the underlying cells and determines their physiological state, including the activities of their uptake and secretion pathways. it will be important to determine to what extent the dyna-dep and dyna-ind iav entry pathways are operational under the conditions prevailing along the respiratory tract. current knowledge on the protein composition of the fluids covering the respiratory epithelium is rapidly expanding by the application of proteomic methods to determine the protein composition of bronchial alveolar lavage fluids (balf). these studies have extended the previous notion that balf is highly similar in composition to serum. for example, just as for the serum proteome more than % of the total protein mass of the balf proteome is accounted for by albumin, immunoglobulins, transferring, a -antitrypsin and haptoglobin. in addition, many other proteins have been identified both in serum and in balf including growth factors that can bind to growth factor receptor tyrosine kinases [ ] [ ] [ ] . thus, balf is likely to harbor, just as serum, the protein factors that can activate signaling pathways that are crucial for the induction of dyna-ind entry of iav. in agreement herewith, macropinocytosis has been described as a functional entry pathway of haemophilus influenzae into primary human bronchial epithelial cells [ ] although the factors involved in signaling the process have not been identified yet. in addition to infecting the respiratory tract, iav has been shown to be able to cause systemic infections involving multiple organs. this has mainly been studied in avian infections [ , ] or by infection of mice with human-derived h n or h n iavs [ ] but is poorly documented for human infections and may have been underestimated thus far. obviously, during potential systemic spreading of iav, the serum-rich conditions that we have demonstrated here to enable the use of alternative entry pathways will be encountered and may contribute to such spreading. mdck, a , df- and hela cells were maintained in complete dulbecco's modified eagle's medium (dmem) (lonza, biowittaker) containing % (v/v) fetal calf serum (fcs; bodinco b.v.), u/ml penicillin, and mg/ml streptomycin. chinese hamster-e cells were maintained at uc in a-minimal essential medium (gibco) supplemented with % (v/v) fcs, u/ml penicillin, and mg/ml streptomycin. cells were passaged twice weekly. influenza a/wsn/ (h n ) (iav-wsn) was grown in mdck cells. briefly, , % confluent mdck cells were infected with iav-wsn at a moi of . . supernatant was harvested after hr of incubation at uc and cell debris was removed by centrifigutation ( min at rpm). virus was stored at uc and virus titers were determined by measuring the tcid on hela cells. the iav-wsn luciferase pseudovirus (wsn-ren) system has previously been described [ ] . briefly, wsn-ren pseudovirus harbors a ha segment in which the ha coding region is replaced by renilla luciferase. the pseudovirus is produced in a mdck cell line that stably expresses the ha of iav-wsn. wr-luc, a firefly luciferase encoding vaccinia virus (strain wr) was previously described [ ] . vsv-fl, a firefly luciferase encoding vsv virus was also previously described [ ] . stocks of bafilomycin a (bafa ), dynasore, cytochalasin d, cytochalasin b, blebbistatin, -aa-geldanamycin, ml- , ml- , pp- , -(n-ethyl-n-isopropyl)amiloride (eipa), ipa- (all obtained from sigma-aldrich), latrunculin a (enzo), jasplakinolide, wiskostatin, nsc (all obtained from calbiochem) and pirl (chembridge) were prepared in dimethylsulfoxide (dmso). all stocks were stored at uc. a kinase inhibitor library composed of kinase inhibitors was obtained from biomol ( a[v . ]). hela cells ( , cells/well in -well plates) were treated with munits of vibrio cholerae neuraminidase (roche) in ml phosphate-buffered saline (pbs) for hr. after washing with pbs cells were infected with iav as described. virus-like particles (vlps) were produced as described [ ] . briefly, t cells were transfected using lipofectamine (invitrogen) with pcaggs-blam (encoding a beta-lactamase reporter protein fused to the influenza matrix protein- ), pcaggs-ha (encoding ha derived from either a/newyork/ / or iav-wsn) and pcaggs-na (encoding iav neuraminidase [na] derived from either a/brevigmission/ / or iav-wsn) and maintained in optimem. supernatants were harvested h after transfection and centrifuged to remove debris. vlps were used for inoculation of cells without further concentration. vlps were incubated for min at uc with trypsin/tpck for activation of ha. mdck or hela cells grown to near confluency in -well plates were inoculated with ul of vlps after pre-treatment of the cells with inhibitors as indicated. infection was synchronized by centrifugation at rpm for min at uc and was performed by further incubation at uc for h in the absence or presence of % fcs and inhibitors as indicated. detection of beta-lactamase activity was performed as described [ ] by loading cells with ccf -am substrate (invitrogen) and subsequent analysis by flow cytometry on a lsrii flow cytometer (becton dickinson). typically , events were collected and analyzed using flowjo . . software. the reporter construct phh-gluc was derived from plasmid phh-fluc [ ] by replacing the firefly luciferase coding region with the gaussia luciferase coding region of pgluc-basic (new england biolabs). unique spei and xbai restriction sites were introduced into phh-fluc using the quikchange xl site-directed mutagenesis kit (stratagene) and oligonucleotides spe ( - gcctttctttatgtttttggcactagtcattttaccg-atgtcactcag), spe ( -ctgagtgacatcggtaa-aatgactagtgccaaaaacataaagaaaggc), xba ( -gtatttttctttacaatctagactttccgcccttc-ttgg) and xba (ccaagaagggcggaaagtctag-attgtaaagaaaaatac). a spei site was introduced by sitedirected mutagenesis in pgluc-basic directly following the start codon of the gaussia luciferase coding sequence. the unique spei -xbai fragment of pgluc-basic was subsequently cloned into the spei-xbai site of phh-fluc resulting in plasmid phh-gluc. cells were seeded in -well plates at a density of , cells/ well and transfected the next day with ng phh-gluc using lipofectamine (invitrogen) according to the manufacturer's protocol. after hrs the transfected cells were treated with inhibitors and infected as indicated. at hr p.i. samples from the supernatant were assayed for luciferase activity using the renilla luciferase assay system (promega) according to the manufacturer's instructions, and the relative light units (rlu) were determined with a berthold centro lb plate luminometer. wr-luc and vsv-fl were used to inoculate hela cells ( , cells/well) at an moi of , in complete dulbecco's modified eagle's medium (dmem) (lonza, biowittaker). after hr the luciferase activity was detected using the steadyglo assay kit (promega). the addition of % (v/v) fcs did not change infection levels for both viruses. cells were fixed with . % paraformaldehyde (pfa) in pbs and subsequently permeabilized with . % triton-x- in pbs. after blocking with normal goat serum iav-infected cells were incubated for h with a monoclonal antibody directed against the nucleoprotein (np) (hb- ; kindly provided by dr. ben peeters). after washing, the cells were incubated with a : dilution of alexa fluor -or -labeled goat anti-mouse igg (molecular probes) secondary antibody for h. nuclei were subsequently stained with topro- and after three washing steps, the coverslips were mounted in fluorsave (calbiochem). actin was stained using phalloidin labeled with alexa fluor . the immunofluorescence staining was analyzed using a confocal laser-scanning microscope (leica tcs sp ). fitc, gfp or alexa fluor were excited at nm, alexa fluor at nm, and topro- at nm. hela cells were grown in -well plates on glass coverslips ( , cells/well). prior to fitc-dextran uptake cells were serum-starved for hr in pbs. fitc-dextran (mw , , sigma-aldrich) was incubated with hela cells (final concentration of . mg/ml) in ml pbs or in pbs containing % fcs in the absence or presence of iav (strain wsn; moi ; concentrated and purified by centrifugation through a to % sucrose gradient with a % sucrose cushion at the bottom) at uc. after min cells were washed times with pbs at uc, fixed with . % pfa in pbs and subsequently permeabilized with . % triton-x- in pbs. slides were stained for examination by confocal microscopy as described above. for quantification of fitc-dextran uptake . hela cells were infected with iav-wsn (moi ) in suspension in a volume of ml in the presence of fitc-dextran ( mg/ml). infections were performed for min in pbs (containing % bsa to reduce unspecific binding of fitc-dextran) or in pbs containing % fcs at uc or at uc (control for binding of fitc-dextran to cells in the absence of endocytosis). mock-infected samples were analysed in parallel. infection was terminated by addition of ml ice-cold pbs followed by three washes with cold pbs and fixation with . % pfa. , cells were analyzed by facs and results were represented as the mean fluorescence which was plotted relative to the uptake in the mock-infection in pbs (after subtraction of background fluorescence obtained at uc). the effect of dynasore and eipa on dextran and transferrin uptake hela cells (grown on glass cover slips) were incubated at uc for hr with mg/ml alexa -labeled transferin (invitrogen) in pbs. after hr the medium was replaced by pbs or pbs supplemented with % fcs containing iav (strain wsn; moi ) and . mg/ml fitc-dextran (sigma; kda) and cells were transferred to uc for min. after min cells were fixed and stained as described above and examined by confocal microscopy. cells were fixed with . % pfa in pbs and subsequently permeabilized with . % triton-x- in pbs. peroxidase was visualized using an aec substrate kit from vector laboratories. iav-positive cells were detected using bright-field light microscopy. two sirna duplexes targeting different sites within the coding sequences of dynamin were obtained from ambion inc ( (dynamin sirna ) and (dynamin sirna )). a scrambled sirna (ambion inc.) was taken along as a control for non-specific effects of the transfection procedure and was used for normalization. one day after seeding in -well plates ( , cells/well), the hela cells were transfected with a final concentration of nm sirna using oligofectamine (invitrogen). h after transfection, the cells were inoculated with the wsn-ren pseudovirus (moi . ) in pbs or in pbs containing % fcs. after h of infection the entry medium was replaced by complete growth medium containing nm bafa to prevent further entry. at h post infection intracellular renilla luciferase expression was determined as described above. each sirna experiment was performed in triplicate. cell viability was not affected as determined by performing a wst- cell-viability assay (roche). functional knockdown of dynamin mrna levels was performed by quantitative rt-pcr. using a taqman gene expression assay for dnm (hs _m , ambion) and using s rna (hs _g , ambion) as a control for normalization. the comparative ct-method was used for quantification of the results [ ] . reduction of dynamin protein levels was determined by western blotting using polyclonal goat-anti-dynamin c (santa-cruz sc- ). a monoclonal against alpha-tubulin (dm a, sigma t ) was used to detect tubulin for normalization. results were quantified by densitometric scanning of the dynamin and tubulin signals displayed in fig. h . hela cells were grown in -well plates on glass coverslips ( , cells/well) for hrs. cells were then transfected ( mg of dna with lipofectamine as described above) with plasmids encoding wild-type or dominant-negative (dn) human mlck fused to gfp [ ] , wild-type or dn rab fused to gfp [ ] , or myoii-tail or myoii-head domain fused to gfp [ ] . hr after transfection cells were inoculated with iav-wsn (moi ) in pbs or in pbs containing % fcs and mm dynasore. hr after infection cells were fixed and stained for examination by confocal microscopy as described above. an unpaired student's t-test was used for detemination of statistically significant differences. the use of the term significant in text refers to a comparison of values for which p, . . studies on the mechanism of influenza-virus entry into cells infectious entry pathway of influenza virus in a canine kidney cell line assembly of endocytotic machinery around individual influenza viruses during viral entry epsin is a cargo-specific adaptor for the clathrinmediated endocytosis of influenza virus influenza virus entry and infection require host cell n-linked glycoprotein influenza virus can enter and infect cells in the absence of clathrin-mediated endocytosis endocytosis of influenza viruses mechanisms of endocytosis molecular mechanisms of clathrin-independent endocytosis endocytosis unplugged: multiple ways to enter the cell virus entry: open sesame virus entry by endocytosis defining macropinocytosis virus entry by macropinocytosis ruffles induced by salmonella and other stimuli direct macropinocytosis of bacteria phosphatidylserine (ps) induces ps receptor-mediated macropinocytosis and promotes clearance of apoptotic cells origin, originality, functions, subversions and molecular signaling of macropinocytosis clathrin-independent endocytosis: a unique platform for cell signaling and pm remodeling pathways of clathrin-independent endocytosis dynamin, endocytosis and intracellular signaling ctbp / bars drives membrane fission in dynamin-independent transport pathways virus-inducible reporter genes as a tool for detecting and quantifying iav replication involvement of the vacuolar h(+)-atpase in animal virus entry role of clathrin-mediated endocytosis during vesicular stomatitis virus into different host cells vesicular stomatitis virus enters cells through vesicles incompletely coated with clathrin that depend upon actin for internalization an enzymatic virus-like particle assay for sensitive detection of virus entry human host factors required for influenza virus replication virucidal activity of hypericin against enveloped and non-enveloped dna and rna viruses modulation of influenza virus replication by alteration of sodium ion transport and protein kinase c activity the specificities of protein kinase inhibitors: an update rab proteins as membrane organizers differential requirements of rab and rab for endocytosis of influenza and other enveloped viruses vaccinia virus strains use distinct forms of macropinocytosis for host-cell entry virus-induced abl and fyn kinase signals permit coxsackievirus entry through the epithelial tight junctions phosphoinositide metabolism during membrane ruffling and macropinosome formation in egf-stimulated a cells membrane ruffling and signal transduction regulation of macropinocytosis by p -activated kinase- an isoform-selective, small-molecule inhibitor targets the autoregulatory mechanism of p -activated kinase the small gtp-binding protein rac regulates growth factor-induced membrane ruffling activation of rho gtpases by cytotoxic necrotizing factor induces macropinocytosis and scavenging activity in epithelial cells ras-related gtpases and the cytoskeleton vaccinia virus uses macropinocytosis and apoptotic mimicry to enter host cells rho gtpases and actin dynamics in membrane protrusions and vesicle trafficking regulation of actin dynamics by wasp family proteins the cdc inhibitor secramine b prevents camp-induced k+ conductance in intestinal epithelial cells rational design and characterization of a rac gtpase-specific small molecule inhibitor chemical inhibition of n-wasp by stabilization of a native autoinhibited conformation role of src-family kinases in formation and trafficking of macropinosomes src triggers circular ruffling and macropinocytosis at the apical surface of polarized mdck cells ) c-src trafficking and co-localization with the egf receptor promotes egf ligand-independent egf receptor activation and signaling modulation of the fcepsilon receptor i signaling by tyrosine kinase inhibitors: search for therapeutic targets of inflammatory and allergy diseases ansamycin inhibitors of hsp : nature's prototype for anti-chaperone therapy geldanamycin inhibits tyrosine phosphorylation-dependent nf-kappab activation distinct endocytotic pathways in epidermal growth factor-stimulated human carcinoma a cells adenovirus triggers macropinocytosis and endosomal leakage together with its clathrin-mediated uptake a clathrin independent macropinocytosis-like entry mechanism used by bluetongue virus- during infection of bhk cells kaposi's sarcoma-associated herpesvirus utilizes an actin polymerizationdependent macropinocytic pathway to enter human dermal microvascular endothelial and human umbilical vein endothelial cells early stages of influenza virus entry in mv- lung cells: involvement of dynamin dynasore, a cell-permeable inhibitor of dynamin a single amino acid substitution in influenza virus hemagglutinin changes receptor binding specificity amiloride inhibits macropinocytosis by lowering submembranous ph and preventing rac and cdc signaling population context determines cell-to-cell variability in endocytosis and virus infection role of the actin cytoskeleton during influenza virus internalization into polarized epithelial cells an emerging role for p -activated kinases (paks) in viral infections histone deacetylase regulates growth factor-induced actin remodeling and endocytosis mucus and mucins mapping the lung proteome in cystic fibrosis proteomics and the lung: analysis of bronchoalveolar lavage fluid proteome analysis of bronchoalveolar lavage in lung diseases infection of primary human bronchial epithelial cells by haemophilus influenza: macropinocytosis as a mechanism of airway epithelial cell entry a mouse model for the evaluation of pathogenesis and immunity to influenza a (h n ) viruses isolated from humans biological heterogeneity, including systemic replication in mice, of h n influenza a virus isolates from humans in hong kong multiorgan distribution of human influenza a virus strains observed in a mouse model expression of the firefly luciferase gene in vaccinia virus: a highly sensitive gene marker to follow virus dissemination in tissues of infected animals carrier cell-based delivery of an oncolytic virus circumvents antiviral immunity mouse hepatitis coronavirus replication induces host translational shutoff and mrna decay, with concomitant formation of stress granules and processing bodies myosin ii light chain phosphorylation regulates membrane localization and apoptotic signaling of tumor necrosis factor receptor- dynamin and rab regulate grk -dependent internalization of dopamine d receptors myosin iia is involved in the endocytosis of cxcr induced by sdf- a cortactin signalling and dynamic actin networks the following persons are gratefully acknowledged for providing us with plasmids. dr. a. pekosz for plasmid phh-fluc; dr. m yanez-mo for miia-egfp ''head'' and ''tail'' constructs; dr. p. gallagher for dn-mlck-egfp; dr. p. van der sluijs for dn-s n-rab a-egfp. we thank dr. m. esteban for providing us with vaccinia wr-luc virus and dr. j. bell for providing vsv-fl virus. dr. b peeters is acknowledged for a gift of monoclonal antibody against iav np (hb- ). confocal images were acquired at the center for cellular imaging (cci) in the faculty of veterinary medicine, utrecht university and we thank dr. r. wubbolts for help and technical advice. key: cord- -c wgg v authors: wu, jiqin; ye, han-qing; zhang, qiu-yan; lu, guoliang; zhang, bo; gong, peng title: a conformation-based intra-molecular initiation factor identified in the flavivirus rna-dependent rna polymerase date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: c wgg v the flaviviruses pose serious threats to human health. being a natural fusion of a methyltransferase (mtase) and an rna-dependent rna polymerase (rdrp), ns is the most conserved flavivirus protein and an important antiviral target. previously reported ns structures represented by those from the japanese encephalitis virus (jev) and dengue virus serotype (denv ) exhibit two apparently different global conformations, defining two sets of intra-molecular mtase-rdrp interactions. however, whether these ns conformations are conserved in flaviviruses and their specific functions remain elusive. here we report two forms of denv serotype (denv ) ns crystal structures representing two conformational states with defined analogies to the jev-mode and denv -mode conformations, respectively, demonstrating the conservation of both conformation modes and providing clues for how different conformational states may be interconnected. data from in vitro polymerase assays further demonstrate that perturbing the jev-mode but not the denv -mode intra-molecular interactions inhibits catalysis only at initiation, while the cell-based virological analysis suggests that both modes of interactions are important for virus proliferation. our work highlights the role of mtase as a unique intra-molecular initiation factor specifically only through the jev-mode conformation, providing an example of conformation-based crosstalk between naturally fused protein functional modules. the function of a protein is often dictated by a single defined fold, which in turn is determined by its amino acid sequences. however, multiple global conformations can be utilized by a protein to fulfill distinct functions under different circumstances. the flavivirus ns protein, a natural fusion of an n-terminal methyltransferase (mtase) and a c-terminal rna-dependent rna polymerase (rdrp), may be such an example. previously reported ns crystal structures exhibit two apparently different global conformations. in the flaviviruses are a large group of positive-strand rna viruses, including dengue virus (denv), west nile virus (wnv), japanese encephalitis virus (jev), zika virus (zikv), tickborne encephalitis virus (tbev), and omsk hemorrhagic fever virus (ohfv). the majority of flaviviruses are mosquito-borne or tick-borne, sometimes causing human encephalitis or hemorrhagic diseases. the recent zikv outbreaks in south and north america, and more recently in southeast asia, have intensified the global threats of flaviviruses, in part due to the capabilities of the virus to cause birth defects through maternal-fetal transmission [ ] . the flavivirus rna genome is - kilo-bases in length, bearing a type cap and lacking a poly-adenine tail. it encodes a large polyprotein that is further processed by viral and host proteases, yielding three structural proteins c/prm/e, and seven nonstructural proteins ns /ns a/ns b/ns / ns a/ns b/ns [ ] . being a unique natural fusion of an n-terminal methyltransferase (mtase) and a c-terminal rna-dependent rna polymerase (rdrp), the ns is the largest and most conserved protein encoded by flaviviruses. the ns mtase catalyzes the guanylyltransfer and both the guanine n and nucleoside -o methylation steps in the capping process, and is a single-domain module adopting a common s-adenosyl-l-methionine (sam)dependent mtase fold [ , ] . the k-d-k-e catalytic tetrad sits in the center of the mtase catalytic cleft, with the methyl donor sam binding site and the cap binding site residing on the opposite sides. the rdrp module is the central molecular machine governing the viral genome replication, and has an encircled human right hand architecture with palm, fingers, and thumb domains surrounding the active site [ , ] . the fingers domain can be further divided into index, middle, ring, and pinky subdomains according to nomenclatures first used in describing the poliovirus (pv) rdrp ( fig a) [ , ] . among the seven viral rdrp catalytic motifs, a/b/c/d/e are within the most conserved palm, and f/g are part of the ring and pinky fingers, respectively. motifs a/b/c/f contain amino acids highly conserved in all viral rdrps, and these conserved residues have highly analogous spatial arrangements around the polymerase active site [ , ] . being an entity bearing two active sites and multiple essential viral enzymatic activities, ns has become a very attractive system in flavivirus research, and understanding the interplay between its mtase and rdrp modules is undoubtedly critical. among the evidence related to mtase-rdrp crosstalk, high-resolution structures of fulllength ns are essential in providing direct and informative readout of the interactions between the two modules. to date, two types of global conformations have been observed in full-length ns structures [ ] . the conformation revealed by the jev ns structure (named jev-mode hereinafter) features a medium size interface (~ Å , for all interface area values presented in this study, the total buried solvent accessible surface from both side of the interface was accounted) with a conserved hydrophobic core [ , ] , and is also observed in recently reported zikv, yellow fever virus (yfv), and denv serotype (denv ) full-length ns crystal structures [ ] [ ] [ ] [ ] [ ] [ ] . in such a conformation, the mtase approaches the rdrp from its backside and interacts with the rdrp middle finger, ring finger, and an index finger helix bearing part of a nuclear localization signal (nls-helix) [ ] (fig a) . the second conformation was observed in two different crystal forms of denv serotype (denv ) ns (named denv -mode hereinafter) [ , ] (fig b) . in this case, the mtase also approaches the rdrp from the backside, but it is related to the jev conformation by an approximately ˚rotation around an axis passing near its center of mass with less than Å translation. the rdrp index and middle fingers are still involved in the interactions in a slightly larger interface (~ - Å ), and the nature of the interactions is instead primarily polar. notably, the ntp binding ring finger (motif f) contacts with the mtase are absent in the denv structures and the ring finger itself and the adjacent motif g residues in the pinky finger are largely disordered (fig b) . motif g participates in rna template binding and has been proposed to participate in the translocation step after every phosphoryl transfer reaction [ , ] . hence, the jev-mode conformation likely represents a state more suitable for polymerase synthesis from the structural perspective. with two monomer conformation modes and eight crystal forms identified, more than ns dimer interfaces can be recognized in the aforementioned ns crystal structures with no obvious conservative features. a couple of studies did focus on some of these dimer interface interactions, even though the primary ns solution state is monomer [ , ] . either by probing the inter-molecular interactions, deleting the mtase domain, or mutating the mtase-rdrp linker, multiple in vitro polymerase assay-based studies together suggest that the mtase regulates the rdrp catalytic activities, albeit to overall moderate extents [ , , , , , ] . however, the rdrp assays established in all these studies, no matter in primer-dependent or de novo (including dinucleotide driven) format, did not demonstrate the formation of a processive rdrp elongation complex (ec), at least for the majority of the polymerase molecules, and all these assays require the manganese ion (mn + ) for catalysis, albeit in combination with the magnesium ion (mg + ) in some cases. in other words, the mutation-derived effect on rdrp synthesis observed in these studies may only reflect overall changes in non-processive rdrp synthesis activity, while specific alteration of either rdrp initiation or elongation cannot be clearly judged. furthermore, none of these studies characterized both the jev-and denv -mode monomer conformations to distinguish their differences in rdrp synthesis, except for our previous jev ns study that only probed the jev-mode conformation [ ] . therefore, the precise mechanism of the regulation and the explicit contribution of either ns conformation remain to be clarified. in this work, we report two crystal forms of denv ns that reveal two conformational states bearing clear analogies to those observed in the jev-mode and denv -mode ns structures, respectively. virological data further support the conservation and the functional importance of both conformation modes. ns constructs bearing mutations specifically probing two modes of mtase-rdrp intra-molecular interfaces were tested in in vitro polymerase assays, and only the jev-mode interface related mutants inhibited polymerase initiation primarily through a three-fold reduction in the michaelis constant of the initiating ntp (k m, ntp ), while polymerase ec properties were not much affected by mutations probing both modes of interactions. collectively, our work demonstrates the conformational conservation and diversity of the flavivirus ns and highlights the specific contribution of the jev-mode conformation to polymerase initiation. with an aim to further understand the conformational diversity of ns and related functional relevance, we crystallized and solved the structures of denv ns in two different crystal forms at . Å (form ) and . Å (form ) resolution (table ) . each structure has two ns molecules in the crystallographic asymmetric unit, and has the two molecules arranged in a dimer through pseudo two-fold symmetries with highly consistent global conformation (root mean square (rms) deviation values for superimposable c-α atoms are . Å and . Å, respectively; chain a as the reference). strikingly, the ns conformations between the two crystal forms are quite different (fig c and d ). using a maximum likelihood superpositioning method [ ] , the rdrp palm and the majority of the fingers domain were identified as the structurally most conserved regions in the superpositioning including these and the representative full-length flavivirus ns structures (fig ) . the denv form conformation is clearly related to the jev-mode conformation with the jev interface partially opened through a pure ˚rotation along an axis passing the vicinity of the highly conserved gtr residues that were proposed to pivot the mtase movement relative to rdrp [ , ] (fig a and c; fig a) . the partial opening of the interface results in the reduction of the interface area to only about Å or % of the jev interface. among the six conserved hydrophobic residues forming the mtase as rdrp initiation factor in ns interface core in the jev structure, only three of them (denv ns residues w , f , and p ) remained as part of the interface. the tip of the ring finger no longer contacts the mtase in the denv form structure, and its electron density becomes weak but still readily traceable and its folding is largely consistent with the jev conformation (fig a and c ; fig a) . however, the motif g region in the pinky finger is largely disordered as observed in the denv structures [ , ] . based on these observations, we propose that the interactions between the mtase residues and and the phenylalanine (residue in denv ns ) in the tip of the rdrp ring finger are essential for maintaining the canonical folding of the ring and pinky fingers, and the folding of the motif g region in the pinky finger is likely dependent on the dynamics of the adjacent ring finger. it is also worth noting that, in addition to ring and pinky fingers, the index finger is also partially disordered in most of the rdrp-only flavivirus ns structures [ , ] . these observations together suggest that mtase interaction likely contributes to the folding of rdrp fingers domain, which in turn could affect polymerase properties including rna binding and subsequent catalytic events. the denv form conformation is instead analogous to the denv -mode conformation but contains previously unidentified features. it is related to the denv -mode conformation by a ˚rotation along an axis near the interface and the mtase-rdrp linker region (residues - in denv ns ) and a translation less than Å (fig b and d ; fig b) . the primarily polar interactions between the nls-helix and the mtase are largely retained (fig b) , and interface area is about Å and is only reduced for about % as compared to the first reported denv -mode interface [ ] . the rotational movement widens the cleft between the relatively conserved residue pair e -r in the mtase and the rdrp middle finger, creating a pocket that allows the binding of a putative sah molecule with high occupancy ( . and . ) in addition to the sah molecule usually bound in the sam binding pocket of the mtase (fig d; fig b and c) . usually, sah can be co-crystallized with the flavivirus mtase at a : molar ratio with the sah co-purified with the mtase after its overexpression in bacterial culture [ , , , ] . it is possible that the observed ns conformation allowed sah co-purification with ns at a higher molar ratio. such a secondary sah binding site has not been observed in numerous mtase-containing flavivirus ns structures. this binding pocket appears to be not tight enough, as the sah molecules bind at moderately different positions in the two ns proteins in the crystallographic asymmetric unit, and specific interactions between the non-carbon atoms of the sah and the side chains of the ns are largely lacking (fig b and c ). nevertheless, based on the fact that this secondary sah binding pocket is created at the mtase-rdrp interface and is reasonably conservative (fig c) , it might have potential as a target to develop small molecule inhibitors against flaviviruses. the two denv ns structures nicely fill the gap of major conformational differences between the jev-mode and denv -mode structures, suggesting a plausible order from jevmode to denv form , then to form , and finally to denv -mode, primarily through rotational movements in more or less consistent directions (fig ; fig a and b ; s movie). three structural elements may play critical roles in the transitioning among these states. the first is the universally conserved gtr sequence at the c-terminal end of the mtase (fig c) . this tripeptide sequence was proposed as a pivoting element in the work of the full-length jev mtase as rdrp initiation factor in ns ns structure and was proved to be functionally important in both jev and denv replications [ , ] . the second element is the -residue mtase-rdrp linker that overall exhibits low sequence conservation in flaviviruses ( fig c) . a comparison of all full-length ns structures demonstrated that the n-terminal half of the linker undergoes a swinging motion with a partial refolding to become helical when transitioning from the jev-mode conformations to the denv -mode ones, while the c-terminal half remains unaffected (fig a and b ). not surprisingly, mutations in the linker region or linker substitutions using sequences of other flavivirus ns have been found to affect ns conformation distribution, ns enzymatic activities, and virus proliferation [ , , ] , possibly by altering the flexibility of the linker. the third element is the nls-helix (residues - in denv ns ) in the rdrp index finger. on one hand, this helix is critical to both the jev-mode and denv -mode of interface interactions by contributing the f and r residues (fig a and b ; fig b) . on the other hand, it is at the central region of a long stretch of conserved sequences (residues - in denv ns ) that may also be related to ns nuclear localization, nuclear export, and interaction with another important viral protein ns [ , , ] , emphasizing its possible importance when not participating in the intra-molecular mtase-rdrp interactions. we propose that the largely rotational movement of the mtase from the jev-mode conformations to the denv -mode conformations may utilize this highly conserved helix as a guiding track (s movie). at the starting and end points of the movement, the conserved hydrophobic residue patch p / x /w (the majority of x are l and m) and the conserved polar residue pair e -r provide the anchoring points on the mtase side ( fig b- d ). on the other hand, both of these stable conformations make the nls-helix inaccessible to other factors, and the helix may only become solvent exposed at certain stages of virus life cycle. previously, we tested the functional significance of the jev-mode conformation using both jev and denv systems [ ] . when arginine/aspartic acid/serine (r/d/s) mutations were introduced at the six hallmark hydrophobic residue sites, virus proliferation was significantly inhibited (the corresponding mutation sites in denv ns were listed in fig e) . in order to understand functional relevance of the denv -mode conformation, we designed five mutations at the ns residues and for each virus system (e a, e d, k a, k r and e a/k a in jev; e a, e d, r a, r k and e a/r a in denv , and correspond to the same mutations with an "m_" prefix in fig e) and compared the mutant constructs with the wild type (wt) viruses (fig ) . the residues - were chosen as the mutation sites because these two residues are highly conserved among the mtase residues that participate in the denv -mode mtase-rdrp interface but are not involved in the jev-mode interface interactions. we first introduced each mutation into a jev infectious clone [ ] . after viral a) a comparison between the jev (top) and the first form of denv (bottom) structures. b) a comparison between the denv (top, two models) and the second form of denv (bottom) structures. c) the binding of the second sah molecule observed in the form of denv structure. the binding pocket is shown as surface representations with conservation scores projected. thinner sticks show the moderately different sah binding mode observed in the other ns molecule in the crystallographic asymmetric unit. composite simulated-annealing (sa) omit electron density maps contoured at . σ are overlaid with the denv models in panels a and b and the sah molecule in panel c. for the denv structures in panels a and b, the two ns molecules in the crystallographic asymmetric unit were superposed and shown as thick and thin representations with the density maps of the thick model overlaid. all structures in panels a and b were superposed but may be presented separately. the coloring scheme is the same as in fig . for panels a and b, the rotational movements correlate the both structure pairs are indicated. https://doi.org/ . /journal.ppat. .g mtase as rdrp initiation factor in ns rna was transfected into baby hamster kidney cells bhk- , viral protein expression and virus production were monitored. the expression level of the viral envelope (e) protein in transfected cells was detected by an immunofluorescence assay (ifa) (fig a) . both the e a and k r mutant viruses produced similar ifa positive cells in comparison with the wt virus ( % ifa positive cells observed at h post transfection (hpt)); the k a and e a/ k a mutants showed only around % positive cells; the e d mutant produced very few ifa-positive cells. virus productions were then quantified by a plaque assay at three time points ( , , and h) post transfection. consistent with the ifa data, the e a and k r mutant rnas yielded similar amounts of viruses as the wt at each time point, the k a and e a/k a mutants moderately impaired virus production, and viruses derived from the e d mutant rna-transfected cells were only detected at and hpt ( fig a) . overall, the results indicated residues and are important for jev proliferation. we also performed similar analyses using a denv infectious clone ( fig b) [ ] . no ifa-positive cells were observed in the r a, r k and e a/r a transfected cells; the e a and e d produced around %- % ifa-positive cells relative to the wt. data from the plaque assay indicated that virus production was blocked by the r a, r k, and e a/r a mutations, while the e a and e d mutations had slightly less effect on virus production at each time point post mtase as rdrp initiation factor in ns transfection comparing with the wt (fig b) . taken together, these virological data suggest that the denv -mode conformation is also functionally important and is likely conserved in flaviviruses, consistent with our structural observation of both conformations in denv ns . we previously tested the jev-mode interface mutants in jev ns using in vitro assays derived from an hcv study [ , ] . while the hcv assays have mg + as the only divalent metal ion mtase as rdrp initiation factor in ns and allow the formation of processive ecs [ ] , the jev assays require mn + for rdrp activity and the stability and reactivity of the assembled complexes are far from optimal [ ] . when the hcv assay format was used in the denv ns , the rdrp enzyme behavior is consistent with the hcv enzyme, and therefore the denv assays established in this study can serve as effective systems to assess rdrp catalytic properties and to analyze whether the interface interactions observed in both the jev-mode and the denv -mode conformations modulate rdrp catalysis. note that dinucleotide-driven assays, such as those in the hcv study [ ] , have been used in multiple polymerase systems to reasonably mimic the de novo initiation process [ , ] . when atp and utp were provided as the only ntp substrates, a gg dinucleotide primer (p ) was extended to yield a -mer product (p ) as directed by a -mer template (t ) after a -min incubation (fig a) . we assessed the reactivity of the p -containing complex by a single-nucleotide extension assay. because the complex was in the precipitate form under the low-salt reaction condition ( mm nacl) but was soluble under high-salt condition (e.g. mm nacl), we removed the excess atp and utp by centrifugation, pellet wash, and pellet resuspension, and then added ctp to allow the single-nucleotide addition to make a -mer product (p ). it turned out that the conversion from p to p was very rapid and was completed immediately after manual mixing on ice without further incubation (" min"; fig a, lane ) . although not accurately determined, the expected catalytic rate constant of the p -containing polymerase complex is at least magnitudes larger than that determined in the jev study ( . min - for wt ns ) [ ] . this observation strongly suggests that the p -containing complex of denv ns has completed the transition from initiation to elongation and is a bona fide ec. therefore, the production of this p -containing ec (ec ) can be used to assess the overall process of initiation followed by the transition to elongation. to test the impact on polymerase catalysis brought by the intra-molecular mtase-rdrp interactions, we made two sets of denv ns mutants. the first set contains equivalent mutations utilized in the jev study to perturb the hydrophobic jev-mode interface [ ] , and the second set that contains mutations at residues - was used to probe the polar denv -mode interface ( fig e) . we compared the ec formation of these mutants with the wt ns at three incubation time points. for the wt ns , only limited -mer accumulation was observed beyond the first time point ( min) , and small amount of misincorporation-related -mer products became obvious at the last time point ( min) (fig b, lanes , , and ; fig c, lanes , , and ). these observations again suggest that: although the ec formation is a slow process due to slow pre-initiation and initiation steps, it likely produces a stable ec that is not turning over to carry out multiple rounds of p synthesis. among the six jev-mode ns mutants, three of them exhibited obvious slower accumulation of p products, in particular at shorter incubation time points (fig b, compare lanes - , - , - to the wt lanes). in contrast, all five denv -mode ns mutants showed very similar trend of p accumulation as the wt enzyme ( fig c) . these data together suggest that perturbing the jevmode but not the denv -mode interactions inhibits the overall process to produce a processive ec. to further dissect the mechanism of inhibition brought by the jev-mode interface mutations, we compared representative ns mutants (r for jev-mode interface and m_ a/ a for denv -mode interface) with the wt enzyme in a p -driven initiation assay to explicitly assess the continuous production of the -mer (p ) when atp was provided as the only ntp substrate, while the p synthesis was monitored in parallel for comparison (fig d) . here we mtase as rdrp initiation factor in ns use a chemically synthesized -mer as a quantitation standard (std; fig d, lanes and ) loaded with an equal molar amount of the t template. for the wt, the r mutant, and the m_ a/ a mutant, the p amount was relatively consistent at the and min time points (fig d, compare lanes , , to lanes , , ) , suggesting that all three constructs had formed stable ecs and did not turn over to accumulate the p products over time. in contrast, the p accumulation proceeded continuously during the same period for all three constructs, indicating that the p -containing complex is an initiation complex (ic) that carried out multiple rounds of synthesis in an abortive fashion (fig d, lanes - , - , and - ). the r mutant had a slower p accumulation than the wt and the m_ a/ a mutant had, clearly suggesting that the perturbation of the jev-mode interface impaired the rdrp initiation process. we next performed two tests regarding the ec properties using the wt, r , and m_ a/ a constructs (fig ) . to test the ec reactivity, we compared the p -to-p conversion under high and low ctp substrate concentrations for these constructs (fig b) . at μm ctp concentration, all three constructs converted the majority of p to p at " -min" time point ( - % converted suggested by intensity-based quantitation) (fig b, lanes , , and ). when ctp was supplied at μm, the conversion became slower, but all three constructs showed consistent progress of conversion ( - % and - % converted at " -min" and at min, respectively) (fig b) . to test the stability of the ec , we used nacl as the challenging agent in a highsalt challenge stability assay similar to those described in previous work characterizing the pv, hcv and the classical swine fever virus (csfv) rdrps [ , , ] . we found that ec formed by all three constructs were quite stable and exhibited comparable inactivation rate constants ( . - . h - , corresponding to half life values of - h) upon a nacl challenge at mm concentration (fig c and d) . these data together suggest that the ec is highly stable and reactive, and these properties were not much affected by both types of mutations. to specifically investigate the enzymatic properties of the wt and the two representative mutants during the conversion of the p to p , we measured the relative catalytic rates under different atp concentrations for each construct (fig a- c) , and these data were used to determine the michaelis constants (k m ) of these constructs (fig d- f) . by optimizing the reaction time points selection for each ntp concentration and each construct, the -mer band intensities were controlled to be within the linear range of the stains-all based staining method, to facilitate quantitation accuracy (refer to our previous analysis in the jev study a diagram of construct t /p used in all polymerase assays and related ntp-driven reactions to generate products with different lengths. middle: reaction flow chart of the p -driven ec formation (ic to ec ) and the subsequent single-nucleotide extension (ec to ec ). right: the ec was in a form of precipitate and was able to extend to ec upon ctp addition under high-salt condition. b-c) the ec formation comparison with the wt ns for the jev-mode (b) and denv -mode (c) mutants. the relative intensity of the -mer was used to estimate the polymerase activities (the wt value for each time point series was set to ). d) comparison of the wt and two representative mutants (r for the jev-mode; m_ a/ a for the denv -mode) in the multiple-turnover p formation and in the single-turnover p formation assays. the pppgga was synthesized when gtp and atp were provided as the only ntp substrates using the p -free t template and was used as a migration marker. note that pppgga migrated at similar position as pgg, but faster than pgga since it contains two extra phosphate groups at the end. a chemically synthesized -mer loaded with an equal molar amount to t was used as a quantitation standard (std, lanes and ) . the average intensity of the std bands was set to . based on previously reported evaluation, the intensity-molar amount starts to deviate from a linear relationship when the relative intensity approaches - under similar experimental settings [ ] . therefore, the intensity reported in lanes - and - are underestimated. the p product migrated faster than the chemically synthesized std rna due to its -phosphate inherited from the pgg dinucleotide. mtase as rdrp initiation factor in ns [ ] ) (fig a- c ). it turned out that the k m value of the r mutant is about three times the values of the wt and m_ a/ a mutant ( μm vs. μm and μm), indicating that the initiating ntp binding is clearly impaired by the jev-mode interface mutations (fig d- f ). higher ntp concentrations were not tested in these trials due to a substrate inhibition effect observed in preparatory experiments. we then use the relative catalytic rates determined at μm for the wt and m_ a/ a mutant, and μm for the r mutant, respectively, to correlate the curve fittings of all three constructs (fig g, see materials and methods). the estimated relative specificity constant (rel. k cat /k m ) were approximately . and . for r and m_ a/ a mutants, respectively (fig g, using the wt as reference) . taken together, these data suggest that perturbation of the jev-mode interface clearly impaired denv rdrp initiation mainly by affecting the initiation ntp binding, while perturbation of the denv mode interface only had a moderate effect. in the multiple-turnover p accumulation process, either the catalysis of the p -to-p conversion and the dissociation of the p product could be rate limiting. hence, the observed k cat related differences in the p conversion initiation assay could be affected by the variation in the p dissociation rates among different constructs. to further validate our judgment, we monitored the p -driven p formation for the wt, r , and m_ a/ a constructs (fig ) . in mtase as rdrp initiation factor in ns this case, the observed difference in the p formation is highly dependent on the catalytic rate (presumably the p -to-p conversion step), while not much related by dissociation rates at subsequent steps due to the single-turnover feature of the reaction and the absence of the intermediate products ( - -mer) in the denaturing gel analyses. when the initiating atp was mtase as rdrp initiation factor in ns supplied at the k m -level concentration of the wt and m_ a/ a mutant ( μm), the overall conversion rate (p -to-p ) of the r mutant was much slower than those of the other two constructs ( . min - vs. . - . min - ). when the atp concentration was lifted to the k mlevel of the r mutant ( μm), the conversion rate of the r was still lower than those of the other two constructs, while the difference between the r and wt/ m_ a/ a constructs became smaller ( . min - vs. . - . min - ). these data validate the judgment derived from the p formation assay and strongly suggest that the jev-mode interface interactions are critical in ns initiation, and in particular, in the initiation ntp binding. by solving the denv ns crystal structures in two different global conformations and characterizing the functional relevance of both conformations, the work presented here helps the ( and μm) were tested. b) the p accumulation was monitored over time for the wt, r , and m_ a/ a constructs and the std samples were used for quantitation (set to ). c) the relative p intensity as a function of time was plotted for all three constructs under two atp concentrations. the overall conversion rate (rate conv ) was estimated by fitting each data set to a single exponential rise model. https://doi.org/ . /journal.ppat. .g mtase as rdrp initiation factor in ns understandings of the conformational diversity and conservation in flavivirus ns , highlighting the important role of the mtase module, a natural fusion partner of the ns rdrp module, in the initiation phase of rna synthesis. interestingly, the mtase only facilitates rdrp initiation through the jev-mode conformation, and this specific mechanism is in agreement with the structural observation that the mtase stabilizes the folding of the ntp-binding ring finger only through this conformation [ , ] (fig a) . both conformation modes seem not to apparently contribute to the rdrp catalysis in the elongation phase, supporting our previous proposal that the rdrp may only need the assistance from the mtase in the unstable initiation phase [ , ] . based on the virological data, both conformation modes are important for virus proliferation. therefore, the denv -mode conformation could contribute to other ns -involved processes. however, whether and how it is related to the catalysis of the mtase, or to the interactions with other viral proteins or host factors remain to be clarified. note that, the index finger nls-helix (residues - in denv ns ), possibly participating in ns -binding or ns shuttling between cytoplasm and nucleus, was occluded by the mtase in both jev-and denv -mode conformations [ , , ] . interestingly, residues in the same helix were found to mediate rantes (regulated on activation, normal t cell expressed and secreted, also known as ccl ) expression in tbev [ ] . therefore, additional functional relevant conformation modes likely exist, as also suggested by the small-angle x-ray scattering and reverse genetics data [ , , ] . based on the conformational variation of the n-terminal half of the linker region observed in crystallography, the mtase is able to reach the fingers side without much difficulty. however, a conformational change of the entire linker, or additional rearrangements of the n-terminal extension (ne, residues - ) of the rdrp is probably necessary to allow the mtase to reach the front of rdrp as we previously suggested [ ] . although from the same virus family and utilizing the same de novo initiation mechanism, the overall structure is quite different for flaviviridae rdrp molecules from representative virus genera. the -kd hcv (the type species of the hepacivirus genus) ns b comprises the rdrp catalytic core and a -residue c-terminal membrane anchor. the -kd pestivirus ns b has a -residue c-terminal membrane anchor and a~ -residue unique n-terminal domain (ntd) specifically modulate the rdrp fidelity through intra-molecular interactions with the rdrp palm [ ]. the flavivirus ns does not have the c-terminal membrane anchor, but is naturally fused to the capping related mtase at its n-terminus. compared to the ntd-rdrp regulation in pesitivirus ns b, the crosstalk between the flavivirus mtase and rdrp seems to be more versatile with respect to conformational diversity and functional relevance, as discussed above. while the flaviviridae rdrps exhibit diversities in global structure and regulatory mechanisms involving the naturally fused regions of the rdrp module, this phenomenon appears to be commonly occurring in viral rdrps [ ] . the rhabdoviridae l proteins contain four additional functional regions including an mtase and a polyribonucleotidyl transferase (prntase) [ ] ; the bunyavirales l proteins include two additional regions with analogy to the pa and pb subunits of the paramyxorviridae rdrp complex that participate in the cap-snatching process to generate a capped primer for rdrp synthesis [ , ]; the alphatetraviridae rdrps contain an mtase and a helicase at the n-terminal region [ ]; the nidovirales rdrps, including the coronaviridae nsp and arteriviridae nsp , contain a~ - -residue n-terminal region with the nucleotidyltransferase (niran) function [ , ] . likewise, viral rdrps may evolve from common ancestors comprising only the catalytic module with relative mtase as rdrp initiation factor in ns independency in carrying out rna synthesis, similar to the pv d pol and hcv ns b. co-evolution with versatile host species and the low coding capacity-driven protein function combination may contribute to the structural and functional diversity of current viral rdrps with respect to regions beyond the rdrp catalytic module. we propose that the functional roles currently offered by these regions, such as the fidelity modulation in pestivirus ns b and initiation enhancement flavivirus ns , are likely a result of their co-evolution with the rdrp module through the establishment of specific interactions. moreover, the rdrp fidelity and initiation enhancement offered by the pesitvirus ns b ntd and flavivirus ns mtase, respectively, is likely not an improvement of a specific function relative to the corresponding rdrp ancestors, but a balance between gaining an extra module and maintaining the levels of key enzymatic properties for the rdrps. large-scale protein conformational changes are amazing events in biological systems, albeit associated with very different characteristics. driven by multiple cycles of phosphoryl transfer reactions and accompanied by dramatic changes in protein-nucleic acid interactions, the nterminal one third of bacteriophage t rna polymerase undergoes a dramatic rearrangement as it makes an irreversible transition from the promoter-bound initiation state to the promoter-free elongation state in the transcription process [ ] [ ] [ ] . by contrast, the observed conformational diversity in flavivirus ns has not yet involved continuous events of nucleotide addition, and the energy barrier between different observed states may be low enough to allow a distribution of several states in solution. as both captured by crystallography in multiple virus species, the jev-mode and denv -mode conformations probably represent relatively stable states of apo ns , thus forming the foundation for further understanding of the ns function when it is participating in enzymatic reactions or interactions with essential viral or host factors. the flavivirus ns is also a great example that the different interaction modes can be established between regions of a single protein, and are related to different functions of the protein and different processes in the life cycle of the corresponding species. in a broader context, the conformation-based function diversity extends the function capacity beyond the traditional consideration of protein folding, and is therefore an important factor when understanding protein function. the full-length wt denv ns gene within the dna clone of tsv strain (genbank: ay ) was cloned into a pet b vector to yield the pet b-denv -ns plasmid. eleven full-length ns constructs with point mutations (fig e) were made by using the quickchange site-directed mutagenesis method and the wt plasmid as the template. ns expressing plasmids were transformed into escherichia coli strain bl -codonplus(de )-ril for expression of ns constructs with a hexa-histidine tag at the c-terminus. cells were grown at ˚c overnight in the nzcym medium containing μg/ml kanamycin (kan ) and μg/ml chloramphenicol (chl ) until the optical density at nm (od ) was . . the overnight culture was used to inoculate l of nzcym medium with kan and chl to reach an initial od around . . the cells were grown at ˚c at rpm to an od of . and then cooled to room temperature (r.t.). isopropyl-β-d-thiogalactopyranoside (iptg) was added to a final concentration of . mm, and the cells were grown for an additional h at r. t. or h at ˚c before harvesting. mtase as rdrp initiation factor in ns cell lysis, subsequent purification and storage procedures were as previously described in the jev ns study [ ] , except that the centrifugation duration to remove cell debris was h, a mm imidazole wash was applied prior to the elution step of the nickel-affinity chromatography, and mm tris (ph . ) was used as the buffering agent in the gel filtration chromatography. tris-( -carboxyethyl)phosphine (tcep) was added to the pooled fractions to a final concentration of mm. the molar extinction coefficient for the denv ns was calculated based on protein sequence using the expasy protparam program (http://www.expasy.ch/ tools/protparam.html). the yield is typically mg of pure protein per l of bacterial culture. the denv ns crystals were grown by sitting drop vapor diffusion at or ˚c using - mg/ml protein sample. crystals grew to its final dimension in about weeks with a precipitant/well solution containing . % (vol./vol.) dioxane, . m bicine (ph . ), . % (wt./vol.) peg , and % (vol./vol.) glycerol for crystal form , and . m nh i, % (vol./vol.) tacsimate (ph . ), and % (wt./vol.) peg for crystal form . crystals were transferred into cryo-stablizer solutions and stored in liquid nitrogen prior to data collection. diffraction datasets were collected at the shanghai synchrotron radiation facility (ssrf) beamlines bl u (crystal form , wavelength . Å) and bl u (crystal form , wavelengths: . Å) at k. typically, at least ˚of data were collected in . - . ˚oscillation steps. reflections were integrated, merged, and scaled using hkl or d � trek [ , ] . the initial structure solution for crystal form was obtained using the molecular replacement program phaser [ ] and separated mtase and rdrp ensembles derived from jev and denv ns structures (pdb entries k m and v q) [ , ] . the final model of crystal form was split into three ensembles (mtase, rdrp thumb with index tip, and the rest of rdrp) in the molecular replacement trial to obtain the initial structure solution for crystal form . manual model building and structure refinement were done using coot and phenix, respectively [ , ] . the , k composite simulated-annealing omit f o -f c electron density maps were generated using cns [ ] . all ns superimpositions were done using the maximum likelihood based structure superpositioning program theseus [ ] . relative domain motions were analyzed by dyndom [ ] . the occlusion of the solvent accessible area by the mtase-rdrp interactions was analyzed by program surfrace with a probe radius of . Å and the -residue linker excluded in the calculation [ ] . the multiple sequence alignment of ns was carried out using available complete ns sequences among the flavivirus species documented by the international committee on taxonomy of viruses (ictv) (http://www.ictvonline.org), and the alignment was then used to generate the sequence logos (http://weblogo.berkeley.edu) and the conservation score projected structural representations. the projection of the conservation score onto structural model was done by the consurf server [ ] . bhk- cells (american type culture collection (atcc), ccl- ) was propagated in dulbecco's modified eagle's medium (dmem) supplemented with % fetal bovine serum (fbs), units/ml of penicillin and μg/ml of streptomycin in % co at ˚c. monoclonal antibody ( g ) against envelope protein of flavivirus was used to detect viral protein expression of both jev and denv . fitc-conjugated goat anti-mouse igg was used as secondary antibody. mtase as rdrp initiation factor in ns the infectious clones of pacyc-jev-sa [ ] and pacyc-denv -tsv [ ] were used as the backbone to construct recombinant jev and denv with different ns mutations, respectively. all mutations were engineered by fusion pcr. the jev ns mutations were engineered at bamhi and xbai restriction sites of pacyc-jev-sa . all denv ns mutations were inserted into to pacyc-denv -tsv by restriction digestion with nrui and clai. all constructs were verified by dna sequencing before they were used in the subsequent experiments. the infectious clone of jev and denv with corresponding ns mutations were linearized with xhoi and clai, respectively, and then subjected to in vitro transcription using a t in vitro transcription kit (thermo fisher scientific). approximately μg of transcribed recombinant genomic rnas were transfected into bhk- cells with reagent dmrie-c (invitrogen). then the cell slides were fixed in cold (- ˚c) % (vol. to vol.) acetone in methanol at r. t. for min. after washing three times with phosphate buffer saline (pbs) (ph . ), the fixed cells were subjected to ifa with g monoclonal antibody to examine viral envelope expression of both jev and denv . at the same time, the supernatants of rna-transfected bhk- cells were harvested as viral stocks for plaque assay to quantify viral titers and examine plaque morphologies. briefly, confluent bhk- cells ( × cells per well, plated day in advance) in -well plates were infected with serially -fold diluted viral supernatants and incubated at ˚c with % co for h before the layer of medium containing % methylcellulose was added. after days of incubation at ˚c with % co , the cells were fixed in . % formaldehyde and then stained with % crystal violet. the viral titer was calculated as plaque formatting unit (pfu) per ml. the -mer template rna (t , fig a) used for de novo polymerase assays was chemically synthesized (integrated dna technologies or dharmacon) and purified by % (wt./vol.) polyacrylamide/ m urea gel electrophoresis. the target rna was excised from the gel, electro-eluted by using an elu-trap device (ge healthcare), ethanol precipitated, dissolved in an rna annealing buffer (rab: mm nacl, mm tris (ph . ), mm mgcl ), and stored at - ˚c after a self-annealing process (a -min incubation at ˚c followed by snap-cooling to minimize inter-molecular annealing). a -phosphorylated dinucleotide primer pgg (p ) (jena biosciecnes) was mixed with t at : or : molar ratio to make the t /p construct. all in vitro polymerase assays were based on the dinucleotide (p )-driven reactions. the standard reaction condition was derived from the jev ns work with the μm of the t rna and μm of ns and with a couple of adjustments [ ] . firstly, a manganese-free reaction condition ( mm tris (ph . ), mm nacl, mm mgcl , mm dtt) (hereinafter referred as reaction buffer) was established. secondly, a -phosphorylated dinucleotide (pgg) and the t rna was mixed at a difference ratio. in the assay to characterize the conversion of p to the -mer product (p ), atp was supplied as the only ntp substrate and a high p : t ratio ( : ) was used to achieve multiple turnovers within a reasonable duration. for the p -containing ec (ec ) formation assay to achieve the p -to-p conversion, atp and utp mtase as rdrp initiation factor in ns were supplied and the p :t ratio was : . for the p -to-p single nucleotide elongation assay, reactions were first carried out as described in the ec formation assay. the reaction mixtures were centrifuged at , g for min, and the pellet was washed twice by the reaction buffer, and was then resuspended in a modified reaction buffer with nacl concentration lifted to mm. ctp was supplemented to the resuspended mixture to allow the singlenucleotide elongation at ˚c, and the reaction was quenched immediately (" " min) or at a certain time point after the addition of ctp. the concentrations of nacl and ctp in the final reaction mixture are mm and μm, respectively, unless otherwise indicated. for all assays, the procedures for denaturing polyacrylamide gel electrophoresis (page), gel staining, and quantitative analyses were performed as previously described [ ] . the stains-all (sigma-aldrich)-based staining method is reasonably accurate when quantitating rna bands with the same length, and in the majority of our experiments we tried to keep the band intensity within the linear range estimated in the previous study by adjusting the range of reaction time points [ ] . for the p -to-p conversion assay, , , , , and μm atp concentrations were used for wt and m- a/ a, and , , , , and μm were used for r . to account for gel-to-gel intensity variations, samples from the same reaction mixture (indicated by the same icon above corresponding lanes in fig a- c) were loaded on different gels to normalize the intensities (e.g. lanes / , / , / in fig a) . the normalized intensity was then used to calculate the relative reaction rates (fig d- f, left) , which in turn were fitted to the michaelis-menten equation (fig d- f, right) . to estimate the relative k cat and specificity constant values of all three constructs, the michaelis-menten curves of all three constructs were normalized based on the measurement of the relative reaction rates of each wt-mutant pair in the same gel (fig g) . for the stability assessment of ec , the p -to-p conversion was first carried out as described above. the reaction mixture was subsequently centrifuged at , g for min, and the pellet was washed twice by the reaction buffer and was then resuspended in a modified reaction buffer with nacl concentration lifted to mm. the resuspension was incubated in ˚c for various duration ( - h), and then ctp was added to reach a final concentration of μm to trigger the p -to-p conversion by ec that survived the incubation. the fraction of -mer intensity values were fitted to a single-exponential decay model to estimate the inactivation rates of all three constructs. supporting information s movie. a modelled ns conformational transition from the jev-mode to the denv mode. the movie starts with the jev ns structure (pdb entry k m), switches to the first form of denv structure (pdb entry kr ), then the second form of denv structure (pdb kr ), and finally the denv structure (pdb entry v q). three rotation axes related to the mtase movement are indicated by black lines, and one axis related to the rdrp thumb movement is indicated by a grey line. note that the non-superimposable regions are not included in the structural models. (mp ) zika virus: new clinical syndromes and its emergence in the western hemisphere flavivirus genome organization, expression, and replication the . a structure of vaccinia protein vp : a bifunctional enzyme that participates in the modification of both mrna ends an rna cap (nucleoside- '-o-)-methyltransferase in the flavivirus rna polymerase ns : crystal structure and functional characterization crystal structure of the rna polymerase domain of the west nile virus non-structural protein crystal structure of the dengue virus rnadependent rna polymerase catalytic domain at . -angstrom resolution structural basis for proteolysis-dependent activation of the poliovirus rna-dependent rna polymerase crystal structure of the full-length japanese encephalitis virus ns reveals a conserved methyltransferase-polymerase interface a structural and primary sequence comparison of the viral rna-dependent rna polymerases a structural overview of rna-dependent rna polymerases from the flaviviridae family a structural view of the rna-dependent rna polymerases from the flavivirus genus perturbation in the conserved methyltransferase-polymerase interface of flavivirus ns differentially affects polymerase initiation and elongation crystal structure of full-length zika virus ns protein reveals a conformation similar to japanese encephalitis virus ns structure and function of the zika virus fulllength ns protein the structure of zika virus ns reveals a conserved domain conformation supramolecular arrangement of the full-length zika virus ns structure of the yellow fever ns protein reveals conserved drug targets shared among flaviviruses ns from dengue virus serotype can adopt a conformation analogous to that of its zika virus and japanese encephalitis virus homologues the interdomain region of dengue ns protein that binds to the viral helicase ns contains independently functional importin beta and importin alpha/beta-recognized nuclear localization signals a crystal structure of the dengue virus ns protein reveals a novel inter-domain interface essential for protein flexibility and virus replication dengue virus nonstructural protein (ns ) assembles into a dimer with a unique methyltransferase and polymerase interface structural basis of viral rna-dependent rna polymerase catalysis and translocation the uncoupling of catalysis and translocation in the viral rna-dependent rna polymerase a crystal structure of the dengue virus non-structural protein (ns ) polymerase delineates interdomain amino acid residues that enhance its thermostability and de novo initiation activities the methyltransferase domain of dengue virus protein ns ensures efficient rna synthesis initiation and elongation by the polymerase domain theseus: maximum likelihood superpositioning and analysis of macromolecular structures the interface between methyltransferase and polymerase of ns is essential for flavivirus replication analysis of flavivirus ns methyltransferase cap binding identification of the critical linker residues conferring differences in the compactness of ns from dengue virus serotype and ns from dengue virus serotypes - crm -mediated nuclear export of dengue virus rna polymerase ns modulates interleukin- induction and virus production tick-borne encephalitis virus nonstructural protein ns induces rantes expression dependent on the rna-dependent rna polymerase activity recovery of a chemically synthesized japanese encephalitis virus reveals two critical adaptive mutations in ns b and ns a functional analysis of two cavities in flavivirus ns polymerase assembly, purification, and pre-steady-state kinetic analysis of active rna-dependent rna polymerase elongation complex rna polymerase of influenza virus. dinucleotide-primed initiation of transcription at specific positions on viral rna structure of a t rna polymerase elongation complex at . a resolution snapshots of a viral rna polymerase switching gears from transcription initiation to elongation the finer things in x-ray diffraction data collection processing of x-ray diffraction data collected in oscillation mode phaser crystallographic software coot: model-building tools for molecular graphics phenix: a comprehensive python-based system for macromolecular structure solution crystallography & nmr system: a new software suite for macromolecular structure determination improvements in the analysis of domain motions in proteins from conformational change: dyndom version . novel computer program for fast exact calculation of accessible and molecular surface areas and average surface curvature consurf : the projection of evolutionary conservation scores of residues on protein structures recovery of a chemically synthesized japanese encephalitis virus revealed two critical adaptive mutations in ns b and ns a we thank dr. pei-yong shi for providing the cloning material for the denv ns gene, dr. xiao-dan li for construction of the denv ns m mutant plasmid, dr. bo shu for x-ray diffraction data collection, liu deng and yancheng zhan for laboratory assistance, dr. key: cord- -d htyfcl authors: gaglia, marta maria; rycroft, chris h.; glaunsinger, britt a. title: transcriptome-wide cleavage site mapping on cellular mrnas reveals features underlying sequence-specific cleavage by the viral ribonuclease sox date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: d htyfcl many viruses express factors that reduce host gene expression through widespread degradation of cellular mrna. an example of this class of proteins is the mrna-targeting endoribonuclease sox from the gamma-herpesvirus kaposi’s sarcoma-associated herpesvirus (kshv). previous studies indicated that cleavage of messenger rnas (mrna) by sox occurs at specific locations defined by the sequence of the target rna, which is at odds with the down-regulation of a large portion of cellular transcripts. in this study, we address this paradox by using high-throughput sequencing of cleavage intermediates combined with a custom bioinformatics-based analysis pipeline to identify sox cleavage sites across the mrna transcriptome. these data, coupled with targeted mutagenesis, reveal that while cleavage sites are specific and reproducible, they are defined by a degenerate sequence motif containing a small number of conserved residues rather than a strong consensus sequence. this degenerate element is well represented in both human and kshv mrna, and its presence correlates with rna destabilization by sox. this represents a new endonuclease targeting strategy, in which use of a degenerate targeting element enables rna cleavage at specific locations without restricting the range of targets. furthermore, it shows that strong target selectivity can be achieved without a high degree of sequence specificity. triggering wide-spread rna degradation is a common strategy that viruses use to decrease host gene expression, also known as host shutoff [ , ] . viral factors from many different families including herpesviruses, coronaviruses and orthomyxoviruses either directly cut rnas or indirectly stimulate rna cleavages in an endonucleolytic fashion [ , ] . cellular rna exonucleases are then recruited to degrade the fragments, resulting in a reduction in rna and consequently protein levels [ ] . despite the fact that the proposed role of most of these host shutoff ribonucleases (rnases) is to modulate immune responses, they are generally thought to have little or no specificity and to affect host messenger rnas (mrnas) indiscriminately. however, increasing evidence suggests that this view may be overly simplistic, and that some of the rnases display selectivity for or against specific targets [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . this type of specificity may provide an additional level of regulation in viral control of the host transcriptome. how this selectivity is achieved and how it is balanced with the widespread shutoff phenotype remain open questions. the sox family of proteins from gamma-herpesviruses is an example of a viral rnase that displays both broad targeting of rnas and a poorly understood level of selectivity. gammaherpesviruses include the human pathogens kaposi's sarcoma-associated herpesvirus (kshv), which causes kaposi's sarcoma as well as lymphomas in immunocompromised individuals and remains a leading cause of cancer-linked death in sub-saharan africa. the sox (orf ) protein is expressed early during the lytic cycle of kshv infection and its expression triggers rna degradation, which is recapitulated by expression of the protein alone [ ] . homologs of sox in the other human gamma-herpesvirus, epstein barr virus (ebv bglf ), and in the model murine pathogen murine herpesvirus (mhv musox) also degrade rna in cells [ , ] . studies in mhv suggest that host shutoff by the sox family of proteins is crucial for viral replication in specific cell types and for systemic spread of the virus and establishment of a latent infection [ ] . transcriptomic studies of mrna levels during kshv or mhv infection and in cells overexpressing sox demonstrate that this family of proteins triggers the degradation of a majority of both host and viral transcripts [ , , ] . however, in-depth mechanistic studies of sox reveal a more complex picture. sox targets mrnas, as opposed to non-coding rna species, a specificity that is related to the association of sox with polyribosomes [ ] . moreover, selected transcripts, like the cytokine interleukin (il- ) [ ] and apoptosis enhancing nuclease (aen) [ ] , are spared from sox-mediated decay. in the case of il- , protection is conferred by the presence of a protective sequence in the ' untranslated region (utr) [ ] , but aen appears to be intrinsically resistant to sox mediated degradation [ ] , without a clear protective element in its sequence. the most unexpected observation, however, is that kshv sox and ebv bglf cut rnas at specific locations that appear to be determined by an unknown targeting element [ , ] . these specific cleavages become apparent upon knockdown of the human rnase xrn , the major '- '-directed rnase in eukaryotic cells, which is responsible for clearing the ' rna fragments generated by these rnases [ ] . this ability of sox to cut at specific locations within mrna yet target the majority of transcripts argues for a degenerate targeting motif. in general, the principles guiding the positioning of rna cleavages by cellular and viral mrna endonucleases are not well understood. in the case of endonucleases, the term "sequence specificity" is sometimes used to refer to preferential cutting at specific dimers, often inferred from in vitro studies (for example in datta et al. [ ] ). however, this specificity cannot explain cutting of rnas at single locations in mrnas. additional specificity can be conferred by localization of the target mrna to a specific site in the cells [ ] or proximity to a "landmark" feature on the rna, such as the ' of the transcript [ , ] or the stop codon location [ ] . the sox targeting system is unprecedented because the sequence of the targeting element alone appears to direct cleavages in mrna, and because the targeting element is longer than nt [ ] . to address how sox specificity is mediated, we applied a degradome sequencing technique called parallel analysis of rna ends (pare) [ ] to map cleavage sites of the sox protein across the human transcriptome. development of a stringent python-based analysis algorithm, which we term pydegradome, allowed identification of sox-dependent cuts at specific locations across the mrna transcriptome. the sequences surrounding these sites contained no strong consensus sequence, but rather a degenerate sequence pattern that nonetheless conferred specificity when analyzed experimentally in endogenous mrna targets. the presence of a more complex targeting motif explains how sox achieves cleavage specificity without sacrificing target breadth, and offers a framework for understanding how additional viral and cellular endonucleases may operate. development of a novel bioinformatics pipeline to detect highconfidence sox cleavage sites across the transcriptome following pare prior analyses of individual mrnas indicated that the kshv rnase sox cuts at specific locations within the rna, in a manner dependent on the sequence surrounding the cleavage site [ ] . by performing ' rapid amplification of cdna ends ( ' race) on the gfp reporter mrna, we found that the gfp mrna was cleaved in the same location regardless of whether sox was transiently expressed in t cells or expressed from the kshv genome in lyticallyreactivated islk. cells (s fig) . this is in agreement with the fact that sox activity does not rely on any additional viral proteins, and that its cleavage activity can be studied in transfected cells [ , , ] . to dissect the specificity of sox cleavage across the mrna transcriptome, we designed an approach to map sox cleavage sites in endogenous mrnas using pare [ , ] . pare is an rna-seq based methodology that allows mapping of the ' ends of uncapped, phosphorylated rna species, such as mrna degradation fragments ( fig a) . as we previously found that the sox degradation intermediates are cleared by the host '- ' exonuclease xrn [ ] , we stabilized degradation intermediates in t cells expressing a gfp-sox fusion [ ] by knocking down xrn (s a fig) . cells expressing gfp alone were used as controls to filter out rna fragments generated by cellular rnases or other processing events. this was important because multiple studies have shown that pare and similar techniques detect a large number of rna fragments in human cells, many of which are of unknown origin [ ] [ ] [ ] . we prepared and sequenced pare experimental procedure and peak finding analysis pipeline. a) diagram of the pare procedure. b) schematic of pydegradome analysis approach, which uses read counts in a control sample to generate a table of thresholds to compare test sample counts to. the table lists thresholds for a particular user-defined confidence level and for a range of ratios between control and test samples. the applicable ratio for each position is computed by multiplying a user-defined multiplicative factor by the ratio of total read counts for the exon in test vs. control samples, thus accounting for variation in rna levels and total mapped reads. read counts in test sample at each position must exceed the threshold to be identified as part of a peak. c) example of plot of read counts ( ' end only) for nt surrounding a sox cut site identified by pydegradome within the '-most exon of the limd nm_ transcript in the four samples. note that y-axis has a logarithmic scale. this example shows the expected distribution for a cut site followed by exonucleolytic degradation due pare libraries from two replicates of sox-expressing or gfp control cells and extracted the ' end of each mapped read, which represents the cleavage site (s table) . conventional analysis of pare and similar degradome datasets relies on detecting cut sites in each condition and then comparing conditions to each other a posteriori, but initial attempts at detecting and validating cut sites indicated that this approach did not provide sufficient discriminatory power to identify sox-specific cleavage sites. in previous studies using pare or similar approaches [ , ] , additional information about the pathways, such as the mirna sequences for mirna cut site studies or the proximity of the site to a stop codon for studies of smg and nonsense mediated decay, was used to further select "true" cut sites. however, such contextual information does not exist for sox cleavage specificity. therefore, we devised a python-based analysis approach that would directly use our control dataset as a baseline, which we termed pydegradome ( fig b) . the analysis uses a bayesian probability framework to determine whether the read counts at a given location differ significantly between control and test samples, taking into account random variations in the number of reads. using bayes' theorem, we determine for each location whether the underlying rate of fragment production in the test sample is a multiplicative factor larger than the control rate at a user-defined confidence level. the user also chooses the multiplicative factor. for a given control read count, we thus compute a threshold that the read counts in the test samples have to exceed to be considered part of a "peak" (fig b) . to improve efficiency when testing thousands of locations, the software builds a reference table of the thresholds for the entire dataset. this approach allowed the identification of locations within the transcriptome where the read counts were statistically higher in the samples from sox-expressing cells than in control samples (peaks). in order to correct for up-or down-regulation of the rnas and for the total number of reads obtained for each sample, the ratio used to determine thresholds was computed from the user-defined multiplicative factor and the ratio of the total number of reads mapping to each exon in test vs. control samples (fig b) . to prevent isolated high read counts from skewing our analysis, we integrated read counts within small windows ( nt) rather than single nucleotides. adjacent windows that passed the cutoff were then combined into a single peak. we optimized the userdefined confidence level and ratio by determining how many peaks were detected when comparing each sox replicate sample to its gfp control to detect sox-specific peaks, or performing the opposite comparison to detect gfp-specific peaks. in addition, we also ran the program to detect peaks specific to only one biological repeat, by comparing the two sox or gfp replicates to each other, as these peaks may represent experimental noise. although we consistently detected more sox-specific peaks than gfp-specific peaks, varying the parameters improved discrimination of sox-specific peaks (s b fig) and reduced detection of "noise" peaks specific to one repeat. based on this optimization, we empirically set the final iteration of the program to detect nt windows with read counts in the test samples that are four fold higher than read counts in the control samples within a confidence level of . %. because these parameters are conservative, we expect that the sox cut sites we detected do not represent a comprehensive list of all sox cut sites, but rather only the highest confidence sites. within each peak, we also determined the position where the read count was highest, and we considered this position the location of putative cut site (with the cleavage occurring ' of this position) (fig b and c ). with similar optimization, this program could be used to identify the ends of degradation fragments in other degradome datasets that contain matching test and control samples. sox cuts sites are abundant and not positioned relative to landmark features of mrna using the approach detailed above, we detected a higher number of peaks specific to sox-containing samples relative to control samples, consistent with broad mrna targeting by sox (fig a; s table) . even when varying the allowed distance between peaks from - nt in sample replicates, the sox samples contained~ - times the number of reproducible ("shared") sox-specific peaks (s c fig). up to % of the sox-specific peaks but fewer than % of the control gfp-specific peaks were shared between the replicates, indicating that many of the sox cleavages reproducibly occur at a given site (fig b) . the read counts at the putative cut site in the two sox replicates were highly correlated ( fig c, spearman's ρ = . , p value < . ), further demonstrating that these peaks correspond to specific sox-mediated cleavages. for downstream analyses, we focused on cut sites that were detected in both the replicates using the . % confidence level and were - nt apart (s table) . example plots of the read counts around identified cut sites are shown in figs c and s a-s f. several virally encoded host shutoff factors that trigger rna degradation, including herpes simplex virus vhs and sars coronavirus nsp , are thought to induce sequence-independent cuts near the ' end of the message [ , ] . to examine whether sox cleavage sites in endogenous mrnas are position-specific, we compared the location of the sox-specific cut sites to those found only in control samples using the human transcript annotation from ensembl grch . in both sox and gfp control samples, more cut sites occur towards the ends of the transcripts, most frequently corresponding to the ' and ' untranslated regions (utrs) of the mrna (fig d and e ). it remains unclear whether this non-specific end bias is due to a general preference for cleavage in non-translated regions or a consequence of the pare approach. we also computed the position of the cuts relative to landmarks such as the transcript start site, start codon, stop codon or annotated ' end. only a fraction of the peaks was located within nt of any of these landmarks in either case (fig f) . although a greater percentage of the sox cut sites occurred within nt of start codons, this still only accounted for % of the cut sites. furthermore, the - % of both sox and gfp peaks near annotated transcription start sites may represent the beginning of full-length decapped mrnas rather than endonuclease cleavage fragments. collectively, these analyses indicate that sox cut sites are not restricted to a particular region of the mrna, although cleavage sites in both sox and control gfp samples may be enriched in areas of the transcripts that are not covered by ribosomes. these findings are consistent with our previous reporter mrna data [ ] . we next selected seven sox cut sites for independent experimental validation ( fig a) . the selection was based on three criteria: ) position more than nt from the annotated ' end of the transcript in order to eliminate potential transcription start sites, ) high number of mapped reads, and ) clear sox-specific peaks in a visual inspections of the read plots (figs c and s a-s f). we used two approaches to validate the cut sites, targeted ' race and insertion in reporter constructs, and found that all sites validated in at least one of the two assays. we detected a ' race fragment that appeared specifically in sox-expressing cells (s g fig) and whose size corresponded to the predicted sox cleavage location for four of the transcripts. (we were unable to detect the rnas for bloc s and srsf using control primers). our second validation approach was designed to test the hypothesis that specific rna sequences or structures flanking endogenous cut sites direct cleavage by sox even when removed from their native context, as we had seen for reporter mrnas [ ] . we inserted nt pare analysis identifies sox-specific cut sites in endogenous rnas. a) number of peaks/cut sites identified specifically in sox or gfp samples. b) fraction of the peaks identified in sox or gfp samples that were detected in both biological replicates ("shared peaks"), relative to the maximum distance allowed between the peaks. c) correlation plot of the heights of peaks found in both sox+ samples. peak height is defined as the highest read count within the peak window, at the position defined as the putative cut site. d) position of the cut sites found in both replicates ("shared cut sites") within the mrnas relative to the length of the transcript. e) position of the shared cut sites relative to the coding region of the mrna (in all samples > % of surrounding the cleavage sites from the mrna targets identified by pydegradome into a gfp reporter ( fig b) . we then co-expressed these constructs with sox in xrn -depleted cells and tested whether the inserted sequence caused a sox-specific cut in the mrna. the gfp reporter we used is normally cut by sox at~nt of the coding region [ ] , generating a degradation intermediate that is~ nt shorter than the full length mrna. we found that the insertion of the sequences from six out of seven of the candidate sox cleavage sites resulted in the appearance of a second rna fragment in sox-expressing cells (fig c and d ). interestingly, the intensity of the additional cleavage products varied between the insertions, suggesting some sequences are better sox targets than others. in particular, in the gfp reporters with insertions from the limd mrna (fig d) , we found that the original cleavage site in the gfp coding region was almost completely abolished in favor of the new cleavage site, as evidenced by the disappearance of the longer degradation intermediate. moreover, insertion of the limd nt sequence in a different position in the gfp mrna also elicited sox cleavage ( fig e) , further demonstrating functionality of the targeting sequence regardless of its broader context. taken together, these data indicate that we have identified bona fide sox cleavage sites in endogenous mrnas, and that these sites contain specific elements that lead to sox targeting. the sox cleavage site likely occurs in an unstructured region of the mrna and is characterized by an a-rich sequence just upstream of the cleavage to identify features that define a sox cleavage site, we searched the sequences surrounding the sox cut sites detected in both biological repeats for structural or sequence similarities, using the cut sites shared by the two gfp samples as a comparison set. first, localfold [ ] was used to determine the likelihood that the nucleotides around the cut are located in unpaired regions (i.e. accessibility). this program is a variation on the vienna algorithm rnafold and is based on the assumption that potential structures are formed locally, which is consistent with the success of our insertion experiments. we found that the nucleotides ' of the sox cut were more accessible (thus presumably unstructured) than surrounding sequences ( fig a) . this pattern was different from the sequences surrounding the gfp sites, suggesting that it is feature specific to sox cleavage sequences. we also computed the log likelihood for different nucleotides at positions around the sox cleavage ( fig b and c ). although no strong consensus sequence emerged, two features stood out from this analysis. first, the position right after the cut site (position ) was preferentially c or t. when we computed the frequency of different nucleotides at the cut site for both soxspecific and gfp-specific cleavages, we found that the pyrimidines c or t were found at % of the sox cut sites, whereas c or a were the most frequent bases at position in cuts specific to gfp control samples ( fig d) . this distribution is not due to a bias in library preparation, as a was the most frequent base at the beginning of both aligned (s a we also found that there were more as and fewer cs in the nt ' to the cleavage site. we computed the fraction of putative sox cut sites that had a dimers or trimers in the nt preceding the cut and found that a stretches were found before~ % of our mapped cut sites (fig the peaks fall within coding transcripts). nd = not determined, because multiple transcript isoforms are present in the annotation and the cut site position would differ between isoforms. na = no coding sequence annotated. f) position of shared cut sites relative to annotated landmarks on transcripts. for all panels in this figure, a scanning window of nt, a multiplicative factor of , a confidence level of . %, were used to predict cut sites. all cuts sites nt apart in the two replicates were used for the analyses in panel c-f (sox-specific peaks: n = , gfp-specific peaks: n = ). doi: . /journal.ppat. .g . the gfp-based reporters were then co-expressed with sox ("+" or "+sox") or an empty vector control ("-") in shxrn -treated e). this was not a general feature of the sequences that produce rna fragments, as only five of the cut sites found solely in the control samples were preceded by an a dimer and none by longer a stretches. these analyses suggest that although sox cut sites are defined by a degenerate sequence pattern, this sequence is enriched for pyrimidines at a cut site adjacent to an unstructured stretch of a residues. experimental analysis of an endogenous sox cleavage sequence confirms role of oligo a sequence and potential structural element to probe the sox targeting element further we examined more in detail the sox targeting element in the validated endogenous mrnas (fig a) . the structure prediction program rnafold [ ] predicted that the - nt surrounding the cut sites in six out of seven of the rnas fold in hairpin structures with oligo-a stretches and the cleavage sites in unpaired loops (figs a and s a). the only exception was mapk ip , which also lacked the oligo-a sequence. these structure predictions mirror the accessibility results from localfold analysis (fig a) , which indicated that the positions from - to relative to sox cut sites are more likely to be unpaired. similarly, we predicted the structures of all nt sequences surrounding the soxspecific cut sites in our dataset and determined how many of the sequences presented either the cut site, an a dimer or both the cut site and an a dimer in an unstructured region. for % of the sox cut sites, at least one of these two features was predicted to be in an unpaired region, with over % of the sequences predicted to have both ( fig b) . we reasoned that if these structural features are important for cleavage, the efficiency of the cleavage could vary depending on the length of the inserted endogenous sequence. fragments of different sizes may not be able to fold into the native structure equally well and the stability of the resulting structures may vary. consistent with this idea, we found that changing the length of the inserted fragments for two different rnas (limd and srsf ) changed the efficiency of sox cleavage, measured by the intensity of the degradation intermediate (figs c, d, s b and s c), although sequences of nt were sufficient to elicit sox cleavage. in particular, when we shortened the limd inserted sequence from nt to nt, nt and nt, the efficiency of cleavage progressively diminished (fig c and d ), consistent with limd sequences adopting a stem-loop structure that becomes destabilized upon sequential deletions of the putative stem region. surprisingly, shortening the inserted sequence for srsf had the opposite effect and increased the efficiency of the cleavage (s b and s c fig). because the same sequences are present in the nt and the nt srsf insertion, this observation cannot be explained by the presence of a targeting sequence alone. instead, we hypothesize that the shorter insertion folds more stably into an autonomous element that is required for targeting by sox. we previously found that mutating a tgaagt sequence nt before the gfp cut site to tgagtg could abolish the cleavage site in gfp (s a fig). the limd site is also preceded by a similar tgaaag sequence predicted to be in an unpaired loop. to test directly whether the a trimer in the limd sequence was required for the positioning of the cleavage, we mutated the tgaaag sequence in our insertion reporter to tgcccg, tggggg or tgtttg. as predicted by our data analysis, we found that mutation of the a trimer prevented the limd sequence from eliciting sox cleavage, indicating that the aaa is an integral part of the sox recognition site (fig e) . moreover, deletion of one of the three as in the nt insertion cells and the gfp mrna from these cells was detected using northern blotting. the arrowheads point to the additional cleavage fragments resulting from the insertions. images are representative of results from at least three experimental replicates. [ ] representation of the frequency of each base in the nt surrounding the cut sites found in both sox samples (n = ). the position of the cut site is reduced the efficiency of cleavage dramatically (fig c and d) , while rnafold structure prediction suggested that this deletion is unlikely to substantially alter the structure of the rna (s b fig). similarly, mutating an a dimer just upstream of the cut site in a srsf insertion construct reduces sox-mediated cleavage (fig g) . while the upstream a dimer likely contributes to sox targeting, we note that it is not always required, as a similar mutation in a pgam insertion construct did not abolish cleavage (s c fig). overall, these data are consistent with the idea that the a-stretch is an important feature of the sox cleavage specificity. lastly, our analysis indicated that the nucleotide g is under-represented at the position immediately following the sox cut site (position , fig b- d ). we found that mutating the nucleotide at position from an a to a g prevented sox-mediated cleavage in two out of three of the rna we tested (limd and srsf , but not the pgam ) (figs f, g and s d). these data suggest that sox activity is inhibited by the presence of a g nucleotide as the residue ' of the cleavage. collectively these data experimentally validate our computational finding and strongly suggest that sox cut sites are defined by a combination of sequence and structural features. a conserved sequence pattern is specific to sox-dependent cut sites although sequences flanking the sox cleavage sites lacked a strong consensus motif, our analysis showed that the frequency of the bases around the cut site diverged from the expected distribution for human rna sequences (fig b and c ). this suggested to us that there is a conserved sequence pattern among sox target sequences. in order to be able to search rna sequences for this variable motif, we derived a position weight matrix (pwm) for the positions - to + from the sox cleavage site ( being the nucleotide ' of the cut site, fig c) . the pwm is a matrix that lists the probabilities (transformed into log likelihood to correct for the background frequencies of the nucleotides) for each of the four bases at each of the positions of the putative motif. the log likelihoods for our pwm were derived from very high confidence sox cut sites identified in both of our experimental repeats using a very stringent confidence level of . % and located at least nt away from a transcriptional start site. the logo in s a fig is a pictorial representation of the pwm. we then used the pwm to confirm that sox cut sites specifically matched the motif by scoring several subsets of potential sox target or control sequences using the pwm. the presence of preferred nucleotides in positions - to + from the cut site results in higher (positive) log likelihood scores, whereas a poorer match to the motif produces a lower (negative) log likelihood score. indeed, the sequences flanking the set of reproducible sox cut sites (identified with a confidence level of . %) were a closer match to the motif compared to those surrounding gfp-specific fragment ends, as shown by the distribution of the log likelihood scores (fig a) . a similar difference was seen when analyzing the sequences surrounding all potential sox and control (gfp) cut sites from each of the two biological repeats. sequences around sox cut sites were a good match (positive log likelihood score) to the motif more frequently than control sequences (fig b) . in both analyses (fig a and b) , we removed the sequences of the high confidence cut sites used to derive the pwm from the analyzed sets. the results of these analyses indicate that although the indicated. d) percentage of sequences with each of the four nucleotides at the cut site (position ) among the gfp or sox specific peaks. e) percentage of sequences surrounding putative cut sites that contain at least one oligo-a stretch within the nt upstream of the cut (for d and e, sox: n = , gfp: n = ). for all panels in this figure, the shared cut sites were determined based on a scanning window of nt, a multiplicative factor of and a confidence level of . %. all cut sites with the same exact position in both sox or both gfp samples that were > nt away from an annotated transcription start site and had sufficient surrounding sequences within the same annotated exon ( nt in a, nt in b and nt in c and d) were included in these analyses. the varying number of sequences used for the analyses in the different panels is a result of the requirement for sufficient flanking sequences in the same annotated exon, but as many sequences as possible were analyzed in each case. doi: . /journal.ppat. .g fig . the sequence features at the sox cut site in limd and srsf , as well as a structural element around the site, are required for sox cleavage. a) predicted structure of the nt surrounding the sox cut site in limd , highlighting the a trimer (asterisk) and the cut site (arrow), as well as the ends of the nt, and nt insertions used in d. b) rnafold was used to predict the structure of all -nt sequences surrounding sox-specific and gfpspecific cut sites (based on a scanning window of nt, a multiplicative factor of and a confidence level of . %). the proportion of cut site where the location of the cut was predicted to be unpaired, that had an unpaired a dimer within nt of the cut or both is plotted. c-g) gfp reporters were co-expressed with sox ("+") or an empty vector control ("-") in shxrn -treated cells. the gfp mrna was detected using northern blotting. the empty arrowheads point to the additional cleavage fragment resulting from insertions, whereas the filled arrowheads point to the normal gfp cleavage fragment. c-d) - nt surrounding the sox cleavage site in the limd mrna were inserted into the gfp reporter at nt . in the nt Δa construct, one of the three as found at positions - to - from the cut site was deleted. a representative blot is shown (c), as well as the quantified intensity of the signal from the different rna species (d), plotted relative to the intensity of the bands from the nt insertion construct. error bars = standard deviation, ** p < . , *p < . (student's t-test). e) the a trimer preceding the sox cut site in limd was mutated to a c, g or t trimer in the limd nt insertion construct. images are representative of results from at least three experimental replicates. f) the a immediately ' of the sox cut site in limd was mutated to a g in the nt insertion construct. g) the a immediately ' of the sox cut site in srsf was mutated to a g (a ! g) and the a dimer preceding the sox cut site was mutated to a c dimer (aa ! cc) in the nt insertion construct. fig . a degenerate motif defines sox cut sites. a position weight matrix (pwm) for nucleotide likelihood in the nt surrounding the sox cut sites was generated from the sequences that contained sox-specific sites with a confidence level of . %. sequences were scored using this matrix, after removing the "parent" sequences where applicable. a) frequency distribution histogram of log likelihood scores for the nt surrounding the gfp-or sox-specific cut sites. gfp: n = ; sox: n = . b) frequency distribution histogram of log likelihood scores for the nt surrounding all cut sites found in the two gfp and sox samples. gfp rep : n = ; gfp rep : n = ; sox rep : n = ; sox rep : n = . c) all human and kshv annotated precise sequence composition may vary, there is a specific element marking sox cut sites that is not observed in control samples. furthermore, our stringent analysis parameters have likely resulted in an underestimation of the number of true sox targeting sites, because even sites detected in only one of our two replicates were generally a good match to the sox targeting motif. we next used the pwm to test whether the prevalence of the targeting element differed between the human and viral mrna transcriptomes. we analyzed annotated transcripts with a nt scanning window, computed a log likelihood score for every possible nt sequence, determined the highest possible motif score for each transcript and plotted the distribution of the scores. in agreement with the widespread mrna cleavage by sox, most of the annotated human transcripts have at least one sequence that is a good match to the motif (log likelihood score > , fig c) . the prevalence of high scoring sequences may explain how sox is able to degrade most transcripts. moreover, we found that kshv transcripts also contained sequences that matched the motif (fig c) , which suggests that sequence specificity is not used by sox to discriminate between host and viral mrnas. this results is consistent with findings from the related gamma-herpesvirus mhv [ ] that show degradation of viral transcripts by proteins of the sox family. we have listed examples of human and kshv rnas with high log likelihood scores in s table. interestingly, within human transcripts, coding rnas tended to contain sequences that were slightly better matches to the motif than non-coding rnas (ncrnas, fig d) , as did spliced rna in comparison to non-spliced rnas (s b fig). the meaning of these small but statistically significant differences is unclear, particularly since the log likelihood scores for all groups were generally high, indicating the presence of good sequence matches to the motif. we also wanted to test whether the sequences around the experimentally determined sox cleavage sites were more likely to match the motif than other locations on the same transcripts. we analyzed the transcripts containing sox cuts sites identified in the pare data using a -nt scanning window as described above. we then ranked the log likelihood scores for all possible -mers in the transcript, and asked how highly ranked the score for the actual cut site was. % of the experimentally observed cut sites ranked within the top % of scores for their rna, indicating that the surrounding sequences were a very good match to the motif compared to other sites in the same mrna ( fig e) . finally, we tested whether the relative degradation of different human transcripts by sox was correlated with how well their sequences matched our degenerate motif. human transcripts were classified as down-regulated by sox or sox escapees based on the data from an rnaseq experiment comparing human mrna levels in cells overexpressing gfp-sox or gfp alone [ ] the best motif score for each rna detected in the rnaseq experiment was then computed using a -nt scanning window as described above. we found that the down-transcripts were scored using the pwm. the frequency distribution histogram for the top scores for each transcript is plotted. d) human transcripts were divided into coding and non-coding based on the annotation in ensembl. the frequency distribution histogram for the top scores for each transcript in the two sets of human rnas is shown. p value (kolgorov-smirnoff test) < . . e) all possible scores for mrnas carrying an observed sox cut site far from the transcription start site (n = , confidence level = . %) were computed and ranked in comparison to all the possible scores for the transcript containing the cut. out of ( %) of the cuts were found at the site with the best score. the cumulative frequency distribution for the percentile of the score at the cut was plotted. f) human rnas were classified into sox targets ("down-regulated rnas") or sox escapees ("escapees rna") based on the results from clyde and glaunsinger [ ] . the frequency distribution histogram for the top scores for each transcript in the two sets is shown. p value (kolgorov-smirnoff test) < . . regulated rnas (fold change < . ) had better motif scores than the escapee rnas (fig f) . similarly, when we plotted the fold change for each gene against the best motif score for that gene, we found that there was a modest but significant inverse correlation between the fold change in mrna levels and the motif scores (spearman's ρ = - . . p value < . , s c fig) . these analyses suggest that the level of down-regulation of mrnas by sox is in part determined by the degree to which their sequence is a good match for the sox targeting motif. we have identified key sequence features of the targeting element that directs the rna endonuclease sox to cleave a significant fraction of the mrna transcriptome. as we had hypothesized from the analysis of individual reporter mrnas [ ] , sox cleavages in endogenous mrnas occur in a sequence-specific manner. although surprisingly large, this element is not defined by strong sequence consensus, but instead contains a small number of conserved residues. structural features may also contribute to motif-driven sox targeting. our data resolve how a sequence-specific nuclease can target such a breadth of targets. sox presents a model of rna targeting in which cleavages are at the same time sequence specific and highly promiscuous. this is achieved through the use of a degenerate sequence/structure pattern that is anchored by key residues to define specific rnase targeting locations. good matches for a loosely defined sequence pattern can be found in all viral and host rnas, enabling cleavage to be simultaneously specific and widespread. although this approach may be less efficient than the location-driven targeting of the cap-proximal region reported for other host shutoff factors that promote mrna cleavage [ , ] , such a mechanism may provide more regulatory opportunities. also, it may explain why sox has less dramatic effects on rna than other viral rnases [ ] . many cellular endonucleases have few described targets and transcriptome-wide targeting analyses of other cellular and viral rnases are limited. it will thus be of interest to apply high-throughput sequencing approaches to isolate degradation fragments in other systems and investigate whether any other viral or cellular rnases use principles similar to those employed by sox to achieve target specificity. notably, recent studies of specificity of rnase l, a host rnase that is activated in response to viral infection and cleaves viral rnas and host rrnas, have also suggested that a combination of sequence and structure is important for targeting [ , ] . however, the requirements for rnase l targeting appear less stringent than those for sox, as the preferred cleavages sites occur at uu/ua dinucleotides in unpaired regions of structured rnas [ , ] . the rna motif underlying sox cleavage specificity could be used for target selectivity, enabling a subset of viral and cellular mrnas to escape cleavage. our observation that the majority of both viral and cellular transcripts contain the sox targeting motif is in agreement with the fact that in mhv , viral mrnas are broadly susceptible to degradation by the sox homolog musox [ ] . however, subsets of viral and cellular transcripts are not susceptible to host shutoff [ , , , ] , and it is likely that at least some of these escape due to the absence of a robust targeting element. indeed, the correlation between the scores of matches to the motif and the level of degradation of host mrnas suggest that sequences within the motif can influence rates of degradation of different rnas. this may result in a more nuanced effect of soxmediated degradation. we also note that the level of degradation seen by steady-state level measurements is likely influenced by additional variables that are unrelated to the efficiency of sox cleavage, including the efficiency of removal of different sequences by xrn and a reduction of transcriptional rate due to a feedback loop triggered by the rna degradation [ ] . therefore the relationship we see may be an underestimation of the contribution of targeting preference. an additional level of regulation for transcript selectivity is also provided by the presence of dominant protective elements like the sox-resistance element we have identified in the ' utr of the il- gene [ , ] , which prevents cleavage of the gfp rna despite the presence of a strong targeting sequence. we expect that both dominant and passive mechanisms of escape from sox-mediated targeting ultimately shape the landscape of host gene expression during sox-mediated host shutoff. a limitation of our analyses is that we are unable to readily explore, both computationally and experimentally, the contribution of structural elements to the sox cleavage site. computational analysis of shared structures is difficult when the sequences involved are not evolutionarily related. moreover, even in well-characterized examples of the same protein binding different rnas, for example in the case of bacterial ribosomal proteins binding both ribosomal rnas and their own ' utrs, the features that are recognized are highly variable [ ] , and the remainder of the structure may serve as a scaffold. nonetheless, our identification of endogenous cut sites makes experimental analysis of a putative sox target structure possible in the future. a major outstanding question is how sox recognizes the targeting motif. in vitro studies indicate that the binding affinity of sox for rna is much lower than its affinity for dna [ ] , which is also processed by sox during viral genome replication in the nucleus. this suggests that sox may not recognize rna targets directly but may instead be recruited by a protein partner. this model is supported by the observation that point mutations that abolish sox host shutoff activity in cells do not affect its rnase activity in vitro [ , ] , pointing at a likely protein-protein interaction. a sox partner protein would have to be a fundamental factor in rna metabolism and/or a rna binding protein with promiscuous specificity, as it must bind a large portion of the cellular rnas. another possibility is that sox directly recognizes its target sequence, and that the apparent low affinity for rna in vitro is due to the fact that a noncognate sequence was used for the binding assay. however, the fact that sox cleaves the gfp rna sequence when gfp is expressed from an rna polymerase ii promoter, but not when it is expressed from an rna polymerase i or iii promoter [ ] argues against this scenario. how this motif potentiates sox targeting, as well as whether it is used as a protein-binding scaffold for other purposes in the cell related to mrna fate remain important questions for the future. cells and xrn knockdown hek t, hek t shxrn and kshv-infected islk- [ ] and islk- shxrn cells were maintained in high-glucose dmem (gibco) supplemented with % fetal bovine serum (hyclone). shxrn cells were generated using ptripz-shxrn (thermoscientific, clone v ths_ /rhs - , targeting sequence: tatggtgagatatactatg). to induce expression of the shxrn , cells were treated with μg/ml doxycycline (fisher) for - days prior to harvesting. lytic induction of kshv was induced in islk- cells [ ] by treating cells with μg/ml doxycycline and mm sodium butyrate for - days prior to harvesting. the same induction also led to anti-xrn shrna expression in the islk- shxrn cells. for the second biological repeat of the pare experiment, cells were transfected twice with the sirna against xrn as previously described [ ] . plasmids pd egfp-n was purchased from clontech. pcdna . -gfp-sox was previously described [ ] . pcdef -sox was previously described [ ] . pd egfp-Δtgaag was previously described [ ] . to test sox-mediated cleavage of endogenous mrna sequences, - nt surrounding putative cleavage sites in the human mrnas to be tested were cloned from hek t cdna using vent polymerase (neb). they were then inserted into the bsrgi site at nt of the gfp coding region in pd egfp-n , as previously done to test gfp sequences [ ] , either by restriction enzyme digest or through a modified version of quikchange mutagenesis [ ] . an ecorv site was also generated at nt of the gfp coding region using quikchange mutagenesis (agilent) and the nt surrounding the cleavage site in limd were inserted at this location. quikchange mutagenesis (agilent) was used to insert out the following mutations: ) mutate the aaa sequence preceding the limd cleavage site to ccc, ggg and ttt in the pd egfp construct containing the nt limd fragment; ) mutate the aa sequence preceding the cleavage site to cc in the pd egfp constructs containing either the nt pgam or the nt srsf fragments; ) mutate the cleavage site from a to g in the pd egfp constructs containing either the nt limd fragment, the nt pgam fragment or the nt srsf fragment; ) mutate the gfp tgaagt to tgagtg. all primers used for cloning are listed in s table. pare library preparation and sequencing hek t cells treated with sirnas against xrn (repeat ) or expressing shrnas against xrn (repeat ) were transfected either with pcdna . -gfp-sox or pd egfp-n . in both cases > % transfection efficiency was observed. one day after transfection total rna was harvested and purified using rnabee (teltest). rna was then treated as described in zhai et al. [ ] to generate pare libraries. briefly, poly(a)+ rna was purified, and rna adapters were ligated to free ' phosphate-bearing rna ends. a second poly(a) purification was used to remove unligated adapter. cdna was synthesized using oligodt directed primers, and the cdna was then amplified times. as the adapter includes an mmei restriction endonuclease site, mmei was used to cut the double stranded amplicons bp downstream of the adapters. ' dsdna adapters were then ligated to the ' end of the amplicons. this created libraries of bp tags corresponding to the ' end of rna fragments flanked by adapters, similar to small rna libraries. libraries were checked on an agilent bioanalyzer and sequenced at the vincent j. coates genomics sequencing laboratory at uc berkeley using a hiseq illumina sequencer. raw data are available on the ncbi gene expression omnibus database as study gse . reads flagged by the casava . program were eliminated and cutadapt [ ] was used to trim away the adapter sequence at the read ' end (sequence: tggaattctcgggtgc-caaggaactccagt). because the pare protocol should produce - nt sequence tags from the ' ends of phosphorylated rna fragments, trimmed reads that were longer than nt or shorter than nt were discarded. the resulting sequences were aligned using tophat . . [ ] using bowtie as recommended for short sequences. no mismatches were allowed (-n option), and only alignments that uniquely mapped to the annotated portion of the genome (-t -x options) were retained, to simplify downstream analysis. for the alignment and subsequent analyses, grch and the ensembl annotation for this genome build were used. these and other analysis were carried out on an imac computer (mid model, . ghz intel core i , gb ram). a bayesian probability framework was used to find peaks that were specific to test samples compared to control samples, which takes into account random variations in the observed number of reads. at a given location and a given experiment, we assume that there is an underlying rate at which reads are produced, and the observed count follows a poisson distribution with mean equal to this rate. in both the control and test data sets, we find that the frequency of reads per location follows a power-law distribution, as is typical for gene expression and deep sequencing data [ , ] , and we therefore assume that the prior distributions for the underlying rates follow this powerlaw distribution, where the power is fitted from the data. at a given location, we then use bayes' theorem to construct posterior distributions for the rates, given the observations of the read counts. we then deem that there is a significant difference between the control and test at that location, if the posterior probability of the test rate being a multiplicative factor larger than the control rate exceeds some confidence level. the multiplicative factor (ratio) and confidence level are chosen by the user. the observed counts vary over a large range, from single digits up to values in the millions, and a key feature of the method is that is can effectively deal with these variations within a unified theoretical framework. in practice, for a given control read count, we can compute a threshold for the test read count, beyond which the difference in underlying rates is significant. the software builds a table of the thresholds using bicubic splines so that many locations can be tested efficiently. the peak finding python scripts are attached as s files. parameters were empirically optimized for the analysis so that a scanning window of nt, a multiplicative factor between test/control read counts of and a confidence level of . % or . % were used to output specific peaks. parameters used in the different analyses are specified in the figure legends. after identifying peaks in single test/control comparisons, the peaks found in the biological repeats were compared. for subsequent bioinformatics analysis of sequences only peaks that were found in both biological repeats were used (figs d, b, s c ). sequences surrounding cleavage sites as defined by chromosomal positions were extracted, using the human genome assembly grch build as a reference. as many sequences as possible were used for each analysis. however, because the short reads do not provide information about mrna isoform and splicing, for all sequence analyses only cut sites that had sufficient flanking sequences within the same annotated exon were used. for motif analysis (figs b, c and s a) and rnafold structure prediction (fig b) nt or nt on either side of the cut site were used. for the accessibility computation (fig a) , localfold.pl [ ] , a modification of the rnaplfold algorithm within the vienna rna package (v . . ) [ ] was used using default settings (window = nt and maxspan = nt) and sequences of nt on each side of the cut site were analyzed. the log likelihood of each base at each position was calculated using background frequencies of nucleotides derived from the human cdna list from the ensembl grch build. weblogo [ ] was used to generate a graphical representation of the sequence motif from aligned sequences. to score matches to the motif, a position weight matrix was generated using log likelihoods for positions - to + relative to the cut site using cut sites that were deemed high confidence in our analysis (i.e. identified using a . % confidence level and position at least nt away from an annotated transcription start site). the log likelihood score was then calculated for all sequences surrounding cut sites that were identified in different subsets of the data. the cut sites used to generate the matrix were always eliminated from the sets that were analyzed. to compute the score matches in human and kshv mrna, the log likelihood score for each nt sequence was calculated in all sequences longer than nt listed in the human cdna fasta repository associated with the ensembl grch build or in a kshv mrna fasta-formatted list (compiled using data from arias et al. [ ] ). the highest score was recorded for each mrna. to separate the human rnas into coding and non-coding their ensembl annotation was used. rnas annotated as "protein coding", "nonsense mediated decay" and "non stop decay" were considered coding, whereas rnas annotated as "antisense", "lincrna", "mirna", "snorna", "processed transcript", "unprocessed pseudogene", "pseudogene", "transcribed unprocessed pseudogene", "transcribed processed pseudogene", "processed pseudogene", and "unitary_ pseudogene" were considered non-coding. the ensembl annotation was also used to determine whether the transcripts were spliced. the motif scores for human mrnas detected in clyde and glaunsinger [ ] were also compared to the level of degradation, that is the fold change in steady-state mrna levels between gfpexpressing and gfp-sox-expressing samples in the cited study. for the analysis in fig f, the transcripts were categorized into "down-regulated" (fold change in sox vs. gfp < . ) and "escapees" (fold change in sox vs. gfp > . ). the structure of the sequences surrounding the validated cut sites was predicted using the rnafold webserver (vienna rna package [ ] ). rnafold v. . . [ ] was used to predict structures around all candidate cut sites, and the results were analyzed to determine whether either of the nucleotides at position - and was predicted to be unpaired. they were also analyzed to determine whether they had an a dimer within nucleotides ' of the cut site that was also predicted to be unpaired. custom python . scripts (s files) were used unless otherwise noted. where noted in the figure legends, the kolgorov-smirnoff test was used to determine whether the distribution of scores were significantly different. total cellular rna was isolated for northern blotting using trizol (life technologies). rna was separated on formaldehyde gels ( x mops buffer, . % agarose, . m formaldehyde) in mops buffer ( mm mops (sigma), mm sodium acetate, mm edta, ph . ) and transferred by capillary blotting onto nitrocellulose membrane (bio-rad) using x ssc buffer ( . m nacl, . m sodium citrate, ph . ). northern blots were probed with p-labeled dna probes made using decaprime ii (ambion), against the ' utr of the gfp reporters. blots were imaged using a fujifilm scanner fla- . quantification of the blots was carried out using imagej [ ] . ' rapid amplification of cdna ends (race) was carried out on μg of total rna using the first choice rlm-race kit following manufacturer's protocol (life technologies). ' race primers are listed in s table. protein harvesting and western blotting protein was isolated for western blots in protein lysis buffer ( mm tris ph . , mm nacl, % triton x- ) containing complete edta-free protease inhibitors (roche), separated on sds-page gels run in tris-glycine buffer and transferred onto pvdf membranes (emd millipore). western blots were performed with mouse anti-xrn antibodies (bethyl laboratories or santa cruz biotechnology, : ) or mouse anti-tubulin antibodies ( : , sigma aldrich). secondary antibodies were used at : dilution and purchased from southern biotech. a) the tgaagt sequence at positions - to - relative to the cut site in the gfp rna (see s b fig) was either partially deleted (Δtgaag) or mutated to tgagtg. the wild-type gfp and the two mutated reporters were co-expressed with sox ("+") or an empty vector control ("-") and the gfp mrna was detected using northern blotting. the arrowhead points at the position of the normal gfp cleavage fragment. b) predicted structures (with rnafold) of the nt surrounding the sox cut site in limd , showing the wild-type sequence on the left (" nt") and the mutated sequence lacking one of the as on the right (" nt Δa"). c-d) gfp reporters were co-expressed with sox ("+") or an empty vector control ("-") in shxrn -treated cells. the gfp mrna was detected using northern blotting. the empty arrowheads point to the additional cleavage fragment resulting from insertions, whereas the filled arrowhead in e points to the normal gfp cleavage fragment. the a dimer preceding the sox cut site in pgam was mutated to a c dimer (c) or the a at position was mutated to g (d) in the pgam nt insertion construct. (eps) s fig. additional analyses of prevalence of the degenerate sox targeting motif. a) weblogo [ ] representation of the frequency of each base in the nt surrounding the cut sites found in both sox samples using confidence level setting of . % and excluding sites near an annotated transcription start site (n = ). b) human transcripts were divided into "spliced" and "not spliced" based on the annotation in ensembl. the frequency distribution histogram for the top scores for each transcript in the two sets of human rnas is shown. p value (kolgorov-smirnoff test) < . . c) the fold change in mrna levels in sox-expressing vs. control cells from clyde and glaunsinger [ ] is plotted against the best motif score for that gene. spearman's ρ = - . , p < . . (eps) s table. number of reads obtained from pare. % mapping (no restrictions) indicates the percentage of reads that map to the human genome if the requirement for unique mapping to a previously annotated region of the genome is removed. (docx) s table. number of peaks detected. the number of peaks detected by using each of the samples as test or control in the pydegradome program (and plotted in fig a) is listed. parameters used for this analysis were a scanning window of nt, a multiplicative factor of , a confidence level of . %. (docx) s table. sox cut sites identified by our analysis. this table lists sox cut sites identified in both replicates with confidence level of . % or in one with confidence level . % and in the second with confidence level . %, and that were nt apart in the two replicates. the table includes the chromosomal position of the cut site, the read count at the cut site in each replicate, the gene name and the confidence level setting used for the identification. it also indicates whether the cut site could be a transcriptional start site (tss) and whether the cut site was used for the analyses in figs and or to generate the pwm for analyses in fig . only cut sites identified in both replicates with confidence level . % were used for the analyses shown in figs and . (xlsx) s table. list of human and kshv transcripts with highest log-likelihood scores of a match to sox targeting motif. (docx) s table. primers used for cloning and ' race analysis. (docx) s files. compressed archive of scripts required for pydegradome analysis. "readme.txt" file with instructions on how to run the analysis, as well as the two scripts required for the analysis are included in the archive. (zip) s files. compressed archive of scripts used for analyses in the paper. "readme.txt" file with instructions on how to run the analyses, and several scripts used to analyze the data. (zip) emerging roles for rna degradation in viral replication and antiviral defense kshv and the pathogenesis of kaposi sarcoma: listening to human biology and medicine a common strategy for host rna degradation by divergent viruses an overlapping protein-coding region in influenza a virus segment modulates the host response coordinated destruction of cellular messages in translation complexes by the gammaherpesvirus host shutoff factor and the mammalian exonuclease xrn . renne r, editor highly selective escape from kshv-mediated host mrna shutoff and its implications for viral pathogenesis deep sequencing reveals direct targets of gammaherpesvirus-induced mrna decay and suggests that multiple mechanisms govern cellular transcript escape an rna element in human interleukin confers escape from degradation by the gammaherpesvirus sox a ribonucleoprotein complex protects the interleukin- mrna from degradation by distinct herpesviral endonucleases sars coronavirus nsp protein induces template-dependent endonucleolytic cleavage of mrnas: viral mrnas are resistant to nsp -induced rna cleavage the ul protein of herpes simplex virus mediates selective stabilization or degradation of cellular mrnas the herpes simplex virus ul gene-dependent destabilization of cellular rnas is selective and may be sequence-specific lytic kshv infection inhibits host gene expression by accelerating global mrna turnover host shutoff is a conserved phenotype of gammaherpesvirus infection and is orchestrated exclusively from the cytoplasm host shutoff during productive epstein-barr virus infection is mediated by bglf and may contribute to immune evasion gammaherpesviral gene expression and virion composition are broadly controlled by accelerated mrna degradation host transcript accumulation during lytic kshv infection reveals several classes of host responses characterization of pa-n terminal domain of influenza a polymerase reveals sequence specific rna cleavage decay of endoplasmic reticulum-localized mrnas during the unfolded protein response the herpes simplex virus vhs protein induces endoribonucleolytic cleavage of target rnas in cell extracts a two-pronged strategy to suppress host protein synthesis by sars coronavirus nsp protein smg promotes endonucleolytic cleavage of nonsense mrna in human cells construction of parallel analysis of rna ends (pare) libraries for the study of cleaved mirna targets and the rna degradome rapid construction of parallel analysis of rna end (pare) libraries for illumina sequencing diverse endonucleolytic cleavage sites in the mammalian transcriptome depend upon micrornas, drosha, and additional nucleases human nonsense-mediated rna decay initiates widely by endonucleolysis and targets snorna host genes identification of smg cleavage sites and a preferred rna cleavage motif by global analysis of endogenous nmd targets in human cells global or local? predicting secondary structure and accessibility in mrnas viennarna package . ribonuclease l and metal-ion-independent endoribonuclease cleavage sites in host and viral rnas rnase l targets distinct sites in influenza a virus rnas viral nucleases induce an mrna degradation-transcription feedback loop in mammalian cells bacterial rna motif in the ' utr of rpsf interacts with an s :s complex crystal structure of a kshv-sox-dna complex: insights into the molecular mechanisms underlying dnase activity and host shutoff the exonuclease and host shutoff functions of the sox protein of kaposi's sarcoma-associated herpesvirus are genetically separable generation of a doxycycline-inducible kshv producer cell line of endothelial origin: maintenance of tight latency with efficient reactivation upon induction integration of pcr fragments at any specific site within cloning vectors without the use of restriction enzymes and dna ligase cutadapt removes adapter sequences from high-throughput sequencing reads tophat : accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions zipf's law in gene expression methods for analyzing deep sequencing expression data: constructing the human and mouse promoterome with deepcage data weblogo: a sequence logo generator kshv . : a comprehensive annotation of the kaposi's sarcoma-associated herpesvirus genome using next-generation sequencing reveals novel genomic and functional features nih image to imagej: years of image analysis we thank albert tai at the tufts genomics core facility for discussion on the analysis, rachel brem and members of the brem lab (buck institute of aging research) and glaunsinger lab for helpful discussions. we thank alicia bicknell and members of the glaunsinger lab and gaglia lab for critical reading of the manuscript. conceived and designed the experiments: mmg. performed the experiments: mmg. analyzed the data: mmg chr bag. contributed reagents/materials/analysis tools: chr. wrote the paper: mmg bag. key: cord- -kkhuzf p authors: sharif-yakan, ahmad; kanj, souha s. title: emergence of mers-cov in the middle east: origins, transmission, treatment, and perspectives date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: kkhuzf p nan two years have passed since the initial description of the middle east respiratory syndrome coronavirus (mers-cov), yet the epidemic is far from being controlled. the high case fatality rate, the recent steep increase in reported cases, and the potential to cause a global pandemic during the upcoming hajj season are serious concerns. although a wealth of information about the pathophysiology, proposed animal reservoir, and intermediate host has been revealed, many questions remain unanswered. we herein review mers-cov, covering its proposed origins, route of transmission, treatment options, and future perspectives. first reported in [ ] , middle east respiratory syndrome coronavirus (mers-cov) is a novel coronavirus and the first lineage c betacoronavirus known to infect humans [ ] . with a case fatality rate of %, an urgent response is needed to prevent a global pandemic [ ] . prior to , coronaviruses were not considered serious human pathogens since they only caused mild upper respiratory tract infections (urtis) [ ] . the first zoonotic introduction of a coronavirus into the human population occurred with the severe acute respiratory syndrome coronavirus (sars-cov) in . sars-cov caused a global pandemic, with , recorded cases and deaths [ ] . mers-cov marks the second known zoonotic introduction of a highly pathogenic coronavirus, probably originating from bats. three lines of evidence currently support this theory: ( ) the very close phylogenetic similarity with the bat betacoronaviruses: btcov-hku and btcov-hku [ ] ; ( ) closely related coronavirus sequences have been recovered from bats in africa, asia, the americas, and eurasia; and ( ) mers-cov uses the evolutionary conserved dipeptidyl peptidase- (dpp ) protein in pipistrellus pipistrellus bats for cell entry [ ] . since human-bat contact is limited, camels have been implicated as probable intermediate hosts. mers-cov appears to have been circulating in dromedary camels for over years [ ] . mers-cov uses the dpp (cd ) receptor to gain entry and effectively replicate in camel cell lines [ ] . neutralizing antibodies for mers-cov have been detected in dromedary camels from oman, canary islands, egypt, jordan, united arab emirates, and saudi arabia [ , ] . the exact mode of transmission from camels to humans remains to be confirmed [ ] . camel milk was investigated as a possible route of transmission, given the common practice of consuming camel milk in the arabian peninsula. the first reported case of mers-cov in yemen occurred in a yemeni pilot who consumed raw camel milk [ ] , and reusken et al. reported the finding of mers-cov in camel milk in qatar [ ] . however, respiratory transmission is currently considered as the most likely route of transmission [ ] . mers-cov has been detected by reverse transcription pcr (rt-pcr) from the nasal swabs of three camels in qatar and was linked to two confirmed human cases with high similarity upon sequencing, suggesting a possible respiratory mode of transmission [ ] . several clusters of mers-cov cases have been reported, mainly among household members and health care workers (hcws), suggesting that transmission is through close contact. the largest cluster reported so far has been in hcws in a hospital in al hasa, kingdom of saudi arabia (ksa), while the largest family cluster has been in three infected brothers from riyadh, ksa [ , ] . the basic reproductive rate for mers-cov has still not been determined with certainty [ ] . using two transmission scenarios, breban et al. reported an r of . and . [ ] . cauchemez et al. reported a similar r at . , but warned that in the absence of infection control measures, r may range from . - . and could lead to a self-sustaining transmission [ ] . propensity for the mers-cov to replicate in the lower respiratory tract may account for the observed limited transmission [ ] . the united states centers for disease control and prevention (cdc) recommends standard contact and airborne precautions with the use of an n- mask when caring for an infected patient [ ] . the cdc defines a laboratory-confirmed case of mers-cov as a patient with a positive pcr from a respiratory sample, and a probable case as a patient who had close contact with a confirmed case but inconclusive laboratory evidence [ ] . the incubation period for the presentation of mers-cov symptoms is - days and it remains unknown whether patients are infectious during the incubation period [ ] . the average age of presentation is years, with a male predominance [ ] . clinically, mers-cov causes symptoms of upper and lower rtis [ ] . the severity of symptoms varies widely. most asymptomatic cases have been discovered through screening after contact with a known case [ ] . presenting signs and symptoms may include highgrade fever, non-productive cough, dyspnea, headache, myalgia, nausea, vomiting, and diarrhea that may precede the respiratory symptoms [ , ] . renal failure has been frequently reported, yet no conclusive evidence of a direct viral invasion of renal tissues exists [ , , ] . notably, most patients who developed complications had coexisting medical co-morbidities [ ] . laboratory findings on admission may include leukopenia, lymphopenia, thrombocytopenia, and elevated lactate dehydrogenase levels [ ] . mers-cov can also cause severe pneumonia with acute respiratory distress syndrome (ards), requiring mechanical ventilation and intensive care admission [ ] . to date, there is still a lack of surgical and pathological information from patients infected with mers-cov, which hampers full understanding of the pathogenesis. lastly, coinfection with other respiratory viruses and with community-acquired bacteria has been also reported in mers-cov patients [ , , ] . as of june , , who officially reported affected patients in countries in three continents. two-hundred and fifty-two patients have died of mers-cov, setting the case fatality rate at % [ ] . the cases so far have been acquired either directly through a probable zoonotic source, or as a result of human-human transmission via close contact. retrospective analysis tracked the first outbreak to a hospital in the city of al-zarqa in jordan in april [ ] . an unexplained observation has been the seasonal variation in reported numbers, with a peak between april and june of each year. the number of cases reported during april alone was alarming, because it was greater than the cumulative number of cases reported since the outbreak began [ ] . the recent increase in the number of infected patients may arguably be attributed to better case detection and active surveillance programs. yet other factors may have contributed to the observed surge, including suboptimal infection control practices in affected hospitals in saudi arabia, as documented in a recent report of the who mission to jeddah [ ] . another explanation for the seasonal variation may be that it coincides with camel birthing season, and younger camels seem to be more often infected than their older counterparts [ ] . the distribution of the total reported cases by country is as follows: . % in the ksa, . % in the united arab emirates, . % in jordan, and % in qatar [ ] . cases have also been reported in kuwait, yemen, oman, iran, lebanon, tunisia, algeria, bangladesh, malaysia, france, italy, germany, the netherlands, united kingdom, greece, italy, and the united states [ ] . furthermore, seropositive camels for mers-cov were detected in egypt, kenya, nigeria, tunisia, and ethiopia, suggesting that there may be mers-cov cases that are undetected in africa [ , , ] . in , , , muslims from countries gathered in mecca to perform the annual hajj, the largest gathering of muslims in the world. the identification of mers-cov in saudi arabia has generated international concern of a global pandemic. as a response, the saudi government requested that elderly and chronically ill muslims avoid hajj in and restricted the number of pilgrims to , , . consequently, no cases were reported during the pilgrimage of that year [ ] . nasopharyngeal specimens collected from , pilgrims revealed no cases of mers-cov nasal carriage [ ] . a prospective cohort study of french pilgrims did not reveal any mers-cov cases [ ] . nevertheless, the potential for spreading of mers-cov during the hajj season (october - ) remains possible, especially since documented transmission occurred this year in patients from iran and malaysia after their return from umrah in mecca. it is worth noting that the two most frequently visited cities during the hajj, mecca and medina, have so far reported and cases respectively [ ] . mers-cov binds to the dpp (cd ) surface receptor using the spike (s) surface protein with subsequent cell entry [ ] . the exact mechanism of entry after receptor binding is still unknown. the s surface protein is composed of a core subdomain that shares similarity with that of sars-cov and a receptor binding subdomain (rbsd) that exhibits significant variation from the sars-cov rbsd. the development of vaccines targeting the rbsd of mers-cov is currently under investigation because they are thought to be safer and more effective than vaccines based on inactivated virus, dna, or viral vectors [ ] . another potential therapeutic approach is the inhibition of the papain-like and/or c-like protease of mers-cov [ ] . to date, no effective therapy or prophylaxis for mers-cov exists. supportive therapy remains the cornerstone of management. current treatment is based on previous experience with the sars-cov, in-vitro studies, and case series. various agents have been tried, including those that block virus entry, inhibit viral replication, or interfere with host immune response [ ] . the international severe acute respiratory and emerging infection consortium (isaric) suggested therapeutic options for treatment of mers-cov infection with various agents alongside continuous evaluation of efficacy, and in the setting of clinical trials [ ] . based on experience with sars-cov, the use of convalescent plasma, hyper-immune globulin, or human monoclonal antibodies that contain neutralizing antibodies may be efficacious and is recommended as first-line treatment when available [ ] . ribavirin and interferon alpha- b both showed promising results, especially when used in combination, both in vitro and in animal studies using rhesus macaques monkeys [ ] . however, these positive results did not translate clinically in an observational study in five patients, all of whom succumbed to the infection [ , ] . repurposing of currently available agents may be an efficient approach. dyall et al. screened various agents with potential therapeutic efficacy [ ] . cyclosporin a, mycophenolic acid, interferon-beta, homoharringtonine, cycloheximide, anisomycin, and emetine dihydrochloride hydrate were found to have the most potent in vitro activity against mers-cov. despite the progress in our understanding of mers-cov, many questions remain unanswered. the definitive origin, exact mechanism of transmission, and the reason behind seasonal variability are still unclear. although most cases have been described in countries of the arabian peninsula, the increasing travel to the region and the hajj season in ksa pose a threat of a potential global pandemic. extensive efforts are required to speed up the development of an effective therapy and vaccine. repurposing of currently available pharmaceutical agents is highly desirable for a more rapid drug development. meanwhile, hcws who encounter patients with respiratory symptoms who have lived or traveled to areas with mers-cov should have a low threshold to consider a diagnosis of mers-cov, with testing and immediate implementation of proper infection control practices to prevent further spread. finally, given the important role that camels may play in transmission of the virus, the common practices in the arabian peninsula of herding and consuming unpasteurized camels' milk should be discouraged until conclusive evidence is obtained that such practices do not contribute to infection. isolation of a novel coronavirus from a man with pneumonia in saudi arabia mers: emergence of a novel human coronavirus middle east respiratory syndrome coronavirus (mers-cov) summary and literature update as of identification of a novel coronavirus in patients with severe acute respiratory syndrome consensus document on the epidemiology of severe acute respiratory syndrome (sars) genomic characterization of a newly discovered coronavirus associated with acute respiratory distress syndrome in humans dipeptidyl peptidase is a functional receptor for the emerging human coronavirus-emc antibodies against mers coronavirus in dromedary camels replicative capacity of mers coronavirus in livestock cell lines middle east respiratory syndrome coronavirus neutralising serum antibodies in dromedary camels: a comparative serological study the emergence of the middle east respiratory syndrome coronavirus (mers-cov) middle east respiratory syndrome coronavirus: epidemic potential or a storm in a teacup? global alert and response: middle east respiratory syndrome coronavirus (mers-cov) -update middle east respiratory syndrome coronavirus (mers-cov) rna and neutralising antibodies in milk collected according to local customs from dromedary camels, qatar middle east respiratory syndrome coronavirus (mers-cov) in dromedary camels middle east respiratory syndrome coronavirus in dromedary camels: an outbreak investigation hospital outbreak of middle east respiratory syndrome coronavirus interhuman transmissibility of middle east respiratory syndrome coronavirus: estimation of pandemic risk middle east respiratory syndrome coronavirus: quantification of the extent of the epidemic, surveillance biases, and transmissibility middle east respiratory syndrome coronavirus (mers-cov) causes transient lower respiratory tract infection in rhesus macaques interim infection control and prevention recommendations for hospitalized patients with mers-cov mers case definition mers clinical update from the idsa center for disease control and prevention cdc epidemiological, demographic, and clinical characteristics of cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study clinical features and viral diagnosis of two cases of infection with middle east respiratory syndrome coronavirus: a report of nosocomial transmission family cluster of middle east respiratory syndrome coronavirus infections a patient with severe respiratory failure caused by novel human coronavirus a family cluster of middle east respiratory syndrome coronavirus infections related to a likely unrecognized asymptomatic or mild case clinical features and virological analysis of a case of middle east respiratory syndrome coronavirus infection epidemiological findings from a retrospective investigation who concludes mers-cov mission in saudi arabia mers-cov enigma deepens as reported cases surge geographic distribution of mers coronavirus among dromedary camels mers coronaviruses in dromedary camels middle east respiratory syndrome coronavirus (mers-cov) summary and literature update as of prevalence of mers-cov nasal carriage and compliance with the saudi health recommendations among pilgrims attending the hajj lack of mers coronavirus but prevalence of influenza virus in french pilgrims after hajj the receptor binding domain of mers-cov: the dawn of vaccine and treatment development proteolytic processing, deubiquitinase and interferon antagonist activities of middle east respiratory syndrome coronavirus papain-like protease what are our pharmacotherapeutic options for mers-cov? international severe acute respiratory & emerging infection consortium (isaric)-clinical decision making tool for treatment of mers-cov v. . an animal model of mers produced by infection of rhesus macaques with mers coronavirus ribavirin and interferon therapy in patients infected with the middle east respiratory syndrome coronavirus: an observational study repurposing of clinically developed drugs for treatment of middle east respiratory coronavirus infection the authors would like to acknowledge sima l. sharara for editing the manuscript. key: cord- -w cn iqp authors: earnest, james t.; hantak, michael p.; li, kun; mccray, paul b.; perlman, stanley; gallagher, tom title: the tetraspanin cd facilitates mers-coronavirus entry by scaffolding host cell receptors and proteases date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: w cn iqp infection by enveloped coronaviruses (covs) initiates with viral spike (s) proteins binding to cellular receptors, and is followed by proteolytic cleavage of receptor-bound s proteins, which prompts s protein-mediated virus-cell membrane fusion. infection therefore requires close proximity of receptors and proteases. we considered whether tetraspanins, scaffolding proteins known to facilitate cov infections, hold receptors and proteases together on cell membranes. using knockout cell lines, we found that the tetraspanin cd , but not the tetraspanin cd , formed cell-surface complexes of dipeptidyl peptidase (dpp ), the mers-cov receptor, and the type ii transmembrane serine protease (ttsp) member tmprss , a cov-activating protease. this cd -facilitated condensation of receptors and proteases allowed mers-cov pseudoviruses to enter cells rapidly and efficiently. without cd , mers-cov viruses were not activated by ttsps, and they trafficked into endosomes to be cleaved much later and less efficiently by cathepsins. thus, we identified dpp :cd :ttsp as the protein complexes necessary for early, efficient mers-cov entry. to evaluate the importance of these complexes in an in vivo cov infection model, we used recombinant adenovirus (rad ) vectors to express human dpp in mouse lungs, thereby sensitizing the animals to mers-cov infection. when the rad -hdpp vectors co-expressed small rnas silencing cd or tmprss , the animals were significantly less susceptible, indicating that cd and tmprss facilitated robust in vivo mers-cov infection of mouse lungs. furthermore, the s proteins of virulent mouse-adapted mers-covs acquired a cd -dependent cell entry character, suggesting that cd is a selective agent in the evolution of cov virulence. introduction enveloped virus-cell entry requires glycoprotein-catalyzed fusion of viral and host cell membranes. these viral fusion glycoproteins are catalytically-inactive on virus particles and become triggered to mediate membrane mergers only in response to cellular and environmental factors. this triggering process ensures that virus-cell entry occurs at the appropriate time and place. the triggering factors include host cell receptors, endosomal acids, and proteases. many viruses require a single, soluble trigger, for example, influenza a virus fusion proteins are triggered by protons within the target-cell endosome [ ] . other viruses require two triggering agents, for example, avian sarcoma leukosis virus fusion proteins are partially advanced into fusion-catalyzing forms after binding to host cell receptors, and then fully execute fusion after being exposed to endosomal protons [ ] . most covs also require two triggering agents, receptor binding and proteolytic cleavage, with the proteolysis taking place on receptor-bound viral ligands [ ] . as many of the cov-cleaving proteases are transmembrane-anchored, it follows that cov-susceptible cells might have the two triggering agents, receptors and proteases, in close proximity. here we considered whether the two cov entry factors are coalesced on cell surfaces to facilitate infection, and whether particular host cell features are required to juxtapose the two entry factors. the cov receptors are all transmembrane glycoproteins. their presence is a defining feature of host cell susceptibility to infection [ ] [ ] [ ] [ ] . proteases, the second required determinants of host susceptibility, are variable in type and subcellular location [ ] , with proteases in the type ii transmembrane serine protease (ttsp) family figuring prominently [ ] [ ] [ ] . ttsp family members, most notably the transmembrane protease serine type (tmprss ), can cleave cov fusion glycoproteins (termed spike [s] proteins), into unlocked, fusion-catalyzing forms [ , , ] at the cell surface and facilitate a rapid, "early" entry. studies examining hiv [ ] and influenza [ ] glycoproteins indicate that multiple adjacent fusion glycoproteins must be activated in order to successfully complete the fusion reaction. assuming similar requirements for cov fusion, it is likely that multiple s proteins need simultaneous receptor engagement and sufficient proteolytic cleavage to form an activated cluster that can pull opposing membranes together. thus, fusion likely occurs in regions of the cell membrane with a relatively high local concentration of these entry factors. recent studies have confirmed that ttsps are concentrated into punctate locations on the cell surface, in association with tetraspanin scaffolding proteins [ ] . tetraspanins comprise a family of proteins with four transmembrane spans and two extracellular loops [ ] . tetraspanins interact with other tetraspanins [ ] and with other membrane-associated proteins [ , ] , including transmembrane proteases [ , ] , to form "webs" of interacting proteins [ ] . there is evidence that these tetraspanin webs are locus points for cov-cell entry, as tetraspanin-specific antibodies protect several cell types from cov infection [ ] . however, it remains unclear if individual tetraspanin proteins facilitate cov entry and what function they have in determining viral entry routes. as there are demonstrations that the tetraspanin cd interacts with the mers--cov receptor dipeptidyl peptidase (dpp ) [ , ] and hints of similar cd interactions with the hcov- e receptor aminopeptidase n (apn) [ ] , we hypothesized that cd is necessary to bring these virus receptors to ttsp-enriched regions on the cell surface. no study to date has determined the relative importance of individual tetraspanins and ttsps to mers-cov infection in the lung environment. indeed, there are human tetraspanins and at least members of the ttsp protease family [ ] as well as several soluble extracellular proteases, such as elastases [ ] , that may be expressed in the lung parenchyma. while studies suggest that tmprss can trigger mers-cov in cell culture [ , ] , it is unclear whether cd or tmprss stand out in vivo as single proviral members of their respective protein families. therefore, we set out to determine whether, and to what extent, mers-cov utilizes cd and tmprss during in vivo infection. to this end, we established a mouse model in which virus-resistant mice are rendered susceptible to mers-cov infection by expression of human dpp (hdpp ). the system utilizes a recombinant adenovirus type (rad ) to transduce the hdpp gene, thereby sensitizing only the ad -transduced lung cells to subsequent mers-cov infection [ ] . the rad -hdpp vectors were engineered to include additional genes encoding the potential virus-promoting factor human tmprss [ ] or potential virus-restricting factors, in the form of shrnas targeting murine tmprss and cd . we considered the rad -hdpp system to be especially valuable, as mers-cov infection can only occur in cells expressing hdpp and, thus, only in cells simultaneously expressing the putative virus-promoting or virus-restricting factors. using the dual-expressing rad vectors, as well as tetraspanin knock-out cell lines, we evaluated the roles for cd and another related tetraspanin, cd , in dictating receptor clustering with proteases and in promoting cov infection. our results indicate that a cov-cell entry portal is a multipartite complex that minimally includes the virus receptor, a virus-activating protease, and one or more tetraspanins. these complexes are responsible for the majority of mers-cov entry in lung cells. furthermore, cd facilitated cell entry by mers-cov spikes that were adapted for lung virulence, but cd provided no support to cell culture-derived, avirulent spike-mediated cell entry. these data establish tetraspanins as factors controlling early entry events in pathogenic cov infections. tetraspanins cd and cd are known to influence enveloped virus entry [ , , ] . therefore, we used crispr/cas technology [ ] to eliminate these tetraspanins from cells, with the expectation that this would affect cell susceptibility to covs. t and hela cells were transfected with cas /guide rnas targeting cd or cd , selected for puromycin resistance, and cloned by endpoint dilution. all ko cell lines grew equivalent to parallel "wt" control clones, and the only observable distinctions were with the cd ko cells, which adhered less tightly to plastic than wt or cd ko cells. western blot analyses of the wt and ko clones confirmed the absence of cd or cd , with maintenance of a control tetraspanin cd (fig a) . interestingly, cd levels were highest in cd ko cells and cd levels were low in cd ko cells, possibly due to heterotypic cd -cd interactions influencing tetraspanin stability. lower-resolution immunofluorescent assays (ifas) of umpermeabilized cells showed similar cell-surface cd in wt and cd ko cells, confirmed the absence of the respective tetraspanins in ko cells, and demonstrated that cd distribution remained unchanged in all cell lines ( fig b) . to determine whether the tetraspanins cd or cd operate in cov entry, we utilized hiv pseudoparticle (pp) transduction methodologies, which allow for a specific focus on the viruscell entry stage. we first sensitized the cells to transduction by overexpressing cov receptors, then transduced cells with the respective covpps. relative to wt cells, cd ko cells were % less susceptible to mers (emc strain) pp transduction (fig c) , and % less susceptible to epp transduction ( fig d) . however, cd ko cells remained fully susceptible to sarspp or mhvpp transduction (fig e and f ). cd complementation restored susceptibility to merspp and epp transductions (fig c and d ). cd ko cells were fully susceptible to all four of the covpps (s fig) . these data identify an individual tetraspanin, cd , as an entry factor for a cov. to determine whether receptor overexpression might have contributed to cd dependence, merspps were also transduced into cells containing endogenous cov receptor levels. consistently, cd was necessary to fully sensitize cells to merspps (s fig), indicating that cd proviral activity was independent of hdpp receptor levels. however, cd was not necessary for merspp transduction into cells overexpressing tmprss , a mers-cov activating protease (s fig). the fact that tmprss obviated the cd requirement indicated a role for cd in proteolytic activation of cov entry. the observation that a single tetraspanin family member, cd , promoted cell entry for some, but not all covs, suggested that cd interacts with one or more mers-cov and e-cov entry factors. we considered whether cd associates with dpp and apn, the mers-cov and e-cov receptors, or with tmprss . furthermore, we considered whether cd does not interact with ace and ceacam, the receptors for cd -independent sars and mhv-covs. this was first investigated through biochemical isolation of tetraspanin-enriched membrane fractions, and detection of tetraspanin-associated receptors and proteases. to this end, cd or cd ko cells overexpressing cov receptors or tmprss were surface-biotinylated, and tetraspanins were liberated from cells using zwitterionic chaps detergent, which solubilizes cell membranes while leaving tetraspanin-mediated protein interactions largely intact [ ] . low-density (ld) fractions, with ρ< . g/ml, were then separated from high-density (hd) chaps-solubilized proteins on sucrose density gradients [ ] . as evaluated by streptavidin pull-down and western immunoblotting, the ld sucrose gradient fractions from chapssolubilized cells contained nearly % of cell-surface tetraspanins (s fig) , but only~ % of the surface-biotinylated plasma membrane proteins [ ] , indicating efficient tetraspanin segregation into ld fractions. strikingly, the ld fractions from wt control cells contained~ % of cell-surface dpp , while ld fractions from cd ko cells completely lacked dpp (fig a, rows and ). complementing cd back into cd ko cells restored ld-associated dpp (fig a, row ) . the presence or absence of cd had no effect on dpp distribution between hd and ld fractions (fig a, rows and ). similar results were observed with the e receptor apn (fig b) . by contrast, cd and cd expression had little effect on the distribution of ace , ceacam, or tmprss , all of which distributed about equally between ld and hd fractions (fig c- e ). these data indicated that dpp and apn positioning into tetraspanin-enriched membranes required cd . the fact that cd repositioned dpp and apn, but not ace or ceacam, correlated with the fact that cd promoted the entry of dpp and apn-utilizing mers and e viruses, but not ace -or ceacam-utilizing sars and mhv viruses (fig ) . the evaluations of membrane fractions suggested that cd might localize the mers-cov and e-cov receptors close to virus-activating tmprss . to determine whether cd facilitates specific interactions between dpp and tmprss , we analyzed intact tetraspanin microdomains in situ. we performed proximity ligation assays (plas), which can determine whether two or more transmembrane proteins are adjacent [ ] . in plas, antibodies differentially tagged with oligonucleotide probes are applied to cells, and their close spacing (< nm) allows for probe hybridization into dna polymerization templates, which provide a locus point for fluorescent dna synthesis [ ] . plas have been used to identify interactions between tetraspanins and their partner proteins [ , ] and we used this method to analyze clustering of two tetraspanin partner proteins. hela cells were chosen for plas because their relatively flat morphology facilitated quantification of fluorescent foci, and because our quantitative reverse transcriptase-pcr measurements revealed endogenous expression of cd , dpp and tmprss (s table) . notably, cd transcripts were plentiful in the hela cells (~ times more abundant than the reporter gene hprt), while dpp and tmprss were scarce (~ times less abundant than hprt, and -to times less than that found in several human airway epithelia-derived cell cultures (see s table) ). thus, we presumed that, with hela cells, we could readily detect a cd -directed coalescence of sparse dpp and tmprss . we performed plas on unpermeabilized cd ko hela cells, using primary antibodies to cd , dpp , and/or tmprss . following secondary antibody incubation and amplification of ligated oligonucleotide templates, punctate fluorescent dnas were detected by confocal microscopy and counted using imaris version . . software. using hdpp and htmprss antibodies, fluorescent foci were rarely observed on the hela-cd ko cells (fig a) , and the cells were only modestly susceptible to merspp transduction ( fig h, leftmost bar) . when cd was replenished in the cd ko cells, foci were~ -fold more abundant (fig d) , and these increased foci correlated with a greater cell susceptibility to merspp transduction ( fig h) . these findings argue that cd sensitizes cells to mers-cov entry by bringing dpp and tmprss into proximity. we considered whether this role for cd applied only when dpp and tmprss levels were low, i.e., at endogenous hela-cell levels. thus, hdpp and htmprss were forcibly overexpressed; with overexpression,~ foci/ cell were observed (fig e) , and this increased to~ foci/cell in the presence of cd ( fig f) . merspp entry into cells correlated with the number of foci present, at least for values up to foci/cell ( fig h) . overall, these results indicated that cd connects dpp and tmprss entry factors, and is necessary for their proximity when they are sparse on cell surfaces. the cd :dpp :tmprss complexes then function as mers-cov entry portals. cd also helped to connect overexpressed dpp and tmprss together, but in this overexpression condition, cd did not increase merspp transduction, perhaps because other tetraspanins come in to bridge the abundant receptors and proteases. these results also revealed cd -directed dpp : tmprss complexes on intact cells in the absence of virus, suggesting that the covs infect through pre-existing complexes. because cd brought dpp in proximity with ttsps, we hypothesized that cd facilitates ttsp-mediated early cell entry at or near plasma membranes, but does nothing to support the late, endosomal route that is enabled by cathepsin proteases. to test this, we inactivated cellular ttsps using camostat [ ] and found that camostat suppressed merspp transduction into wt cells by~ %, but did not affect transduction into cd ko cells ( fig a) . cd complementation modestly restored merspp sensitivity to camostat. furthermore, cd had no effect, as merspp entry into cd ko and cd -positive cells were equally suppressed by camostat ( fig a) . these data were consistent with cd specifically enabling ttsp-directed, early virus entry. without cd , the merspp entry route may be directed to a late, endosomal stage in which cathepsins provide fusion-activating triggers. to test this, we blocked late entry in wt and cd ko with μm bafilomycin a (baf), an inhibitor of endosome acidification, or with μm e d, a cysteine protease inhibitor. in wt cells, baf did not significantly decrease merspp entry, while e d decreased entry~ -fold ( fig b) . however, in cd ko cells, these inhibitors were far more antiviral, decreasing entry -and -fold, respectively. complementing cd back into the cd ko cells restored the wt phenotype in which the inhibitors were only weakly antiviral (fig b) . these differential effects of the inhibitors were not observed in cd ko or cd -overexpressing cells (fig b) . we conclude that cd is necessary for ttsp-mediated mers early entry. we advanced to evaluating mers-cov entry factors in vivo. of note, a previous study has demonstrated that camostat inhibits sars-cov spread in mouse lungs [ ] , suggesting that the virus exhibits dependence on serine proteases, probably ttsps, for its entry in vivo. however, the importance of specific ttsps, or for tetraspanins, is unknown for any in vivo cov infection. here we established infections in the mouse lung under conditions in which putative cov entry factors were reduced. to do this, we developed dual-expressing recombinant adenovirus (rad ) vectors expressing both human dpp , which sensitizes mouse cells to mers-cov infection [ , , ] , and shrnas that knock down tmprss or cd mrnas. in initial experiments, these rad vectors were transduced into mouse lung epithelial type (let- ) cells, a line derived from c /bl mouse alveolar type cells [ ] . after -days, the cells were analyzed for the presence of hdpp , tmprss , and cd by western blot (fig a) . relative to the control rad -gfp transductions, all single and dual-expressing rad -hdpp transductants contained recognizable dpp and tmprss , and those ad vectors expressing shrnas reduced the levels of endogenous cd proteins (fig a) . due to endogenous tmprss protein levels being too low for detection on immunoblots, we used qrt-pcr to quantify tmprss transcripts. let- cells transduced with rad -hdpp -shtmprss had only % of the transcripts of cells transduced with rad -hdpp -empty vector (fig b) . this level of tmprss transcripts indicated an efficient knockdown of tmprss in the approximately % of cells that were successfully transduced. these results indicate that the different rad vectors, transduced into cells derived from mouse alveolar epithelia, consistently express equivalent levels of hdpp , while simultaneously increasing or decreasing tmprss or cd . to determine whether the rad -transduced let- cells were susceptible to mers-cov s protein-directed virus entry, the cells were inoculated with recombinant vsvs encoding firefly luciferase [ ] and pseudotyped with mers-cov s proteins. as expected, hdpp expression established susceptibility to vsv-merspp transduction (fig c) . tmprss co-expression from the ad vectors increased susceptibility to merspps by~ -fold, while shtmprss and shcd both restricted merspps by~ fold (fig c) . these results indicated that cd and tmprss act as entry factors in mouse lung-derived let- cells, and suggested that the dualexpressing ad vectors might be effective tools for identifying viral entry factors in the mouse lung. to identify the role of cd and tmprss in vivo, the ad vectors were instilled intranasally into mice which were, after days, challenged with mers-cov. lungs were harvested days post-infection (d.p.i.) and mers-cov titers were measured as pfu/gram of tissue. relative to mers-cov titers in rad -hdpp transduced animals, the mers-cov titers in rad -hdpp -shcd transduced animals were~ -fold lower (fig d) . furthermore, the mers-cov titers in rad -hdpp -shtmprss transduced mice were reduced by~ -fold. interestingly, overexpression of tmprss by the rad -hdpp -tmprss vector had no effect on mers-cov titers in the lungs, presumably because the lung environment has sufficient endogenous murine tmprss to facilitate efficient mers-cov infection. these data indicate that cd and tmprss act as mers-cov susceptibility factors in the lung parenchyma and that their role in entry is slightly more pronounced in vivo than in in vitro let- mouse alveolar cell cultures. indeed, these data show that cd and tmprss are responsible for~ % of mers-cov titers in vivo. virulent mers viruses utilize cd -dependent early entry mers-cov, a camel and human virus [ , ] , has recently been adapted for robust growth and virulence in hdpp + mouse lungs [ , ] . this adaptation process was initiated by intranasally infecting mice with avirulent, vero cell culture-adapted (cca) mers-covs and then serially passaging viruses through hdpp + mouse lungs. relative to cca mers-covs, the mouse-adapted (ma) viruses have distinct s protein changes [ ] (s table) . we considered whether these ma changes fixed into s proteins adapt viruses to utilize cd -facilitated early entry. to address this question, we produced vsv-based merspps, pseudotyped with the cca or ma s proteins. these cca and ma merspps were transduced into cd -replete or cd -knocked down (cd kd) let- cells. the cd -replete and cd kd cells were equally susceptible to cca s-mediated pp entry. however, the same cd kd cells had % and > % reduced susceptibility to ma and ma s-driven pp entry, respectively (fig a) . thus, it appears that in vivo passage in mouse lungs adapts mers-covs to a cd -facilitated cell entry pathway. the ma viruses' utilization of cd for entry correlated with their relatively rapid entry kinetics [ ] . furthermore, cd -facilitated entry correlates with ttsp utilization (fig ) , and ttsp utilization correlates with rapid cov entry into cells [ ] . therefore, we hypothesized that cd is a determining factor in cov-cell entry kinetics. to test this, cca and ma merspps were transduced into cd -replete or cd kd let- cells. to measure pp entry kinetics, the transduction process was abruptly halted at defined time points with a nontoxic protease inhibitor cocktail that prevents s-directed fusion, but has no effect on transduced reporter gene expression [ ] . this strategy allows fluc reporter accumulations to indicate the extents of virus entry taking place within timed intervals. we found that cd had no influence on the rate of cca s-directed virus entry. in both cd -replete and cd -kd cells, half-maximal entry was complete within min (fig b) . a - min half time for virus entry is found for several viruses requiring endocytosis prior to genome delivery [ ] . however, cd strongly influenced ma s-mediated virus entry. the ma and ma pps reached % entry in cd -replete cells in and minutes, but were delayed to and minutes, respectively, in cd kd cells (fig c and d) . these data were corroborated with the tetraspanin ko cell lines (s fig). thus, we conclude that cd utilization and rapid cell entry correlate with mouse adaptation and mers virulence in mouse lung infections. in this study, we demonstrated that the mers coronavirus enters cells using an entry complex that includes a receptor, a protease and a tetraspanin. the tetraspanin operates by bringing the receptor and protease(s) into proximity, such that viral spikes, once attached to receptors, are quickly and efficiently cleaved into fusion-activated forms. these ternary complexes pre-exist on virus-target cells and can theoretically have highly variable subunit composition. presently, there are three known human cov receptors, human transmembrane serine proteases, and human tetraspanins, and therefore there are thousands of potential combinations of receptor, protease and tetraspanin that might provide a coronavirus entry platform. for the mers coronavirus, a particularly effective complex includes the hdpp receptor, the protease tmprss , and the tetraspanin cd . this was discovered, in large part, through creative use of recombinant adenoviruses (rads). the approaches used here extended from the finding that a transducing rad expressing the mers-cov receptor hdpp sensitized laboratory mice to mers-cov infection [ ] . by incorporating rna silencing genes into rad -hdpp , we were able to simultaneously establish mers-cov susceptibility, through hdpp expression, and potentially restrict mers-cov, through shrna-mediated suppression of candidate proviral factors. thus, the dual-expressing rad vectors revealed cd and tmprss as relevant proviral factors, operating to support the primary hdpp susceptibility factor. it is notable that dual-expressing adenovirus vectors can potentially be utilized to identify any mers-cov host factor. indeed, they can be utilized to identify host factors in any virus-host system that requires an exogenously supplied susceptibility determinant. furthermore, the adenovirus transduction process bypasses the need to establish partially humanized mice for studies of human coronavirus infections, and actually has distinct advantages over transgenic animals, in that shrnas reduce pro-or anti-viral factors solely in coronavirusinfectable cells, making for reliable measurements of changes in virus susceptibility. we expect that dual-expressing rad vectors will be excellent general tools to rapidly identify in vivo proand anti-viral host factors. the hdpp :cd :tmprss complexes promoted an "early" mers-cov entry. cov s proteins require simultaneous receptor engagement and proteolysis before catalyzing virus-cell membrane fusion [ , , ] , a process demanding that tmprss be closely juxtaposed to hdpp . we suggest that cd tetraspanins position tmprss next to the receptor-bound s proteins, perhaps in association with cholesterol, a lipid having profound effects on both tetraspanin structural interactions [ ] and cov entry [ ] [ ] [ ] [ ] [ ] . precisely how tmprss abuts against hdpp to access s proteins is not clear, although the structures of three cov s proteins [ , ] and hdpp [ ] indicate that the proteolytic cleavage sites would be displayed at the outer edges of each s trimer. furthermore, it is likely that proteolytic cleavage of several adjacent s proteins is needed to activate membrane fusion, as cooperative "pulling" by several viral fusion proteins is frequently required for virus entry processes [ , , ] . therefore, the tetraspanin-enriched environment, in which dpp and tmprss are collected together, likely permits rapid and simultaneous cleavage of multiple, closely-spaced virion s proteins, generating clusters of activated s proteins that drive the membrane fusion process. without cd , the hdpp and tmprss are not held closely together (fig ) . in this condition, mers-cov still infects hdpp -positive target cells (fig ) , but it takes a slower "late" endosomal route, which we and others find to be around % less efficient than early entry (figs , and ) [ ] . in the late entry route, virus-associated s proteins are first endocytosed and then cleavage-activated by furin proprotein convertases [ , ] , cathepsin l [ , , , ] , and/or cathepsin b [ ] . however, the protease-enriched endo-lysosomal environment [ ] can also generate inactivating cov s protein cleavages, as evidenced by c-terminal s protein fragments, kda and smaller, that must be inactivated fusion domain fragments [ , ] . therefore, in the late entry route, there may be a short time span between a cathepsin-activated fusogenic state and a permanently inactivated, excessively proteolyzed state, accounting for inefficient entry. inefficient late entry may also be explained by differences in lysosomal and plasma membranes, which have unique lipid profiles [ ] and therefore may be differentially susceptible to s -catalyzed fusion. finally, late entry is restricted by interferon-induced gene products, notably interferon-induced transmembrane (ifitm) proteins [ , ] , but early ttsp-facilitated entry is not [ ] . all of these virus-restricting conditions may combine in vivo to make cd -facilitated "early" cell entry the predominant route for mers-cov infections. that the ttsp-facilitated entry route is predominant in vivo is supported by the recent finding that serine protease inhibitors reduce sars-cov infection in mouse lungs [ ] . additionally, clinical hcov- e isolates use a rapid ttsp-facilitated entry route, unlike labadapted hcov- es [ ] . more recently, similar patterns were observed for mers-covs. mouse lung-adapted mers-covs take a rapid tmprss -mediated cell entry, while cell culture-adapted (cca) mers-covs are avirulent and enter cells through the slower and less efficient endocytic route [ ] . here we demonstrated that the virulent ma mers-cov s proteins utilized cd during cell entry, while avirulent cca viruses did not. this new finding suggests that cov receptors and proteases alone are not the selective agents in cov adaptation. rather, the covs adapt to the ternary receptor-tetraspanin-protease complexes. in the case of mers--cov, the key adaptive s mutation facilitating the usage of the ternary complexes was at position (s table) . in the mers-cov s protein cryo-em structure [ , , ] , this residue is part of a peptide that connects two of the helices comprising the fusion domain. the change from n to t may ease restrictions to conformational change in the s trimer, thereby exposing cleavage sites to the nearby cd -associated transmembrane protease, with cleaved spikes then converting to fusion-active forms. finally, these findings may shed light on general roles for tetraspanins in virology. four cov receptors (dpp , apn, ace , and ceacam) were found in tetraspanin-rich membrane fractions (fig ) , and our previous report indicated that tetraspanin antibodies block several cov infections by interfering with receptor-associated cov access to surface proteases [ ] . even antibodies binding to cd suppressed mers s-mediated entry [ ] , indicating that several tetraspanins, including those that are not required per se for clustering hdpp and tmprss , organize into cell-surface "webs" [ ] and enclose the cov entry factors. here, there may be parallels with several tetraspanin-facilitated viruses, including influenza a (iav) [ ] and canine distemper (cdv) [ ] ; the retroviruses hiv [ , ] , feline immunodeficiency virus (fiv) [ ] , and human t-lymphocytic virus (htlv- ) [ ] ; herpes simplex virus (hsv- ) [ ] ; hepatitis c virus [ ] ; and several human papillomaviruses (hpvs) [ ] . for these viruses, tetraspanins facilitate viral entry (covs, iavs, hcv, hpvs) syncytia formation (cdv, hiv, fiv, htlv- ), or promote viral exit (iavs, hsv- and hiv), by unclear mechanisms. conceivably, a common mechanism may involve tetraspanin-mediated clustering of host factors. for example, tetraspanin cd is both an hcv receptor [ ] and a linker of the hcv co-receptors scavenger receptor class b i (sr-bi) [ ] and claudin- [ ] , whose complexing promotes viral endocytosis (reviewed in [ ] ). another example is with the tetraspanins cd and cd , which do not directly interact with hpvs, but rather hold several coreceptors together to permit hpv binding and endocytosis (reviewed in [ ] . therefore, many of the proviral activities ascribed to tetraspanins may relate to their ability to cluster transmembrane proteins, as we have found for the pro-mers-cov activity of cd . given that several viruses depend on tetraspanin webs, it may be useful to consider ways to target entry-blocking drugs to these locations and thereby increase their antiviral efficacy. c bl/ mice were purchased from the national cancer institute and housed in the animal care facility at the university of iowa. the mers-cov (emc strain) was provided by drs. bart haagmans and ron fouchier (erasmus medical center). hek t and hela cells were obtained from dr. edward campbell (loyola university chicago) and maintained in dulbecco's modified eagle media (dmem) supplemented with % fetal bovine serum (fbs, atlanta biologicals), mm hepes, mm sodium pyruvate, . mm non-essential amino acids, u/ml penicillin g, and μg/ml streptomycin. let- cells were obtained from bei resources and were maintained in dmem supplemented with % fbs, u/ml penicillin g, and μg/ml streptomycin. cells were maintained in a humidified environment at ˚c and % co . hae cultures were isolated and maintained as described previously [ ] . this study was carried out in strict accordance with the recommendations in the guide for the care and use of laboratory animals of the national institutes of health. animal experiments were approved by the institutional animal care and use committee at the university of iowa (protocol # ). codon-optimized mers-cov s containing a c tag was purchased from genscript and subsequently cloned into pcdna . + between the ecori and noti restriction sites. pcdna . - e-spike-c and pcdna . -hapn plasmids were provided by dr. fang li, (university of minnesota). pcdna . -sars-spike-c and pcdna . -ace -c plasmids were provided by dr. michael farzan (scripps research institute). c-terminal flag-tagged human dpp plasmid pcmv -entry-hdpp (ncbi reference sequence nm_ ) was purchased from ori-gene. pcaggs-tmprss -flag was previously constructed [ ] . the pnl . -hivluc plasmid was provided by the nih aids research and reference library. pcmvsport -hcd was purchased from open biosystems. pspcas -bb- a-puro was a gift from feng zhang (addgene plasmid # ). monoclonal mouse antibodies against cd (clone m-l ), cd (clone h c ), and cd (clone js- ) were obtained from bd pharmingen. rabbit anti-flag was obtained from sigma aldrich. mouse anti-rhodopsin antibodies were obtained from millipore. rabbit anti-cd (apn) antibodies were obtained from abcam. mouse anti-cd (clone m-a ) was obtained from bd biosciences. rabbit anti-tmprss (clone epr ) was obtained from abcam. secondary antibodies were purchased from invitrogen and include goat-anti-rabbit-alexafluor , goat-anti-mouse-alexafluor , and goat-anti-mouse-alexafluor . donkey-anti-goat, goat-anti-mouse, and goat anti-rabbit hrp conjugated antibodies were purchased from thermo scientific. recombinant adenovirus vectors were produced as previously described by the university of iowa gene transfer vector core [ ] . to generate tmprss -expressing adenoviruses, htmprss containing a c-terminal flag tag was cloned into the pad cmv shuttle vector between xhoi and ecori restriction sites. to generate shrna-expressing adenoviruses, gene blocks containing an shrna targeting either the coding region of cd (target sequence: ccgattcgactctca gaccaa) or the ' utr of tmprss (target sequence: acactagagtggatgaatgt ctgga), flanked by the u promoter and rnapoliii termination sequence, were purchased from genscript. these gene blocks were subcloned into the pacad k-npa e shuttle vector between the kpni and ecori restriction sites. shuttle vectors were linearized and transfected into hek cells along with a linearized ad backbone containing an rsv promoter -driven hdpp in the e region. homologous recombination in hek cells yielded recombinant adenovirus encoding both the shrna and hdpp . titers of purified recombinant adenoviruses ranged from − pfu/ml. isoflurane-anesthetized mice were transduced intranasally with . x pfu of the indicated ad virus in μl of dmem. days posttransduction, mice were infected intranasally with pfu of mers-cov in a total volume of μl dmem. at d.p.i., mice were euthanized by isoflurate inhalation followed by cervical dislocation. lungs were removed into pbs and manually homogenized. virus was plaqued on vero cells. cells were fixed with % formaldehyde and stained with crystal violet d.p.i. all work was performed in the university of iowa biosafety level (bsl ) laboratory. hiv pseudoviruses were produced as previously described [ ] . briefly, t cells were cotransfected with pnl . -hiv-luc and pcdnas encoding appropriate glycoproteins. after two days, supernatants were collected, centrifuged at , x g at ˚c for minutes to remove debris, and stored in aliquots at - ˚c. vsv pseudoviruses were produced by the methods of whitt, [ ] . briefly, t cells were transfected with plasmids encoding viral glycoproteins. two days later, cells were inoculated for h with vsvΔg-luciferase [ ] , rinsed extensively and incubated for one day. supernatants were collected, centrifuged at , x g at ˚c for minutes to remove cellular debris, and stored in aliquots at - ˚c. pseudovirus transductions were carried out by incubating target cells with pseudoviruses for h at ˚c. following initial incubation, unadsorbed viruses were removed by washing thrice with pbs. complete media was placed on the cells and incubated for h for vsv or h for hiv at ˚c. at the end of transduction periods, cells were dissolved into cell culture lysis buffer ( mm tris-phosphate [ph . ], mm dtt, mm , -diaminocyclohexane-n, n,n ,n -tetraacetic acid, % glycerol, % triton x- ) and luciferase levels were measured by addition of firefly luciferase substrate ( mm d-luciferin, pspcas -bb- a-puro was digested with esp i (fermentas) for h at ˚c. the digested plasmid was purified and ligated with annealed guide dnas specific for cd or cd . tetraspanin-specific pspcas -bb- a-puro plasmids were transfected into t cells. after h, cells were selected with μg/ml puromycin for h. selected cells were serially-diluted to isolate clonal populations and clones were selected by western blot. adherent t cells (~ / cm ) were rinsed with ice-cold pbs, incubated for min at ˚c with pbs- mg/ml ez-link sulfo-nhs-lc-biotin (pierce), then for min at ˚c with pbs- mm glycine. cells were rinsed with pbs, then incubated for min at ˚c in mes buffer ( mm mes [ph . ], mm nacl, mm cacl , mm mgcl ) containing % -[( -cholamidopropyl)dimethylammonio]- -propanesulfonate (chaps) detergent (calbiochem cat # ) or % triton x- detergent (sigma). cell lysates ( /ml) were removed from plates and emulsified by cycles of extrusion through g needles. nuclei were removed by centrifugation, lysates mixed with equal volumes of % w/v sucrose in mes buffer, placed into beckman sw tubes, and overlaid with ml of % w/v sucrose, then with . -ml of % w/v sucrose, both in mes buffer. samples were centrifuged with a beckman sw rotor at k x g for h at ˚c. fractions were collected from air-gradient interfaces. biotinylated proteins in gradient fractions were bound to streptavidin agarose beads (pierce). non-reducing western-blotting procedures were used to identify the distributions of proteins in gradient fractions, as described previously [ ] . hela cells were transfected with indicated plasmid dnas and a gfp reporter, incubated for two days, and then lifted from tissue culture plates using . % trypsin. cells were transferred to microscope coverslips coated with fibronectin. cells were allowed to adhere for h. cells were then fixed for minute at ˚c with . % paraformaldehyde in . m piperazine-n,n bis( -ethanesulfonic acid) buffer (ph . ). coverslips were washed with pbs and pla was performed using duolink proximity ligation assay (sigma-aldrich) using primary antibodies against tmprss and cd . images were captured using a deltavision microscope (applied precision) equipped with a digital camera (coolsnap hq; photometrics), using a . -numerical aperture x objective lens. images were deconvoluted with softworx deconvolution software (applied precision). pla foci were detected and quantified using imaris version . . (bitplane scientific solutions). t cells were transfected with dpp and either an empty vector or complementing tetraspanin. h after transfection, cells were plated in a -well plate. merspps were added to cells at ˚c for hour to allow viral binding. media was removed and replaced with ˚c media and the plates were moved to an incubator. at sequential time points following the shift to ˚c, a protease inhibitor cocktail was added to cells such that the final concentration was μm camostat, μm proprotein convertase inhibitor, and μm e d. these drugs were left on cells overnight before cells were lysed and luciferase was measured as described above. luciferase levels were compared to that of cells treated only with dmso control. t cells were transfected with dpp and an empty vector or the complementing tetraspanin. cells were pre-treated for h with μm camostat, μm bafilomycin, or μm e d before transduction with merspps in the presence of the inhibitors. after h, cells were washed to remove drugs and unadsorbed virus. luciferase assays were performed as described above. quantitative reverse transcriptase-pcr cellular rna was isolated using the rneasy mini kit (qiagen) and or ng was reverse transcribed using an iscript cdna synthesis kit (bio-rad). quantitative pcr was performed using power sybr green (thermo fisher) and primers specific to human cd , dpp , tmprss , or hprt. statistical comparisons were made by two-tailed student's t-test. error bars in the figures indicate the standard error of the data. non-linear regression analysis was used to fit a curve to the entry kinetics data and obtain the time of % infection. this analysis was performed using minitab software. cleavage of influenza virus hemagglutinin by airway proteases tmprss and hat differs in subcellular localization and susceptibility to protease inhibitors retroviral entry mediated by receptor priming and low ph triggering of an envelope glycoprotein two-step conformational changes in a coronavirus envelope glycoprotein mediated by receptor binding and proteolysis cloning of the mouse hepatitis virus (mhv) receptor: expression in human and hamster cell lines confers susceptibility to mhv angiotensin-converting enzyme is a functional receptor for the sars coronavirus human aminopeptidase n is a receptor for human coronavirus e dipeptidyl peptidase is a functional receptor for the emerging human coronavirus-emc tmprss activates the human coronavirus e for cathepsin-independent host cell entry and is expressed in viral target cells in the respiratory epithelium the spike protein of the emerging betacoronavirus emc uses a novel coronavirus receptor for entry, can be activated by tmprss , and is targeted by neutralizing antibodies evidence that tmprss activates the severe acute respiratory syndrome coronavirus spike protein for membrane fusion and reduces viral control by the humoral immune response cleavage and activation of the severe acute respiratory syndrome coronavirus spike protein by human airway trypsin-like protease estimating the stoichiometry of human immunodeficiency virus entry influenza-virus membrane fusion by cooperative fold-back of stochastically induced hemagglutinin intermediates coronavirus and influenza virus proteolytic priming takes place in tetraspanin-enriched membrane microdomains lateral organization of membrane proteins: tetraspanins spin their web. the biochemical journal functional domains in tetraspanin proteins. trends in biochemical sciences ewi- and ewi-f link the tetraspanin web to the actin cytoskeleton through their direct association with ezrin-radixinmoesin proteins direct extracellular contact between integrin α β and tm sf protein cd functional interplay between tetraspanins and proteases. cellular and molecular life sciences: cmls the sheddase activity of adam /tace is regulated by the tetraspanin cd . cellular and molecular life sciences: cmls proteomic analysis of the tetraspanin web using lc-esi-ms/ms and maldi-fticr-ms cd negatively regulates cd expression and inhibits cd -mediated enhancement of invasive potential of malignant mesothelioma cells tetraspanins: push and pull in suppressing and promoting metastasis type ii transmembrane serine proteases middle east respiratory syndrome coronavirus infection mediated by the transmembrane serine protease tmprss rapid generation of a mouse model for middle east respiratory syndrome interacting regions of cd and two of its partners, ewi- and ewi- wint, and their effect on hepatitis c virus infection dual function of cd in influenza virus uncoating and budding genome engineering using the crispr-cas system tetraspanin functions and associated microdomains evaluation of prototype transmembrane superfamily protein complexes and their relation to lipid rafts direct observation of individual endogenous protein complexes in situ by proximity ligation two-color, rolling-circle amplification on antibody microarrays for sensitive, multiplexed serum-protein measurements hepatitis c virus infection propagates through interactions between syndecan- and cd and impacts the hepatocyte glycocalyx plasma membrane tetraspanin cd complexes with proprotein convertase subtilisin/kexin type (pcsk ) and low density lipoprotein receptor (ldlr), and its levels are reduced by pcsk protease inhibitors targeting coronavirus and filovirus entry mouse dipeptidyl peptidase is not a functional receptor for middle east respiratory syndrome coronavirus infection receptor variation and susceptibility to middle east respiratory syndrome coronavirus infection characterization of innate responses to influenza virus infection in a novel lung type i epithelial cell model. the journal of general virology generation of vsv pseudotypes using recombinant deltag-vsv for studies on virus entry, identification of entry inhibitors, and immune responses to vaccines evidence for camel-to-human transmission of mers coronavirus a mouse model for mers coronavirus-induced acute respiratory distress syndrome mouse-adapted mers coronavirus causes lethal lung disease in human dpp knockin mice clinical isolates of human coronavirus e bypass the endosome for cell entry high-content analysis of sequential events during the early phase of influenza a virus infection inhibitors of cathepsin l prevent severe acute respiratory syndrome coronavirus entry proteolytic processing of middle east respiratory syndrome coronavirus spikes expands virus tropism differential effect of cholesterol on type i and ii feline coronavirus infection requirements for ceacams and cholesterol during murine coronavirus cell entry importance of cholesterol-rich membrane microdomains in the interaction of the s protein of sars-coronavirus with the cellular receptor angiotensin-converting enzyme murine coronavirus requires lipid rafts for virus entry and cell-cell fusion but not for virus release human coronavirus e binds to cd in rafts and enters the cell through caveolae cryo-electron microscopy structure of a coronavirus spike glycoprotein trimer pre-fusion structure of a human coronavirus spike protein structure of mers-cov spike receptor-binding domain complexed with human receptor dpp modeling how many envelope glycoprotein trimers per virion participate in human immunodeficiency virus infectivity and its neutralization by antibody host cell entry of middle east respiratory syndrome coronavirus after two-step, furin-mediated activation of the spike protein inhibition of proprotein convertases abrogates processing of the middle eastern respiratory syndrome coronavirus spike protein in infected cells but does not reduce viral infectivity activation of the sars coronavirus spike protein via sequential proteolytic cleavage at two distinct sites cathepsin l functionally cleaves the severe acute respiratory syndrome coronavirus class i fusion protein upstream of rather than adjacent to the fusion peptide endosomal proteolysis by cathepsins is necessary for murine coronavirus mouse hepatitis virus type spike-mediated entry proteases and proteolysis in the lysosome the spike protein of the emerging betacoronavirus emc uses a novel coronavirus receptor for entry, can be activated by tmprss , and is targeted by neutralizing antibodies membrane lipids: where they are and how they behave ifitm proteins inhibit entry driven by the mers-coronavirus spike protein: evidence for cholesterol-independent mechanisms distinct patterns of ifitm-mediated restriction of filoviruses, sars coronavirus, and influenza a virus cryo-em structures of mers-cov and sars-cov spike glycoproteins reveal the dynamic receptor binding domains cd , a tetraspan transmembrane protein, renders cells susceptible to canine distemper virus assembly differentially alters dynamics and partitioning of tetraspanins and raft components formation of syncytia is repressed by tetraspanins in human immunodeficiency virus type -producing cells inhibition of feline immunodeficiency virus infection by cd antibody operates after virus entry and is independent of virus tropism interaction of cd tetraspanin proteins with htlv- envelope glycoproteins inhibits cell-to-cell fusion and virus transmission egress of hsv- capsid requires the interaction of vp and a cellular tetraspanin membrane protein cell entry of hepatitis c virus requires a set of co-receptors that include the cd tetraspanin and the sr-b scavenger receptor tetraspanin cd mediates papillomavirus type endocytosis binding of hepatitis c virus to cd inhibition of hepatitis c virus infection by anti-claudin- antibodies is mediated by neutralization of e -cd -claudin- associations the tetraspanin cd in papillomavirus infection a transmembrane serine protease is linked to the severe acute respiratory syndrome coronavirus receptor and activates virus entry a simple method for the rapid generation of recombinant adenovirus vectors labeling hiv- virions with two fluorescent proteins allows identification of virions that have productively entered the target cell the authors thank drs. bart haagmans and ron fouchier for providing mers-cov; drs. lijun rong, fang li, and michael farzan for plasmid dnas; and dr. michael whitt for recombinant vsvs. the authors acknowledge the expertise of the university of iowa gene transfer vector core for ad production. we thank dr. edward campbell for providing expertise with confocal microscopy. we thank dr. tim o'brien for performing statistical analysis of our data, especially the non-linear regression analysis. key: cord- - reu yz authors: reguera, juan; santiago, césar; mudgal, gaurav; ordoño, desiderio; enjuanes, luis; casasnovas, josé m. title: structural bases of coronavirus attachment to host aminopeptidase n and its inhibition by neutralizing antibodies date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: reu yz the coronaviruses (covs) are enveloped viruses of animals and humans associated mostly with enteric and respiratory diseases, such as the severe acute respiratory syndrome and – % of all common colds. a subset of covs uses the cell surface aminopeptidase n (apn), a membrane-bound metalloprotease, as a cell entry receptor. in these viruses, the envelope spike glycoprotein (s) mediates the attachment of the virus particles to apn and subsequent cell entry, which can be blocked by neutralizing antibodies. here we describe the crystal structures of the receptor-binding domains (rbds) of two closely related cov strains, transmissible gastroenteritis virus (tgev) and porcine respiratory cov (prcv), in complex with their receptor, porcine apn (papn), or with a neutralizing antibody. the data provide detailed information on the architecture of the dimeric papn ectodomain and its interaction with the cov s. we show that a protruding receptor-binding edge in the s determines virus-binding specificity for recessed glycan-containing surfaces in the membrane-distal region of the papn ectodomain. comparison of the rbds of tgev and prcv to those of other related covs, suggests that the conformation of the s receptor-binding region determines cell entry receptor specificity. moreover, the receptor-binding edge is a major antigenic determinant in the tgev envelope s that is targeted by neutralizing antibodies. our results provide a compelling view on cov cell entry and immune neutralization, and may aid the design of antivirals or cov vaccines. apn is also considered a target for cancer therapy and its structure, reported here, could facilitate the development of anti-cancer drugs. the coronaviridae is a large family of enveloped, plus-rna viruses. they are involved in respiratory, enteric, hepatic and neuronal infectious diseases in animals and humans that lead to important economic losses [ , ] , as well as to high mortality rates in severe acute respiratory syndrome cov (sars-cov) infections [ ] . the covs are a numerous group of coronaviridae. they have been clustered in the coronavirinae subfamily, which includes three approved genera, alpha-, betaand gammacoronavirus, as well as a tentative new genus, the deltacoronavirus [ ] . representative cov species in each genus are alphacoronavirus (comprising transmissible gastroenteritis virus (tgev), porcine respiratory cov (prcv) and related canine and feline covs), human coronavirus (hcov- e and hcov-nl , genus alphacoronavirus), murine coronavirus (including mouse hepatitis virus (mhv), genus betacoronavirus, cluster a), severe acute respiratory syndrome-related coronavirus (sars-related cov, genus betacoronavirus, cluster b), avian coronavirus (including infectious bronchitis virus (ibv), genus gammacoronavirus), and bulbul-cov (tentative genus deltacoronavirus) [ ] . cov particles display characteristic large surface projections or peplomers ( - nm) comprised of homotrimers of the spike glycoprotein (s), a type i membrane protein [ , ] . the peplomers have a globular portion connected by a protein stalk to the transmembrane domain [ ] . the globular region is formed by the n-terminal s region, whereas the stalk corresponds to the membrane-proximal s region, which mediates virus fusion to host cells and adopts a helical structure characteristic of class i virus fusion proteins [ ] . determinants of cov tropism locate at the s region [ , ] , which mediates attachment of cov particles to cell surface molecules, initiating virus entry into cells and infection. there is considerable variability in receptor usage among the covs. most alphacoronavirus such as tgev and hcov- e use apn [ , ] , whereas the related hcov-nl uses a distinct cell entry receptor, the human angiotensin converting enzyme (ace ) [ ] ; sars-cov also recognizes the ace receptor [ ] . sars and nl cov bind to common regions of the ace protein, although the structures of their receptor-binding domains (rbds) are quite distinct [ , ] . mhv uses the cell adhesion molecule ceacam a [ ] ; a recent crystal structure showed that the mhv rbd adopts a galectin-like fold [ ] . the use of alternative receptors that confer extended tropism has been described for sars-cov, mhv and tgev [ , ] . the mammalian apns (cd ) are type ii cell surface metalloproteases whose large glycosylated ectodomain has a zinc metal ion at the active site [ ] . apn is linked to many cell functions, leading it to be termed the ''moonlighting enzyme'' [ ] . animal models confirmed a role for this cell surface enzyme in angiogenesis [ ] . peptides and inhibitors that target apn showed a link between this protein and tumor growth and invasion [ , ] . apn is a target for cancer chemotherapies; drugs that bind this protein have been developed to treat tumors, some of which are in clinical trials [ ] . as mentioned above, apn is also a major cov cell entry receptor [ , , ] . cov recognition of apn is species-specific, and specificity is associated with n-linked glycosylations in the apn protein [ ] . cell tropism and immune neutralization have been extensively studied in some porcine alphacoronavirus, such as the enteropathogenic tgev and porcine respiratory cov (prcv), a nonenteropathogenic virus derived from tgev [ ] . both viruses use porcine apn (papn) for cell entry. the apn-binding domain in tgev, prcv and other alphacoronavirus locates at the c-terminal portion of the s region [ , , ] , which bears epitopes recognized by cov-neutralizing antibodies [ , , , ] . most tgev-neutralizing antibodies cluster at antigenic site a [ , ] , comprised within the rbd at the s region ( figure a ) [ ] ; the other antigenic sites defined in the tgev s region (b through d) are outside the rbd ( figure a ) [ ] . to date, there is no structural information available on antibody neutralization and apn recognition by alphacoronavirus. we determined crystal structures of the prcv rbd in complex with the papn ectodomain, and the tgev rbd in complex with the neutralizing monoclonal antibody (mab) af [ ] . the rbd adopts a b-barrel fold, with a distinct protruding tip engaged in papn recognition. the structures show how these porcine alphacoronavirus recognize its cell entry papn receptor and how immune neutralization of these covs is achieved by antibody targeting of receptorbinding residues in the s protein. the mechanisms used by tgev to escape immune neutralization and the evolution of receptor recognition in the cov family are discussed. apn-binding domain and epitopes for neutralizing mabs overlap in tgev and prcv s proteins apn receptor recognition and envelope s antigenicity are well documented in tgev and related prcv. the papn-binding figure . apn-binding domain and epitopes for neutralizing mabs overlap in tgev and prcv s proteins. a. scheme of tgev and prcv s proteins showing the s , s , transmembrane (t) and cytoplasmic (cy) regions. location of the c, b, d and a antigenic sites [ ] , and the papn rbd (bar with n and c-terminal residues) [ ] are shown. length is indicated for mature s regions. b. a short, soluble s protein variant containing the tgev rbd region binds to cell surface papn. binding of a bivalent sa-fc fusion protein (sa) to bhk-papn (open histograms), alone (left) or in the presence of the site a-specific ac mab (right), as analyzed in facs. filled histograms correspond to an unrelated fc fusion protein. c. binding of site a-specific tgevneutralizing mab to the sa protein. mab binding to plastic-bound sa protein, monitored by optical density (od). site a mabs are specific for the aa ( bb ), ab ( de ) and ac ( ac , af ) subsites [ ] . an anti-ha mab that binds to the ha tag in the sa protein was used as control. d. site a-specific mabs prevent sa protein binding to papn. binding of the sa protein to bhk-papn cells in panel b was monitored alone or in the presence of site a (shown in c) or site d-specific ( dg ) mab, and the binding ratio determined (see materials and methods). c, d. mean and standard deviation for three experiments. doi: . /journal.ppat. .g the cell surface aminopeptidase n (apn), a membranebound metalloprotease target for cancer therapy, is a major cell entry receptor for coronaviruses (covs), agents that cause important respiratory and enteric diseases. in some covs, the virus envelope spike glycoprotein (s) mediates attachment of the virus particles to the host apn protein and cell entry, which is blocked by antibodies that prevent cov infections. the crystal structures of the s proteins of two porcine cov in complex with the pig apn (papn) or with a neutralizing antibody shown here, reveal how some cov bind to its cell surface apn receptor and how antibodies prevent receptor binding and infection. the report uncovers a unique virus-receptor recognition mode that engages a glycan n-linked to the papn ectodomain, revealing structural determinants of the receptor-binding specificity in covs. neutralizing antibodies target viral residues used for binding to the apn receptor and entry into host cells, showing that efficient cov neutralization requires immune responses focused toward key receptor binding motifs in the virus envelope. these structural insights, together with the structure of the apn ectodomain, provide a compelling view of relevant cell membrane processes related to infectious diseases and cancer. domain was mapped within residues to of the mature tgev s polypeptide [ ] , whereas tgev mab-resistant (mar) mutants defined four antigenic sites (c, b, d and a) [ , ] ( figure a) . antigenic sites c and b are not present in the prcv s protein. antigenic site a determinants are located within the papn-binding domain at the c-terminal moiety of the tgev and prcv s regions ( figure a ) [ , ] . we recently reported the modular dissection of the n-terminal s region of tgev and prcv, and the preparation of soluble s length variants with single antigenic sites [ ] . we produced a recombinant short s protein fragment termed sa, which comprises only residues to of the tgev s protein that binds cell surface papn ( figure b ) and displays conformational epitopes for the three antigenic a subsites (aa, ab, and ac) ( figure c ). antibodies clustered at the aa ( bb ), ab ( de ) and ac ( af and ac ) subsites blocked binding of the soluble sa protein to papn ( figure d ). the sa protein therefore includes the papn-binding domain of tgev and epitopes for site aneutralizing mab. we applied x-ray crystallography to s protein variants containing the rbd of the related tgev and prcv, and have identified how these alphacoronavirus bind to the cell surface papn and its inhibition by neutralizing antibodies. we attempted crystallization of the soluble papn-binding sa protein derived from the tgev s, alone and in complex with several neutralizing mabs. crystals were prepared with the sa protein in complex with the fab fragment of the af mab [ ] ; the structure of the complex was determined and refined using diffraction data extending to . Å resolution (materials and methods; table ). the asymmetric unit of the crystals contains two antibody-rbd complexes, one of which is shown in figure . residues pro to val of the tgev s protein, previously identified as the papn-binding domain ( figure a ) [ ] , were well defined in the crystal structure. they folded in a single domain structure, the rbd of tgev (figure a) . the rbd adopts a bbarrel fold formed by two b-sheets with five b-strands each (scheme in figure s a ). n-and c-terminal ends are on the same side of the domain (terminal side), which presumably lies close to other s protein domains; at the opposite side, two b-turns (b -b and b -b ) form the tip of the barrel (figure a) , where the mab binds to the rbd. the immunoglobulin (ig) variable domains of the mab heavy (v h ) and light (v l ) chains contact the b -b , b -b and b -b regions of the tgev rbd ( figure b ), burying a virus protein surface of , Å . the buried surface of the af mab is , Å , with equal contribution by the v h ( %) and v l ( %) ig domains. complementarity determining regions (cdr) of the antibody heavy (h ) and light (l and l ) chains, the n-terminus of the light chain and the c, c and c b-strands of the v h domain contact the viral rbd tip ( figure b ). the cdr-h of the af mab is relatively long, with two-residue insertion (tyr h and asp h ) relative to other homologous h loops in reported mab structures (table s ). the rbd b -b hairpin with tyr at its tip is at the center of the interacting surface and penetrates between the v l and v h ig domains of the af mab ( figure b and c). similar antibodyantigen recognition is described for some peptides and is common for small hapten molecules [ , ] . the rbd b -b region contributed % of the rbd surface buried by the af mab, and docked between the af mab variable domains ( figure b ). the b-turn is fully buried between the mab ig domains ( figure c ), forming a contact network with mab residues ( figure d ). the rbd residue tyr at the bottom of the pocket contacts mab residues trp h and tyr h , whereas its hydroxyl group is hydrogen bonded to the side chain of gln l and main chain carbonyl of tyr h ( figure c and d). these structural findings on af recognition of the rbd b -b region correlate with af mab binding to peptides (mkrsgygqpia ) that include this hairpin region [ ] . the rbd b -b and b -b regions are at the periphery of the epitope ( figure b ); their contribution to interaction with af is smaller than that of the b -b region, representing respectively , % and % of the rbd surface buried by the mab. they contact either the v l or v h ig domains ( figure b ). rbd residues leu and trp at the b -b loop contact the n-terminus, cdr-l and cdr-l of the v l domain, whereas the b -b loop contacts the long cdr-h loop ( figure b and c). to characterize cov attachment to its apn receptor, we attempted crystallization of the papn ectodomain in complex with tgev and prcv s protein variants comprising their rbds (materials and methods). crystals were obtained only with a mixture of a prcv s protein (s h) and the papn. using these crystals, we determined the structure of the prcv rbd-papn complex by molecular replacement using previously solved structures of the tgev rbd shown in figure ( % sequence identity) and of the papn ectodomain (materials and methods and table ). the asymmetric unit of the crystals contained two macromolecular rbd-papn complexes ( figure a ). the prcv rbd adopts a b-barrel fold like the tgev rbd ( figure s ). each papn molecule was engaged by the tip of a single prcv rbd molecule, which bears two exposed aromatic residues (tyr and trp) ( figure a , in red), and they bound to a membrane-distal region of the papn ectodomain ( figure a ). the rbd n-and cterminal ends and the remaining cov s are also distant from the papn, and are unlikely to contact the receptor molecule. based on a cryo-em structure of the sars-cov s [ ] , the rbd must be also at the viral-membrane distal side of the s and therefore, the receptor binding edge must be accessible for cov binding to the apn receptor. the papn is a type ii membrane protein and the n-terminal end of the ectodomain must be near the cell membrane ( figure a ). the n-terminal residues of the crystallized papn ectodomain are largely disordered in the structure and they might form a flexible region close to the cell membrane. the papn ectodomain is composed of four domains ( figure a ). domain i (orange) is made of b-strands, domain ii (yellow) adopts a thermolysin-like fold bearing a zinc ion at the catalytic site, domain iii (red) is a small b-barrel domain, and the c-terminal domain iv (green) is composed of alpha-helices (domain boundaries are shown in figure s ). the papn molecule structure is closely related to that of the human endoplasmic reticulum aminopeptidase- [ , ] (root-mean-square deviation of . Å for residues sharing % sequence identity, based on dali server). domain ii bearing the enzyme active site is the most related domain ( % identity), whereas domain iv is the most distinct ( % identity). the zinc ion is coordinated to conserved residues at the papn active site in domain ii ( figure s ). the active site conformation is similar to that of other aminopeptidases ( figure s ). the papn crystallized in complex with the prcv rbd had an open conformation [ , , ] , in which domain iv was , - Å from domains i and ii; this creates a central cavity in which the zinc ion at the catalytic site is highly accessible ( figure a ). the mammalian apns are cell surface metalloproteases that form membrane-bound dimers [ ] . the crystallized papn ectodomain also behaved as a dimer in solution ( figure s ). the papn dimeric assembly showed in figure a buried a large accessible surface (, Å ) in each monomer. the dimerization surface comprises residues spread across domain iv, which are distinct from those recognized by cov ( figure s ). similar dimeric assemblies were observed in two crystal structures determined for the papn ectodomain alone (not shown), crystallized using distinct conditions. the papn molecular assembly shown here might thus be representative of the dimer described for mammalian apn on membrane surfaces [ ] . in the crystals of the prcv rbd-papn complex, the rbd tip contacts a membrane-distal region of the papn ectodomain ( figure a ). the conformations of the receptor-binding loops (b -b and b -b ) at the tips of the two prcv b-barrel domains in the structure are identical ( figure s b ), suggesting very similar rbd-papn interactions in both complexes of the asymmetric unit. the virus-receptor interaction buried , Å of the virus protein, % of which corresponded to the b -b region ( figure b ) and % to the b -b turn ( figure c ). the size of the papn surface buried by the rbd was similar (, Å ), and included papn residues ranging from alpha helix (a ) to (a ) in domain iv, and a few domain ii residues ( figure s , table s ). the end of the papn helix a and helix a contacted the b -b region of the rbd ( figure b ). the tyr side chain (tyr in tgev), which protrudes at the b-turn in prcv and tgev rbds ( figure b and d) , is almost fully buried in the complex, locating between the first n-acetyl glucosamine (nag ) linked to papn asn , the end of helix a , and the first half of helix a ( figure b ). the hydroxyl group of the rbd tyr was hydrogen bonded to side chains of papn residues glu and trp , and contributed to virus-receptor binding specificity. the preceding rbd gly residue was at the papn proximal side of the b-turn, hydrogen bonded to the papn asn main chain; at the opposite side, the rbd gln side chain formed a network of hydrogen bond interactions with papn nag and asn side chain ( figure b ). the n-acetyl moiety of the glycan also interacted with rbd residues at the b and b strands ( figure b , table s ). the papn n-linked glycan and surrounding residues that contact the cov rbd b -b region in the structure were identified as one of the apn determinants of the cov host range [ ] . the second relevant virus-receptor interacting region engaged a b-turn at the beginning of the rbd b -b loop ( figure c and d). the unique rbd trp residue, which protrudes at the turn, docked in a papn cavity formed by the coils that precede helices a in domain iv and a in domain ii ( figure c and s ). the bulky side chain of the rbd trp residue packed against papn residues his and pro , and its imino group was hydrogen bonded to the main chain carbonyl of asn ( figure c ). the rbd trp as well as the rbd tyr at the b-barrel tip in tgev and prcv appear to be central residues in the virus-receptor interaction, as they contact with many papn residues and contribute also to binding specificity by mediating polar interactions with the papn (table s ) . to confirm the contribution of the prcv or tgev rbd bbarrel tip in papn receptor recognition, we analyzed binding of wild type and mutant tgev rbd proteins to cell surfaceexpressed papn ( figure a ). mutations in the three regions (b -b , b -b and b -b ) that build the receptor binding edge of the b-barrel decreased rbd binding to papn, whereas mutations outside the receptor-binding region (v ngly) had no effect on receptor recognition. deletion of the papn asn glycosylation site also abolished tgev rbd binding to cell surface-expressed papn ( figure b ). deletion of the homologous glycan in feline apn similarly prevents cell infection by feline, canine and porcine covs, all of which share the glycan-binding tyr residue in the b -b turn (see below), whereas addition of this glycan to human apn is sufficient to render it a tgev receptor [ ] . we determined the crystal structures of the related tgev and prcv rbds bound to two distinct ligands. the rbds adopt bbarrel structures with small differences in the ligand binding loops (figures s ). in the rbd, each of the two highly twisted b-sheets that build the b-barrel is formed by five b-strands ( figure a ). the bent b-strand (b ) crosses both b-sheets and has a b-bulge at asn ( figure a , magenta). at one side of the b-barrel, all bstrands are antiparallel ( figure a, cyan) , whereas on the opposite a dali search of structural homologs showed the greatest similarity (z score of ) with the rbd of the ace receptorbinding hcov-nl (root-mean-square deviation of . Å for residues), the other alphacoronavirus rbd whose structure is known [ ] . the cores of the tgev and hcov-nl b-barrel domains are structurally similar, but the loops at the tips ( figure b and d). the tip region of the hcov-nl rbd is the ace receptor-binding edge and has a ''bowl''-shaped conformation ( figure c ) that differs from the tgev rbd protruding edge. aromatic residues protrude from the b-turns at the tip of the bbarrel in tgev, whereas they are partially buried at the center of the ''bowl''-shaped edge in hcov-nl ( figure b and c). the distinct rbd tip conformation in ace -binding hcov-nl and in apn-binding tgev might be a determinant of their distinct cell entry receptor specificities. the degree of sequence identity in the rbd region among members in the species alphacoronavirus (, % identity) suggests a structure closely related to that of tgev, including conforma-tion of the receptor-binding loops (b -b and b -b ) at the bbarrel tip ( figure ). therefore, tgev, prcv, ccov and fcov must recognize the apn receptor in similar fashion. in contrast, the receptor-binding loops at the tip appear to have a different conformation from tgev in the hcov- e rbd, which also binds to the apn. in this cov, the b -b region has two cys, as in hcov-nl , and lacks the apn-binding tyr residue in alphacoronavirus , although it preserves the two gly residues found in the tgev b-turn ( figure ). the b -b loop in hcov- e is markedly shorter than in tgev, but it also has a trp residue. sequence identities between the rbd of tgev and ibv (gammacoronavirus) or the bulbul-cov (tentative deltacoronavirus) are relatively large (, %), and similarities are found mostly in bstrands and at the rbd c-terminal half ( figure ) . these data indicate a conserved rbd fold between alphacoronavirus and gammaor deltacoronavirus. there is less sequence similarity between the alphaand betacoronavirus rbd regions (, %), which correlates with notable structural differences between their detail of the rbd b -b region with the exposed tyr residue interacting with the papn. side chains of rbd and papn residues engaged in the interaction are shown as sticks with carbons in magenta or green, respectively. nag glycan n-linked to papn asn is shown with carbons in yellow and the electron density map, determined without the glycan, shown as a blue mesh contoured at sigma. c. detail of the rbd b -b region with the trp residue interacting with the papn. in b and c, rbd residues are numbered following the tgev sequence shown in d, and intermolecular hydrogen bonds are shown as dashed red lines. d. structure-based sequence alignment of the tgev and prcv rbds. b-strands are marked with bars. tgev sequence is numbered. in red, af mab-(for tgev) and papn receptor-binding residues (for prcv) identified by the structures. residues absent in the rbd structures are in grey, and the thrombin recognition sequences at the end of recombinant porcine cov rbds are in lowercase letters. doi: . /journal.ppat. .g rbds [ , , ] . the rbds of the sars and mhv betacoronavirus adopt folds unrelated to the b-barrel shown for alphacoronavirus. the most tgev-neutralizing mabs, including af , recognize antigenic site a in the s protein, divided into the aa, ab and ac subsites [ ] . to further characterize site a antigenic determinants in the tgev rbd, we mutated rbd residues targeted by the af mab ( figure ) and some surrounding residues, and analyzed binding to other site a-specific mabs. the antigenicity of residues in the b -b region, in the center of the epitope for af ( figure c ), was determined by monitoring mab binding to rbd mutants with tgev residue substitutions gly (g d), tyr (y a) and gly (g d) ( figure a ). all three substitutions abolished rbd binding by the ac subsite-specific mabs af and ac . the y a rbd mutant was recognized by aa-( bb ) and ab-specific ( de ) mabs ( figure a) , and mab de also bound the g d mutant. in contrast to the antibody binding profile of the y a rbd mutant, ala substitution of the tgev trp residue (w a), a papn-binding residue in the b -b loop at the periphery of the rbd epitope for af ( figure c ), did not affect binding by the ac-specific mabs ( af and ac ), whereas rbd recognition by bb and de mabs was greatly reduced ( figure a ). deletion of the b -b turn (lwd a mutant) reduced ac mab binding to the rbd markedly, with a partial reduction in af binding ( figure a ); this indicates that mab ac recognizes a broader epitope, which correlates with its higher tgev neutralization activity [ ] . replacement with ala of rbd residues thr and asn at the b -b hairpin, which contacts the af mab in the rbd- af structure ( figure c) , reduced binding by all site a-specific mab ( figure a ). this might be a result of a conformational effect induced on the nearby b -b region of the rbd. results for antibody binding to rbd mutants showed that site a epitopes extend across the tgev rbd tip, although there are some differences among the three a subsites ( figure b ). the epitopes recognized by aa-and ab-specific mabs bear the exposed tgev trp residue at the b -b loop, whereas epitopes for the acspecific mabs center on tyr in the b -b turn. none of the mab tested simultaneously targeted the two aromatic side chains (tyr and trp) at the tip of the tgev rbd that bind to the papn. subsite-specific residues defined by mar mutants (lys for aa, arg for ab and gly for ac) might be located at the periphery of their respective epitopes ( figure b ). ab and ac subsites appear to be relatively far apart, with the aa epitope in an intermediate position. the rbd tip, shown here as the papn-binding edge of the domain (figure ) , is the main s protein determinant of antigenic site a, recognized by the most effective neutralizing antibodies of tgev and related cov infections [ , ] . here we show how a group of covs attaches to the cell surface apn metalloprotease for entry into host cells, and how some covneutralizing antibodies prevent infection. the rbd-receptor complex structures determined for alphacoronavirus indicate that the conformation of the receptor binding edge in the envelope s proteins probably determines their receptor-binding specificity. the cov that bind apn analyzed here have protruding receptorbinding motifs that engage recessed surfaces on the receptor. this mode of receptor recognition is essentially opposite to that reported for cov binding to the ace receptor, where recessed receptor-binding motifs in the viral rbd cradle exposed surfaces of the ace ectodomain [ , ] . in the case of papn, an nlinked glycan is also engaged in the virus-receptor interaction. the inherent flexibility of this glycan might facilitate the initial contact of the cov tyr residue with apn amino acids, and subsequent virus-receptor interactions could lock the bound tyr between the glycan and an a-helix ( figure b ). the glycan n-linked to asn in papn is also conserved in canine and feline apn proteins ( figure s ), as are the viral s protein residues that interact with this glycan in the rbd b -b and the b -b regions ( figure ). this unique glycan-virus interaction must thus be conserved among the different covs in the species alphacoronavirus , in accordance with the glycan requirement reported for cell infection by ccov, fcov, and tgev/prcv [ ] . the lack of this glycan in human apn ( figure s ) and the absence of the interacting tyr residue in the b -b region of hcov- e rbd ( figure ) imply distinct virus-apn local contacts in humans. as shown for the alphacoronavirus group, however, hcov- e probably has a protruding receptor-binding edge in the envelope s, responsible for its apn-binding specificity. the structure of the rbd- af complex, together with structure-guided rbd mutagenesis and mab binding data, demonstrated that the receptor-binding region is a major antigenic determinant in the envelope s protein of cov that bind apn. potent tgev-neutralizing antibodies, such as the ac mab [ ] , target key apn-binding residues in the s (figure ) , preventing infection. data from antibody neutralization-resistant tgev mar mutants nonetheless show that some substitutions can be accommodated in the receptor-binding region of alphacoronavirus, which confer the ability to escape immune neutralization, while preserving . substituted residues at the rbd tip that contact papn in the prcv rbd-papn structure are shown in figure , except for the v ngly mutant, with a glycan at rbd position in the b -b b loop, outside the rbd tip (see figure d) . b. tgev rbd binding to cell surface papn glycosylation mutants. relative binding of the sa protein and the anti-ha mab to ha-tagged papn proteins with (papn) or without the glycan linked to asn (n a and t v). mean and standard deviation for three experiments. doi: . /journal.ppat. .g the receptor-binding affinity necessary for cell entry [ , ] . our results thus demonstrate that the receptor-binding region in alphacoronavirus is under selective pressure from the immune system, as described for other viruses [ , , , ] . it is tempting to speculate that immune pressure on exposed receptor-binding residues in the cov s could lead to conformational changes in receptor-binding edges of cov rbds. this would result either in changes in the apn-recognition mode observed with hcov- e and tgev, or in conformational changes in the rbd tip that lead to a receptor specificity switch for cell entry, as observed for hcov-nl [ ] . virus use of recessed binding regions, as for hcov-nl , is a well-defined strategy for hiding conserved receptorbinding residues from antibodies [ , ] . like hcov-nl , sars-cov uses a recessed, although broader ace -binding surface, which can accommodate mutations that permit crossspecies receptor recognition [ ] . it remains to be understood why, despite major changes in the receptor-binding region, all these cov use metalloproteases as cell entry receptors. in the course of our studies, we also determined the crystal structure of the cell surface apn, an important target for cancer therapies. the domain architecture of apn resembles that of related aminopeptidases [ , , ] . here we show a unique dimer configuration for the apn, mediated by its domain iv, the most divergent domain among m aminopeptidases [ ] . the implication of these structural findings for apn biology will require further biochemical analysis. knowledge of the structure is leading to research on the mechanism of action of numerous anti-tumor compounds that target mammalian apn [ ] ; these studies will be fundamental for improving drug specificity. the detailed view of the apn-cov interaction shown here might also lead to development of small molecules to block cov infection. we have identified the receptor-binding region as the major antigenic site in the alphacoronavirus envelope s, which could guide the design of immunogens that boost cov-neutralizing immune responses to key motifs for virus cell entry. design of soluble s proteins variants of tgev and prcv has been described [ ] . the sa protein containing the rbd of tgev was derived from the sc strain, and contains residues b-strands are marked (bars) above or beneath their sequences. tgev sequence is numbered. ace receptor-binding residues reported for hcov-nl [ ] , as well as papn receptor-binding residues for tgev (supplementary table s ) are colored as in c. residues absent in the rbd structures are in grey, and the thrombin recognition sequence at the end of the tgev rbd is in lowercase letters. doi: . /journal.ppat. .g to of the tgev s, an n-terminal influenza hemagglutinin ha peptide, and either a flag mab epitope (monovalent sa-flag variant) or the human igg fc portion (bivalent sa-fc variant) at the c-terminal end. the engineered soluble papn contains residues to (ectodomain) of the cell surface protein fused to ha and flag tags at the n and c terminus, respectively [ ] . the soluble s protein crystallized in complex with the papn was derived from the prcv hol strain (s h in [ ] ), and contains the n-terminal residues of the prcv s protein and same c-terminus as the tgev-derived sa protein [ ] . a recombinant membrane bound papn with an ha tag at the cterminal end was engineered for cell surface expression. thrombin recognition sequences were introduced between the tags and the viral or papn protein sequences. proteins were produced in transiently transfected t or stably transfected cho-lec . . . (cho-lec) cells as described [ ] , and concentration in cell supernatants determined by elisa. proteins prepared in cho-lec cells were used in crystallization experiments. hybridoma cells secreting the tgev s mabs were grown in dmem supplemented with % fcs in roller bottles. proteins secreted to culture supernatants were initially purified by affinity chromatography. all protein samples were further purified by size exclusion chromatography in hepes-saline buffer ( mm hepes, mm nacl) ph . . the fab fragment of the af mab was prepared by papain digestion of the purified antibody. the reaction was terminated by the addition of e (sigma) and the fab fragment purified by size exclusion and ion exchange chromatography using hepes-saline buffer ph . . the polypeptide chains of the ig variable domains of the af mab were determined by sequencing of their cdna prepared from reverse transcribed mrna purified from hybridoma cells. binding of anti-tgev s or -ha (control) mab to wild type and mutant sa proteins was tested in -well plates, using purified mab or hybridoma supernatants. the sa-fc fusion proteins in serum-free (opti-mem, invitrogen) cell supernatants were bound to plastic, and mab binding monitored by optical density (od nm ). at least four sa-fc protein concentrations ranging from to mg/ml were used in duplicate and average binding determined in each experiment. binding ratios were determined after correction for background binding. apn binding assays were also carried out with the sa-fc fusion protein comprising the tgev rbd. bhk-papn cells constitutively expressing cell surface papn were used for binding experiments comparing wild type and mutant rbds, whereas transiently transfected t cells were used for analysis of rbd binding to papn glycosylation mutants. binding was monitored as the percentage of stained cells with the fc fusion proteins and fitc labeled anti-fc antibodies by fluorescence-activated cell sorting (facs), as shown in figure b . the percentage of cells stained was determined for each protein sample and corrected for background staining. papn binding ratios for wild type and mutant rbd proteins shown in figure a were determined from the percentage of bhk-papn cells stained with same concentration of wild type and mutant sa-fc proteins. the binding ratios for wild type and mutant papn glycosylation mutants shown in figure b were determined from the percentage of sa-fc stained t cells expressing similar amounts of ha-tagged papn proteins. cell surface expression of the papn-ha protein was determined with the ha ac mab. the tgev rbd in complex with the af fab fragment was crystallized using the size exclusion-purified complex of a table ). crystallization of the papn ectodomain in complex with porcine cov s proteins was carried out with mixtures of the receptor protein and several tgev and prcv protein variants comprising the receptor-binding region (sa, s h and s h in [ ] ). crystals appeared only in trials performed with an equimolar mixture of papn and the s h protein derived from the prcv s at a final protein concentration of mg/ml, and with a crystallization solution of % peg- k, . m lithium sulfate and . m tris buffer ph . . crystals were transferred to crystallization solution containing % ethylene glycol and frozen for diffraction data collection at the id beamline (prcv rbd-papn in table ). the structure of the tgev rbd- af fab fragment was initially determined by the molecular replacement (mr) method using the phaser program [ ] , and two search models having either the variable or constant regions of the pdb id aif mab structure. the af fab model structure was built manually following electron density maps determined from the mr solution, after improvement with the dm program [ ] . the af fab structure was refined with the program phenix.refine [ ] , which provided an excellent electron density map for building residues to of the tgev s, as well as four residues of a thrombin recognition site at the c-terminus. final structure refinement of the complex was carried out with data extending to . Å resolution (statistics in table ). three cycles of solvent correction, refinement of individual coordinates and atomic displacement parameters combined with tls were applied in each step of structure refinement with phenix.refine, which was alternated with manual adjustment of the model to the electron density maps. all residues are in allowed regions of the ramachandran plot. sa protein residues included in the structure of the tgev rbd are shown in figure d . the structure of the prcv rbd-papn complex was resolved by the mr method using the papn structure determined alone (manuscript in preparation) and the tgev rbd structure as search models. mr solutions were obtained for the two papn molecules (chains a and b) of the asymmetric unit and for one rbd molecule (chain e). the three molecules were adjusted manually and refined with the phenix.refine program. the second rbd molecule (chain f) bound to papn molecule b was built manually into the electron density map. the residues nterminal to the prcv rbd in the s h protein were largely disordered or degraded during crystallization, and are absent in the structure. the complex structure was refined with the program phenix.refine applying solvent correction, ncs, refinement of individual coordinates and atomic displacement parameters combined with tls ( table ). the current model comprises residues to of the papn ectodomain with a zinc metal ion at the papn enzyme active site, and residues to of the prcv s, homologous to the tgev s residues to that defined the tgev rbd structure ( figure d ). all the residues are in allowed regions of the ramachandran plot. coordinates and structure factors have been deposited in the protein data bank with id codes f m (tgev rbd- af ) and f c (prcv rbd-papn). buried surfaces and residues at the molecular complex interfaces were determined with the pisa server (http://www. ebi.ac.uk/msd-srv/prot_int/pistart.html). only residues with at least % of their surface buried at interfaces in the two independent molecules of the crystal asymmetric units are shown. figure d was prepared with ligplot (http://www.ebi.ac.uk/ thornton-srv/software/ligplot/), figure a with ribbons [ ] and the other structure representations with pymol (pymol.org). structural alignments were carried out with modeller using a gap penalty of [ ] . accession numbers of the alphacoronavirus s proteins mentioned are q pkz (tgev), q (ccov), p (fcov), p figure . determinants of tgev s antigenic site a. a. binding of tgev-neutralizing, site a-specific mabs to rbd mutants. relative binding (%) of mutants to wild type sa protein is shown for tgev sspecific mabs (top; described in figure c ) and a control anti-ha antibody (see materials and methods). rbd regions in which mutations locate are shown (bottom; see also figure d ). mean and standard deviation of data from at least three experiments. b. antigenic site a in the tgev rbd and epitopes for antibodies. surface and ribbon representation of the rbd with the af contact regions colored as in figure b . three antibody-binding residues (tyr , trp and asn ) in the loops at the rbd tip, as well as tgev lys , arg and gly residues associated with aa, ab and ac subsites [ ] , respectively. lines indicate epitopes for mabs specific for each of the three antigenic subsites: aa in yellow, ab in green and ac in red. doi: . /journal.ppat. .g (hcov- ), q q s (hcov-nl ), b vdw (bulbul-cov) and q q p (ibv). the prcv hol s protein sequence is reported in reference [ ] . sequence identities among s proteins were determined with psiblast (http://www.ebi.ac.uk/tools/sss/ psiblast/). accession number for the papn protein is p . figure s structures of tgev and prcv rbds. a. secondary structure elements of the rbd structures. b-strands are shown with arrows and colored in blue and cyan, a b-bulge at the b-strand is shown in magenta, helix with a red cylinder, coils with black lines, and disulphide bonds with green lines. b. stereo view of the superimposed asymmetric unit rbd structures of tgev (blue and cyan), complex with the af mab, and of prcv (green and red), complex with the papn protein. view as in figures a and a . locations of n and c terminal ends are indicated in lowercase letters. (tif) figure s mammalian apn ectodomains. sequence alignment of the porcine, canine, feline and human apn proteins with conserved residues highlighted in red. secondary structure elements of the papn structure determined in complex with the rbd of prcv are shown above the sequences. cov-binding residues and those engaged in papn dimerization are highlighted in blue and green, respectively, whereas those at the papn catalytic site are in yellow. residues coordinating the zinc ion are marked with an asterisk, and the n-linked glycosylation site recognized by cov is marked with a triangle at the papn asn . the beginning of each of the four apn domains is indicated. (tif) figure s aminopeptidases active site. side chains of residues at the catalytic site of four structurally aligned zinc aminopeptidases based on domain ii are shown with stick representation, and with the coordinated zinc ion as a cyan sphere. human erap- (pdb code xdt) is shown in green, aminopeptidase n of e. coli (pdb code hpt) in magenta, aminopeptidase n of neisseria meningitidis (pdb code gtq) in blue, and papn in yellow. the glutamic acid located in the gamen motif is labeled in blue and those located at the conserved hexxhx e motif are in red (sequence in figure s ). (tif) figure s dimerization of the papn ectodomain in solution. size exclusion chromatography of the soluble papn ectodomain. continuous line shows optical density (od) at nm for the elution volume. papn protein was run through a superdex / column (ge healthcare) with hepessaline buffer ph . . exclusion volume and size (kda) of molecular weight markers are indicated. determined molecular weight for the single recombinant glycosylated papn ectodomain is about kda, whereas the protein elutes with a volume corresponding to , kda. (tif) table s sequence of homologous cdr-h loops in known mab structures. sequence of homologous heavy chain cdr-h loops to that of the af mab, identified by a blast search among protein structures, whose pdb codes are shown. (tif) table s intermolecular contacts in the prcv rbd-papn complex structure. rbd and papn residues in close contact (# Å ) in the two complexes of the crystal asymmetric unit, computed with the program ncont [ ] . rbd residues from the b -b , b -b and b -b regions at the tip of the bbarrel domain are shown, with those engaged in hydrogen bonding in red. tgev/prcv numbering is given for the rbd residues. (tif) the molecular biology of coronaviruses encyclopedia of virology coronaviruses post-sars: update on replication and pathogenesis virus taxonomy: ninth report of the international committee on taxonomy of viruses assembly of coronavirus spike protein into trimers and its role in epitope expression architecture of the sars coronavirus prefusion spike the coronavirus spike protein is a class i virus fusion protein: structural and functional characterization of the fusion core complex crystal structure of mouse coronavirus receptor-binding domain complexed with its murine receptor human aminopeptidase n is a receptor for human coronavirus e aminopeptidase n is a major receptor for the entero-pathogenic coronavirus tgev crystal structure of nl respiratory coronavirus receptor-binding domain complexed with its human receptor angiotensinconverting enzyme is a functional receptor for the sars coronavirus structure of sars coronavirus spike receptor-binding domain complexed with receptor cloning of the mouse hepatitis virus (mhv) receptor: expression in human and hamster cell lines confers susceptibility to mhv the moonlighting enzyme cd : old and new functions to target impaired angiogenesis in aminopeptidase n-null mice the neovasculature homing motif ngr: more than meets the eye novel aminopeptidase n (apn/cd ) inhibitor f can suppress invasion of hepatocellular carcinoma cells as well as angiogenesis aminopeptidase n (cd ) as a target for cancer chemotherapy mutational analysis of aminopeptidase n, a receptor for several group coronaviruses, identifies key determinants of viral host range genetic evolution and tropism of transmissible gastroenteritis coronaviruses major receptor-binding and neutralization determinants are located within the same domain of the transmissible gastroenteritis virus (coronavirus) spike protein identification of a receptor-binding domain of the spike glycoprotein of human coronavirus hcov- e residues involved in the antigenic sites of transmissible gastroenteritis coronavirus s glycoprotein mechanisms of transmissible gastroenteritis coronavirus neutralization four major antigenic sites of the coronavirus transmissible gastroenteritis virus are located on the amino-terminal half of spike glycoprotein s antigenic modules in the n-terminal s region of the transmissible gastroenteritis virus spike protein crystal structures of an antibody to a peptide and its complex with peptide antigen at . a crystal structures of a quorum-quenching antibody crystal structures of the endoplasmic reticulum aminopeptidase- (erap ) reveal the molecular basis for n-terminal peptide trimming structural basis for antigenic peptide precursor processing by the endoplasmic reticulum aminopeptidase erap structure of aminopeptidase n from escherichia coli suggests a compartmentalized, gated active site reconstitution of purified amphiphilic pig intestinal microvillus aminopeptidase. mode of membrane insertion and morphology the canyon hypothesis: hiding the host cell receptor attachment site on a viral surface from immune surveillance evolution subverting essentiality: dispensability of the cell attachment arg-gly-asp motif in multiply passaged foot-and-mouth disease virus structural basis of immune evasion at the site of cd attachment on hiv- gp structure of the measles virus hemagglutinin bound to the cd receptor pushing the boundaries of molecular replacement with maximum likelihood the ccp suite: programs for protein crystallography phenix: a comprehensive python-based system for macromolecular structure solution ribbon models of macromolecules comparative protein modelling by satisfaction of spatial restrains we thank the esrf for provision of synchrotron radiation facilities through bag-madrid projects, as well as the swiss-sls facility, n. cubells for technical help and c. mark for editorial assistance. key: cord- - cbh u z authors: honce, rebekah; schultz-cherry, stacey title: they are what you eat: shaping of viral populations through nutrition and consequences for virulence date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: cbh u z nan humans have coexisted with viral pathogens for tens of thousands of years, influencing both their emergence and evolution. however, the pervasiveness of the western diet and disparities in food access and security have altered how we as hosts interact with our viral pathogens. malnutrition, the state of having insufficient, excess, or imbalanced sources of energy, is well known to attenuate immune responses. could nutrition also actively shape how viruses evolve? malnourishment is a global, intersectional issue, and it may soon force a revision of our understanding of how viruses evolve within their hosts (fig ) [ ] . first theorized over decades ago [ ] , a quasispecies population structure has been documented in plant, animal, and human pathogens [ ] [ ] [ ] . a viral quasispecies describes the mutant but related genomes that collectively infect, replicate, and spread among hosts. traditionally, the theory has been applied to rna viruses. because of their short generation times, small genomes, and the inherent lack of proofreading in most rna replication, single nucleotide variants (snvs) emerge at a rate of roughly to more mutations per nucleotide copied compared with dna viruses [ ] . nonsynonymous snvs are continuously accrued and purged from the viral genome. this flux generates a related "swarm" of viruses, which have little effect on the consensus sequence but may show phenotypic differences. mutations with phenotypic consequences are generally deleterious; very few mutations have any fitness benefit. however, if beneficial mutations arise, they may relate to host range, drug resistance or vaccine escape, and replicative capacity [ , ] . both beneficial and the common deleterious mutations balance the structure of the viral swarm through complementation, interference, and cooperation [ ] [ ] [ ] . within a single host, tissue-specific subpopulations may vary in virulence without affecting consensus sequence or phenotype [ , ] . importantly, the consensus sequence should not be considered the "fittest sequence," because selection, competition, and genetic drift act upon the entire viral swarm. therefore, fitness of the swarm exceeds clonal sequence fitness, highlighted by work in vesicular stomatitis virus [ ] and bacteriophage systems [ ] . viruses are obligate intracellular parasites that require a host cell to complete their life cycle. barriers to replication exist within and between susceptible hosts, which restrict viral population diversity to quell infections [ ] . in these wide-ranging environments, a a a a a a heterogenous viral swarm containing isolates with differing abilities to infect, transmit, and survive environmental and immunological onslaughts may safeguard viral existence. however, this genetic plasticity has bounds, with an evolutionarily beneficial middle ground between high-and low-fidelity replication [ , ] . the "goldilocks" approach maximizes fitness by avoiding lethal mutagenesis while ensuring amenability to selective pressures [ ] . too low fidelity leads to error catastrophe and collapse of the viral population; conversely, a highly clonal population may be extinguished by host defenses [ ] [ ] [ ] [ ] . numerous theories have questioned the biological relevance of a quasispecies and challenged its significance [ , ] . however, boosting genetic diversity-to a point-is theorized to increase virulence. a viral swarm may be better equipped to face bottlenecks imposed by infecting hosts, environmental persistence, and transmission. even within a single host, blockades due to infection barriers and the immune response diminish sequence variation, leaving a relatively homogenous population until replicative errors replenish the mutant pool [ ] . so, do viruses harboring higher genetic diversity initially fare better in establishing an infection and displaying virulent phenotypes? in studies with classical swine fever virus, higher genetic diversity correlated with virulence [ ]; however, this conclusion has been challenged [ ] . in other animal viruses, diversity increases precede the selection of virulent genomes [ ] . parallel conclusions have been made for human pathogens. in hepatitis c virus (hcv)-positive patients, high viral diversity prior to transplantation correlated with higher liver fibrotic scoring year post-transplantation [ ] . continued genetic evolution of hcv correlated with progressing hepatitis, whereas resolution was associated with genetic stasis of hcv population [ , ] . a model low-fidelity rnadependent rna polymerase (rdrp) poliovirus variant demonstrates that increasing genetic diversity may not always yield fit populations [ , ], yet high-fidelity rdrp mutants producing nearly clonal populations display reduced fitness in vivo [ ]. selection pressures ranging from host antiviral responses to pharmaceutical interventions mold the viral swarm. upon infection, immune responses restrict genetic diversity by limiting spread and replication, eloquently demonstrated using a model poliovirus rdrp [ , ] exogenous control of infections can affect viral swarm composition. as hosts, we have exploited the high mutation rates of viruses by redirecting viral evolution toward error catastrophe via pharmaceutical interventions [ , ] . interestingly, high-fidelity foot-and-mouth disease viral variants possess a higher level of resistance to pharmacologics but are attenuated in vivo, suggesting that the resulting restricted quasispecies hampers adaptability in the presence of drug or host pressures [ ] . also, antiviral treatment can lead genetic diversity gains that may precede selection of drug-resistant genotypes, as has been observed with oseltamivir [ , ] . globally, in people are undernourished and in are overweight or obese, with innumerable others suffering from micronutrient deficiencies [ ] . consequently, it is of utmost importance to understand whether host nutrition actively shapes how viruses evolve because many hosts do not mirror the actively studied "wild-type" condition. previous work has identified micronutrient deficiencies that may increase pathogen virulence through acquisition of minor variants. in mineral-and vitamin-deficient mice, genetic mutations arise in coxsackie b and influenza virus populations that promote virulence even in well-nourished hosts [ ] [ ] [ ] [ ] [ ] . in our work with influenza virus, we determined that nutrient excesses can drive virulence through population diversification [ ] . experimental evolution of ca/ virus through two models of murine obesity resulted in a viral population displaying increased virulence upon inoculation of a wild-type host. this phenotype was not strain specific; an avirulent h n virus was, upon passage in obese hosts, able to productively infect immunocompetent mice. we observed a significant increase in viral diversity and subsequent virulence after a single round of infection, with the phenotype persisting in obese-derived viral populations across passages [ ] . interestingly, arbovirus-infected obese or protein-deficient mice showed higher morbidity but lower viral diversity, and both malnourished models transmitted virus less efficiently, highlighting that the effects of nutrition may vary based on the natural life cycles of viral families [ ] . it is yet to be determined how malnourishment may impact transmission of a respiratory, as compared with a vector-borne, virus. both undernourishment and obesity are two sides of the same coin and are implicated in blunting immune responses and increasing susceptibility to infection [ , ] . in our studies with influenza virus, we linked the emergence of a more diverse and virulent viral population with blunted interferon responses in obese hosts. interferon treatment of obese mice restricted the emergence of a diverse quasispecies and attenuated the virulence of the resulting viral population, strengthening the claim that a robust innate immune response restricts subsequent infection severity, possibly through reduced viral replication and acquisition of a genetically diverse viral population [ , , ] . dietary metabolites also influence cellular metabolism and can push the body to a state of metainflammation; this prooxidant environment may also directly influence the genetic composition of the viral population [ ] . nutritional excess or deficiency may dampen the host immune responses and alter cellular metabolism, indirectly fostering an advantageous environment for viruses to explore the sequence space (fig ) . the dearth of host responses to infection-particularly innate immunity-and the baseline malnourished state facilitates greater viral replication, permits the diversification of the viral swarm, and potentially allows for the emergence of advantageous mutations. other indirect consequences of poor nutrition may also be involved. blunting of immune responses may alter viral tropism and viral-or immune-induced pathology, thus remodeling the microenvironment in which the virus attacks the host. also, nutrition is increasingly appreciated as an influence on the gut microbiome (reviewed in [ ] ). interestingly, perturbations to the microbiome-both respiratory and gut-dampen interferon responses to respiratory virus infection [ ] [ ] [ ] . however, to our knowledge, no empirical studies connect the obese microbiome to modulating enteric or respiratory viral populations. pathogen virulence is a complex interplay of both host and pathogen properties. host nutritional status has long been considered a risk for infection susceptibility and severity and is now implicated in shaping viral evolution. continued studies on the molecular consequences of obesity and malnutrition at the macro-and micronutrient levels will reveal which host defenses are impaired through malnutrition and how they control quasispecies development and viral pathogenesis. similarly, as we gain insight into how hosts influence quasispecies formation and pathogen virulence, we too can exploit these features for host benefit [ , ] . the global ubiquity of malnutrition is shifting our population toward a more susceptible state. this will undoubtedly influence how pathogens behave within and between hosts. continued study of how quasispecies evolution relates to other human, animal, and plant pathogens will indeed usher in a greater understanding of host-pathogen interactions and provide novel insights into how pathogens impact hosts and hosts impact pathogens. global nutrition report: action on equity to end malnutrition selection and self-organization of self-reproducing macromolecules under the constraint of constant flux subclonal components of consensus fitness in an rna virus clone analysis of newcastle disease virus quasispecies and factors affecting the emergence of virulent virus hepatitis c virus quasi-species dynamics predict progression of fibrosis after liver transplantation rna virus mutations and fitness for survival viral quasispecies evolution. microbiology and molecular biology reviews: mmbr generalized selection to overcome innate immunity selects for host breadth in an rna virus. evolution; international journal of organic evolution evolutionary transition toward defective rnas that are infectious by complementation quasispecies diversity determines pathogenesis through cooperative interactions in a viral population the effects of a deleterious mutation load on patterns of influenza a/h n 's antigenic evolution in humans hidden virulence determinants in a viral quasispecies in vivo bottleneck-mediated quasispecies restriction during spread of an rna virus from inoculation site to brain nucleotide sequence heterogeneity of an rna phage population prolonged influenza virus shedding and emergence of antiviral resistance in immunocompromised patients and ferrets oseltamivir expands quasispecies of influenza virus through cell-to-cell transmission rapid genomic evolution of a non-virulent coxsackievirus b in selenium-deficient mice results in selection of identical virulent isolates selenium deficiency increases the pathology of an influenza virus infection host nutritional selenium status as a driving force for influenza virus mutations from avirulent to virulent: vitamin e deficiency in mice drives rapid genomic evolution of a coxsackffi b virus host nutritional status: the neglected virulence factor obesity-related microenvironment promotes emergence of virulent influenza virus strains host nutritional status affects alphavirus virulence, transmission, and evolution protein energy malnutrition decreases immunity and increases susceptibility to influenza infection in mice the effect of malnutrition on the susceptibility of the host to viral infection copper deficiency increases the virulence of amyocarditic and myocarditic strains of coxsackievirus b in mice the human microbiome and obesity: moving beyond associations the microbial metabolite desaminotyrosine protects from influenza through type i interferon nasally administered lactobacillus rhamnosus strains differentially modulate respiratory antiviral immune responses and induce protection against respiratory syncytial virus infection microbiota regulates immune defense against respiratory tract influenza a virus infection attenuation of rna viruses by redirecting their evolution in sequence space key: cord- -uqw ra authors: stenglein, mark d.; jacobson, elliott r.; chang, li-wen; sanders, chris; hawkins, michelle g.; guzman, david s-m.; drazenovich, tracy; dunker, freeland; kamaka, elizabeth k.; fisher, debbie; reavill, drury r.; meola, linda f.; levens, gregory; derisi, joseph l. title: widespread recombination, reassortment, and transmission of unbalanced compound viral genotypes in natural arenavirus infections date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: uqw ra arenaviruses are one of the largest families of human hemorrhagic fever viruses and are known to infect both mammals and snakes. arenaviruses package a large (l) and small (s) genome segment in their virions. for segmented rna viruses like these, novel genotypes can be generated through mutation, recombination, and reassortment. although it is believed that an ancient recombination event led to the emergence of a new lineage of mammalian arenaviruses, neither recombination nor reassortment has been definitively documented in natural arenavirus infections. here, we used metagenomic sequencing to survey the viral diversity present in captive arenavirus-infected snakes. from infected animals, we determined the complete or near complete sequence of genome segments that grouped into l and s genotypes. the majority of snakes were multiply infected, with up to distinct s and distinct l segment genotypes in individual animals. this s/l imbalance was typical: in all cases intrahost l segment genotypes outnumbered s genotypes, and a particular s segment genotype dominated in individual animals and at a population level. we corroborated sequencing results by qrt-pcr and virus isolation, and isolates replicated as ensembles in culture. numerous instances of recombination and reassortment were detected, including recombinant segments with unusual organizations featuring intergenic regions and superfluous content, which were capable of stable replication and transmission despite their atypical structures. overall, this represents intrahost diversity of an extent and form that goes well beyond what has been observed for arenaviruses or for viruses in general. this diversity can be plausibly attributed to the captive intermingling of sub-clinically infected wild-caught snakes. thus, beyond providing a unique opportunity to study arenavirus evolution and adaptation, these findings allow the investigation of unintended anthropogenic impacts on viral ecology, diversity, and disease potential. several mechanisms generate viral genetic diversity [ ] [ ] [ ] . all work by producing populations of viral genomes, a small fraction of which may exhibit an adaptive advantage in a new host species, different tissue, or in the face of drug or immune pressure. replication of rna viral genomes by error-prone polymerases results in relatively high mutation frequencies, and rna viruses replicate as collections of closely related variant genotypes [ , [ ] [ ] [ ] [ ] . viral genomes can shed content or acquire new loci from their hosts by horizontal gene transfer. in coinfected cells, recombination between different viral strains or species can produce chimeric progeny [ ] [ ] [ ] [ ] . and in cells coinfected by segmented viruses, reassortment generates virions containing shuffled mixtures of segments from the parental genotypes [ , ] . understanding basic mechanisms of viral adaptation is essential in order to better combat, prevent, and predict viral diseases. for example, the ability of pandemic influenza virus strains to efficiently replicate in and be transmitted between humans frequently results from de novo mutation and reassortment [ ] . the continuous emergence of drug-resistant genotypes is a major hindrance to the effective treatment of human immunodeficiency virus- and other pathogens [ , ] . and, recombination between individually attenuated strains present in the oral poliovirus vaccine results in neuropathic progeny strains, and complicates eradication efforts [ ] . viruses in the family arenaviridae have bi-segmented single-stranded rna genomes with a characteristic organization and gene repertoire [ ] [ ] [ ] [ ] [ ] . the larger genome segment (l) is about kb in length and encodes the viral rna-dependent rna polymerase (l) and a small zinc-binding ring domain protein (z). the smaller segment (s) is about half as long and encodes the glycoprotein precursor (gpc) and nucleoprotein (np). on each segment, the two viral genes are in opposite coding orientations and are separated by an intergenic region (igr) that is predicted to form stable hairpin structures. two major lineages of arenaviruses have been described: those that primarily infect rodents and those that infect snakes. the rodent arenaviruses (proposed genus mammarenavirus) typically establish chronic mild infections in their natural hosts but can be transmitted to humans and other mammals [ ] . severe disease such as lassa hemorrhagic fever can result from these zoonotic infections. the snake arenaviruses (proposed genus reptarenavirus) were first identified in us cases of inclusion body disease, a progressive and sometimes fatal disease best described in members of the boidae and pythonidae families (boas and pythons) [ , ] . the identification and study of snake arenaviruses in captive snakes in europe corroborated and extended this finding [ ] [ ] [ ] . one major difference between the snake and mammalian arenaviruses is the provenance of their gpc genes, with the snake virus gene being more closely related to the glycoprotein gene of filoviruses and some avian retroviruses [ , ] . several mechanisms of arenavirus evolution have been described [ , [ ] [ ] [ ] . like all rna viruses, arenavirus genome replication is relatively error prone, and arenaviruses replicate as collections of related variants in vivo [ ] . recombination is thought to have given rise to the ancestral s segment of a clade of the new world rodent arenaviruses [ ] [ ] [ ] . recombination and reassortment between co-infecting arenaviruses has been observed in the laboratory [ ] [ ] [ ] . and, it has been suggested that arenaviruses detected in snakes in europe might have undergone recombination [ , ] . however, arenavirus recombination and reassortment have not been confirmed in natural infections involving extant species. in this study we document and investigate viral genetic complexity of an unanticipated extent and form in naturally infected captive snakes. we determined the complete or near complete sequences of viral genome segments using metagenomic sequencing. sequencing results were corroborated and extended by discriminating quantitative reverse transcriptase pcr (qrt-pcr) and by tissue culture isolation experiments. we detected widespread recombination and reassortment. we also observed an unbalanced accumulation of multiple distinct viral genotypes in individual infections. these findings provide an opportunity to study basic mechanisms of virus evolution and fitness through the identification of genetic determinants underpinning their action. in order to further characterize the genetic diversity of the snake arenaviruses, we gathered frozen case and control tissue samples from around the u.s.a. that were collected between and (s table) . we screened these samples for snake arenavirus rna using qrt-pcr with degenerate primers targeting the glycoprotein gene. a total of samples tested positive by qrt-pcr for viral rna. clinical data of varying detail was available for samples. histopathology was available for of the samples and detection of viral rna was well correlated with histopathology-based ibd diagnosis (table ) . however, many infected snakes displayed no overt clinical signs (s table) . thus, detection of viral rna was correlated with detection of inclusions in tissue sections, but not with obvious clinical measures in a straightforward fashion, consistent with previous reports [ , , , ] . we performed metagenomic sequencing to determine complete viral genome sequences. samples from animals, including the pcr-positive samples, were sequenced. samples from of the arenavirus-positive snakes were sequenced to a depth sufficient for assembly of complete or near complete viral genome segment sequences. in many cases, the assemblies included predicted terminal sequences (s fig, [ , ] ). in total, just over million reads contributed to the assembly of genome segment sequences ( l and s sequences) totaling . mb. genome segment assemblies were validated by re-mapping paired-end reads [ ] . assemblies were well supported, with -fold median coverage (s fig) . coverage levels were generally lowest in the intergenic regions (igr), likely as a result of the predicted hairpin-forming sequences in these regions (s fig) . in cases where coverage levels in igrs fell below reads, pcr was used to confirm assembly continuity. genome segments with less than % global pairwise nucleotide identity were classified into distinct genotypes (figs and and s fig) [ ] . a total of s and l genotypes were delineated by this criterion, and were designated s -s and l -l . within genotypes, l segment sequences shared a mean value of % pairwise nucleotide identity, and between genotypes, sequences shared % identity (s a fig) . for s segments, these values were % and % (s b fig). multiple alignments of the four coding sequences were used to create bayesian phylogenies to visualize the inferred evolutionary relationship of these genotypes (figs - and s fig) . in of infected snakes, viral genotypes consisted of a single s and a single l segment genotype (fig and s table, snakes # - ) . for example, snakes # - , the annulated tree boas from the california academy of sciences described in an earlier report, harbored s genotype and l genotype . this s /l genotype corresponds to the virus we referred to as "cas virus" (casv; [ ] ). in snakes # - , segments of genotype s and l were detected, a genotype corresponding to "golden gate virus" [ ] . numerous reassortant genotypes were evident among the singly infected snakes (fig ) . for example, the l segments present in snakes # and were nearly identical at . % pairwise identity, but their s segments shared only . % pairwise identity (genotypes s and s ), indicating a more distant common ancestor. similarly, the s segments in snakes # and were . % identical but the l and l segments in these snakes were only . % identical. it was not possible in most cases to determine which s/l pairs were ancestral, nor when precisely reassortment had occurred, but it was clear that reassortment had produced these permuted pairings. however, in the majority of animals ( of snakes), we observed viral genotypes that were substantially more complex than single s/l pairs. these snakes harbored multiple s and l genome segment sequences (fig ; snakes # - ). the sets of viral genotypes ranged from that which would be expected for a straightforward co-infection by virus strains in snake # , to more complex combinations such as that in snake # , which contained the sequences of s and distinct l segments. the animal with the most viral genotypes was snake # , which contained genotypes ( s and l) that could combine to produce virions with unique s/l pairs (assuming coinfection of individual cells). the distinct l and s segment sequences within individual snakes shared on average only % and % pairwise nucleotide identity respectively, a level of variation not consistent with sequencing error or intrahost diversification. snakes that were housed together often shared similar combinations of genotypes, and in these cases the sequences of these segments were closely related (figs and and fig ) . the accumulation of viral genotypes within individual animals was not balanced. in all cases there were more l than s segment genotypes (fig and s fig). on average, there were more than twice as many l segment genotypes as s genotypes per animal (mean values of . l and . s genotypes per multiply infected animal; s fig) . in fact, of animals with multiple l sequences harbored only a single s genotype. the s genotype was dominant in individual animals and at a population level (fig and fig ) . this genotype was first detected in a snake from collierville, tn, and a partial s sequence was reported previously [ ] . s segments of this genotype were detected in of snakes ( %). these sequences shared % average global pairwise nucleotide identity. re- individual snakes are infected by complex unbalanced sets of viral genotypes. these tables depict the fractional abundance of s and l genotypes detected in individual animals. each row corresponds to an individual animal. each column corresponds to a particular s or l segment genotype. phylogenies on top of the tables were created using representative sequences from each genotype and a neighbor joining clustering method. shading of cells indicates the fractional abundance of that genotype in the indicated animal, which was calculated as the proportion of sequencing reads mapping to that genotype divided by the total number of arenavirus-mapping reads from that animal. recombinant segments are depicted with a triangle. all shaded boxes correspond to coding-complete assemblies, except for those indicated with a circle. groups of animals harboring similar virus genotype combinations that were housed together are indicated with brackets. neg snake is a sample from an uninfected snake and hela is a sample from total hela cell rna. doi: . /journal.ppat. .g markably, the s segment genotype was found in combination with of the l segment genotypes described in this study. in addition to reassortant genotypes, we also identified recombinant s segments and recombinant l segments ( table) . we used the rdp software to detect and statistically evaluate support for recombination events ( table ; [ ] ). recombination events were well supported by rdp analysis and by read coverage levels over recombination junctions (s fig and table ). we also confirmed segment continuity by pcr amplification across junctions. while this analysis provided clear evidence of recombination, it was not always possible to determine which genotypes were parental and which were recombinant. however, it appeared that many of the recombinant segments coexisted in snakes with one of their parental genotypes. for example, in snake # , s segment sequences were evident. one of these was a canonical s genotype. the other segment, designated s , shared % average pairwise nucleotide identity with the s segment's gpc gene, but only % identity in the np gene ( fig a) . the s np appeared to derive from a segment similar to the s np found in snake # . similar patterns were observed for s segments in snakes # , , , , and , and for l segments in snakes # , , , , , , , , and (figs and and fig b) . these may be situations where the parental and progeny genotypes persistently replicated together following a recombination event. alternatively, the genotypes could have been acquired in independent transmission events. some recombination events resulted in unusual genome organizations. for example, the l genotype found in snakes # and # consisted of a full z coding sequence and a partial l coding sequence from one parental segment concatenated to a partial z and full l from a second segment (fig ) . this segment was predicted to contain intergenic hairpin-containing regions ( xigr) separating the l/z pairs. similar double-igr segments and partial cds were observed in recombinant s segments as well (fig ) . an offset template switching event during genome replication may have generated these xigr segments (s fig; [ ] ). we used several independent approaches to corroborate the original metagenomic sequencing. first, we completely re-sequenced samples, using independent library preparations, and derived essentially identical results. second, we developed a panel of pcr primer pairs that discriminated between distinct viral genotypes, and performed qrt-pcr on a subset of samples and genotypes. in all cases, qpcr-based genotyping mirrored sequencing results (s fig). third, we used tissue culture isolation as another means of determining viral genotype and to confirm that sequences corresponded to infectious virus (see below). the introduction of an already infected snake (# ) into the proximity of an uninfected snake (# ) in a private collection enabled us to monitor viral transmission (fig ) . a blood sample from snake # tested negative for snake arenavirus rna by qrt-pcr and deep sequencing. snake # was then not exposed to other snakes until september , when its owners obtained a second snake, # . snake # arrived with stomatitis and was anorexic. nevertheless, after a -week quarantine, snakes # and # were placed in the same enclosure. snake # continued to refuse to feed and died two weeks later. we obtained the body of snake # and a blood sample from snake # taken in november , and an additional blood sample from snake # from january . we determined that multiple genotypes were transmitted from snake # to snake # during their cohabitation (fig ) . snake # 's viral genotype consisted of s and l genotypes (s , / l , , , , , ; fig b) . the november snake # blood sample was arenavirus positive, but the only genotypes detected by sequencing were s and l . the january snake # sample was still positive, but now l genotypes , , and were also detectable in the blood. analysis of the viral sequences recovered from the two snakes revealed that they were closely related ( . - % identity). this data supports the transmissibility of compound unbalanced snake arenavirus genotypes in the context of cohabitation. we performed tissue culture isolation and passaging experiments to investigate whether compound viral genotypes were competent to initiate productive infections. we applied homogenates from samples to cultures of boa constrictor-derived jk cells and monitored levels of virus rna by qrt-pcr using genotype-discriminating primers. in all cases, we detected replication of all of the viral genotypes identified by our metagenomic sequencing (fig ) . for example, snake # contained viral sequences of genotype s /l , . when a liver homogenate from this snake was applied to a jk culture, replication of all of these segments was detected (fig a) . similarly, when a homogenate from snake # was used as inoculum, replication of all expected viral genotypes was observed (s and l , , , , ; fig b) . the distinct l segments exhibited approximately equal replication efficiencies in these experiments, and the populations could be passaged to uninfected cell cultures. thus, sequences of multiple viral genotypes recombinant genome segments with unusual organizations. recombinant s and l genome segments with unusual double-intergenic regions ( xigr) and/or partial coding sequences are depicted as cartoons. plotted below each cartoon are coverage levels (the number of sequencing reads supporting each base in the assembly) and predicted free energy of folding (i.e. predicted rna secondary structure; -Δg) of nt sliding windows. approximate locations of recombinant breakpoints are indicate with triangles and dotted lines. partial coding sequences are indicated. some of these segments, or very closely related versions thereof, were detected in multiple animals, as indicated. in these cases, cartoons and plots are based on the segment from the snake listed in bold font. doi: . /journal.ppat. .g corresponded to replication competent virus, and multiple viral genome segments replicated as stable ensembles in culture. we also investigated whether an l segment with an unusual x igr organization was competent for replication. snake # l contains a partial z region and predicted igr hairpins (fig ) . to track this segment during infection, we performed qrt-pcr using primer pairs: one pair that targeted the l gene of this segment and one pair that spanned the recombination junction (fig c) . throughout the experiment, near equivalent amounts of template were detected using these primer pairs, suggesting that most copies of this segment maintained their unusual structure. we performed endpoint dilution experiments to determine the genotypes of individual virus particles. we prepared dilution series from liver homogenates from snakes # and # and inoculated jk cells in well plates. after - days, we transferred supernatants to new plates and stained cells with anti-np ab to determine wells positive for the presence of virus. positive wells were then genotyped using discriminating qrt-pcr. in most cases, rna from a single s and a single l genotype were detected in individual wells infected with the most dilute inocula (fig ) . in of ( %) wells at these highest dilutions, more than one l genotype was detected. this could be the result of stochastic co-infection, clumped virus particles, or virus particles packaging more than one l segment. these results were consistent with the model that most virus particles packaged a single l segment, although we could not exclude the possibility that a minority of particles may package additional segments. intrahost variation for individual genotypes was also evident. for most genome segments, multiple sites with minor allele variants were detected (s fig). the frequency of variants in most of these cases was less than variant sites per kb (i.e., %; s fig) . in several cases, a higher frequency of variant sites was observed, for example the s segment of snake # , which averaged variant sites per kb ( . % variants sites). this could have resulted from a greater degree of intrahost variation and divergence or from infection by viruses with closely related genotypes whose sequences were too similar to separate using short read assembly. in this study, we surveyed the genetic diversity of arenaviruses infecting captive snakes in the usa. we used metagenomic sequencing and de novo assembly to determine genome sequences of viruses infecting snakes. we found that most snakes were multiply infected by unbalanced ensembles of s and l segment genotypes. in total, we assembled l and s segment sequences that grouped into l and s genotypes. this expands the known diversity within this group of viruses by several fold. the high level of multiple infection has apparently given rise to numerous recombinant and reassortant genotypes, altogether compromising hundreds of unique viral combinations. we also discovered recombinant genotypes with non-canonical genome organizations, including those harboring apparently superfluous content. metagenomic sequencing results were corroborated by pcr-based approaches, and extended by tissue culture isolation experiments. these findings highlight the utility of performing unbiased whole genome sequencing to determine pathogen genotypes. indeed, our initial pcr-based screening correctly identified infected animals, but completely failed to uncover the genetic complexity present in the infections. although natural infection by multiple arenaviruses has not been previously documented, this phenomenon has been reported for other viruses. for example, infection involving up to influenza viruses has been documented in humans and wild birds [ , ] . and, up to or distinct genotypes of torque teno virus or papillomavirus have been identified in individual human samples [ ] [ ] [ ] . in plants, a virus isolate from citrus trees persistently infected by citrus tristeza virus was found to include several genotypes [ ] . shared characteristics of hostpathogen interaction may enable such highly multiple infections. these include persistent, sub-clinical viral replication, the absence of barriers to superinfection, the lack of an immune response capable of clearing infection, and a high prevalence of infection. virus populations replicate as stable ensembles in culture: (a) liver homogenate from snake # was applied to cultures of jk cells and replication was monitored by measuring supernatant viral rna levels using qrt-pcr and genotype-specific primers. levels of distinct s and l genotypes detected are indicated and are normalized to the amount of s rna detected in the first time point. points and error bars represent mean and standard deviation of two independent experiments. (b) as in (a), but a liver homogenate from snake # was used as inoculum. (c) the xigr l segment detected in snake # replicates stably in culture. same experiment as (b), but qrt-pcr used primers that targeted two different regions of the l segment as depicted in the inset cartoon. doi: . /journal.ppat. .g although we detected many instances of snake tissues containing multiple viral genotypes, our results do not prove that individual cells in these animals were multiply infected. however, the detection of recombinant and reassortant genotypes suggests that at least in some cases cells are multiply infected. although multiple infection per se is not unprecedented, several aspects of these findings are. one is the apparent disconnect between the dynamics of the two viral genome segments, both in individual animals and at a population level. in individual animals, the accumulation of s and l segments was unbalanced: in all multiply infected animals, there were more l than s segments. in the most extreme case (snake # ), a single s genotype was paired with an most or all virus particles contain one l genotype. end-point dilution experiments were performed to determine the viral genotype of individual virus particles. serial dilutions were prepared and applied to jk cells in -well plates. after - days, supernatant was reserved and wells were stained with anti-np antibody to identify infected wells. qrt-pcr using discriminating primers was used to genotype the virus in individual well supernatant. each row corresponds to one well and each column to an s or l genotype. the fractional abundance of s and l genotypes detected in individual wells are indicated as in fig as are the dilution used to inoculate the genotyped well. negative wells ("neg"), not staining with anti-np antibody, from the highest dilutions served as negative controls. the amount of each genotype detected in the inoculum is also indicated. inoculum from snake # liver was used in (a) and from snake # liver in (b). doi: . /journal.ppat. .g ensemble of l genotypes. it is possible that within animals the s and l segments inhabit different fitness landscapes. another, not mutually exclusive, possibility is that differential replication kinetics or packaging efficiencies of the two segments may underlie the observed imbalance. additional experiments in vitro and in animals will clarify this issue. the population level dominance of the s genotype was also unexpected and is worthy of additional investigation. genotype s segments were present in / infections ( %) and in of these, no other s segment was detected. one possible explanation is that the s genotype replicates more efficiently within animals, or is more efficiently packaged and transmitted than other competing s segments. alternatively, the high frequency of this genotype may be a stochastic effect, or may be proportional to viral genotypes in natural virus populations, from which these viruses in captive snakes presumably originate. alternatively, it is possible that the l genotypes observed here were originally paired with s genotypes in free-ranging hosts. if this were the case, then s genotypes are unaccounted for. testing of wild-caught snakes could reveal the "missing" s genotypes and original s-l pairings and would reveal whether the s genotype has indeed risen to dominance in the context of captive animals. whether a similar degree of multiple infection is possible in mammalian arenaviruses is an open question, and one that may be relevant to the possible emergence of new mammalian arenavirus strains with pathogenic potential. it may be that there are larger species barriers for mammalian arenaviruses than there are for snake arenaviruses. another possibility is that an ecological situation analogous to captive snake breeding has never been created for rodents. alternatively, characteristics of the mammalian arenavirus host-pathogen interaction may prevent multiple infection. indeed, superinfection exclusion has been documented in mammalian arenavirus tissue culture experiments [ ] [ ] [ ] [ ] [ ] [ ] . and, cross-protection between mammalian arenaviruses has been documented in vivo [ ] [ ] [ ] . assuming that superinfection accounts for at least some of the genotype accumulation observed here, then no such mechanisms are operating in these snakes. laboratory experiments with mammalian arenaviruses and other segmented viruses could test the generality of this phenomenon. the discovery of " xigr" genome segment configurations was also unanticipated. it would be reasonable to predict that these segments would exhibit decreased fitness or be unstable during replication, given that they carry superfluous content. however, two lines of evidence suggested that these xigr segments are capable of transmission and are stable over multiple rounds of replication. first, several of these segments were detected in co-housed snakes (l in snakes # and ; l in # , , and ; l in # - ). presumably, each of these segments was initially generated via recombination in a single infected snake and then transmitted. second, in tissue culture these segments replicated stably and could be passaged and isolated (figs c and b) . more extensive passage experiments in animals and culture will reveal whether maintenance of the xigr configuration is disfavored over the long term. these novel segment configurations also raise the possibility for the creation of payloadcontaining arenavirus genome segments. for example, the l segment found in snakes # and # contain intergenic regions and bases of extraneous incomplete coding sequence. if a suitable reverse genetic system were developed, this regions could be replaced with an internal ribosomal entry site and the base nanoluc luciferase gene or other payloads [ ] . such a tagged virus could be used for example in in vivo pathogenesis studies as an alternative to trisegmented recombinant arenaviruses [ ] . the nomenclature and taxonomy of the snake arenaviruses will likely have to be reconsidered in light of these findings [ ] . we propose a nomenclature like that used for influenza a virus (iav) subtypes, where new s and l genotypes are simply enumerated [ ] . we would also propose following the taxonomic scheme for iav subtypes, which belong to a single species, influenza a virus. in this case, snake arenavirus genotypes could be grouped into one or possibly more species. recombinant genotypes were not limited to those described in this study. discordance between gpc-and np-based phylogenies including sequences from viruses detected in snakes in europe suggested possible s-segment recombination [ , ] . the increased phylogenetic resolution enabled by this study confirmed the recombinant nature of the s and l segments of boa av nl and the l segment of uhv- [ , ] (table , figs and ) . it is possible that snake importation and husbandry practices have inadvertently created an ecological context that has enabled this phenomenon. boa constrictors with different colorations ("color morphs") are highly valued by collectors and breeders. such colorations arise in nature as local adaptations and wild-caught snakes are commonly imported for breeding purposes. an estimated , boa constrictors per year were imported into the usa alone between - [ ] . mammalian arenavirus species have co-evolved with their distinct, geographically isolated rodent hosts, and it may be that snake arenavirus strains have coevolved similarly in the wild. it is plausible that apparently healthy snakes persistently infected by various arenavirus species have been imported and intermingled in high-density breeding operations. this possible anthropogenic disruption of pathogen ecology is reminiscent of the influenza virus diversity generated in live animal markets [ ] . sampling of viral diversity in free-ranging snake populations are needed to clarify the impact of human activities on the evolution of these viruses and to further assess the disease potential of the resulting recombinant and reassortant genotypes. in the absence of barriers to superinfection, an incalculable number of novel viral genotype configurations, made even more numerous by frequent intra-segment recombination and an error-prone polymerase, could rapidly evolve and accumulate within individual animals and in breeding facilities. in theory, this situation could be exacerbated by the introduction of mammalian arenavirus-infected rodents as feedstock [ ] , although it is unknown whether recombinant or reassortant mammalian/reptile arenaviruses are possible or viable. regardless, further investigation of high-density reptile breeding and feed rodent facilities should be considered. samples were collected between and from california, washington, arkansas, tennessee, louisiana, georgia, and florida. veterinarians in private practice or at university teaching hospitals collected samples. samples were submitted to the university of florida or the university of california san francisco for further processing and storage. blood samples were collected by cardiac or tail vein puncture and frozen until further processing. tissue samples were collected during necropsy and frozen until further analysis or placed in formalin for histopathology. for histopathology, samples were preserved in % formalin, paraffin embedded, sectioned, and stained with hematoxylin and eosin. board-certified pathologists blinded to infection status of samples examined h&e stained sections. rna extracted from tissues ( ng) or tissue culture supernatant was denatured for min at °c then cooled on ice and added to μl rt reactions containing pmoles random hexamer oligonucleotide (mds- ), × reaction buffer, mm dithiothreitol, . mm (each) deoxynucleoside triphosphates (dntps), and u superscript iii (life technologies). reaction mixtures were incubated for min at °c then for min at °c and then for min at °c. cdna was diluted : in mm tris ph , . mm edta. qpcr reactions contained μl diluted cdna, . μm each primer, mm tris ph . , mm kcl, . mm mgcl , . mm each dntp, % glycerol, . % np- , . % tween- , . μl taq dna polymerase, and x sybr green (life technologies) in each μl reaction. primer sequences are listed in s table. degenerate primers targeting the glycoprotein gene (mds- and - ) were used to screen for infection. qrt-pcr to screen for individual s and l genotypes was performed using panels of primers that were designed to discriminate between the different sequences. primer pair efficiencies were determined using template dilution series [ , ] . rna was extracted from tissues as previously described [ ] . sequencing libraries were prepared as previously described [ ] . paired-end x bp sequencing was performed on an illumina hiseq in the center for advanced technology at ucsf., producing an average of . x read pairs per sample. a stepwise pipeline was used to process sequencing data. first, data was demultiplexed. then, bases were trimmed of the end of reads and base off the end. next, low quality read pairs with any base window with an average quality score below were discarded. then, reads sharing > % global nucleotide identity (likely pcr duplicates) were collapsed using the cd-hit-est software version . [ ] . adapter sequences were trimmed from the ends of reads. then, host-derived sequences were filtered as previously described [ ] . viral genome sequences were assembled from the remaining reads an iterative strategy was used to assemble genome segment sequences. first, from each dataset, post-filtering reads were aligned using bowtie to a database composed of all alreadydescribed snake arenavirus genome segments sequences [ ] . alignment parameters were set stringently (minimum alignment score of in local mode alignment) so that only reads closely matching already described sequences aligned. alignments were converted into bam format using samtools software and inspected in geneious software [ , ] . sites differing from reference sequences were corrected to generate new draft genome sequences. remaining virus-derived reads (determined by blastx, as described below) that didn't align to an existing virus sequence were used to seed assemblies using the price targeted de novo assembler [ ] . price contigs were added to the set of genome segment sequences and the process was reiterated until all reads were accounted for as described below. once a complete set of genome segment sequences was assembled, we used bowtie to remap all reads from each dataset to the set of genome segment sequences derived from that dataset. these alignments were manually inspected and were used to generate coverage metrics. for each aligned base, coverage was only counted if the preceding and succeeding bases also aligned. this "continuous coverage" metric is more conservative than a simple coverage metric and was implemented to identify possible incorrect assemblies. bam files from these alignments, as well as fastq files of raw sequencing data for all snakes, have been deposited in the ncbi short read archive (sra; accession srp ). genome segment sequences have been deposited in genbank with accessions kp -kp . we employed the following analysis strategy to confirm that we were accounting for the full viral genetic complexity in our datasets, i.e. that we were not overlooking any viral genome segments. we used the blastx tool to align translated reads from each dataset to a database containing all available snake arenavirus protein sequences, including the ones from this study. because blastx alignments are based on protein sequence similarity, it is possible to use them to detect sequences with relatively distant homology. thus, the number of reads with blastx alignments to arenavirus protein sequences (with e-value - ) determined a minimum expected number of virus sequences in each dataset. then, we used the bowtie aligner to stringently map reads from each dataset to the coding sequences of the assemblies generated from that dataset as described above. this allowed us to confirm that the assemblies accounted for all of the arenavirus-derived sequences in each dataset. to calculate the fractional abundance of individual genotypes, we divided the number of read pairs mapping to that genotype by the total number of arenavirus-mapping reads from that dataset. we performed phylogenetic analyses to infer evolutionary relationships between viral genotypes. we created multiple sequence alignments of the coding sequences for each of the viral gene coding sequences, using mafft version v . with default parameters [ ] . these alignments were trimmed using the gblocks software version . b using default parameters except allowing up to half gaps in columns (parameters:-t = d-b = h [ ] ). we used these trimmed alignments and the jmodeltest software v . . to identify a best-fit nucleotide substitution model (gtr; [ , ] ). we ran this software with parameters:-s -f-i-g -aic-bi-c-aicc-dt-p-a-w. we used mrbayes . . to create bayesian phylogenies from these alignments, using commands lset nst = rates = propinv and mcmc ngen = [ ] . these phylogenies were visualized using figtree software (http://tree.bio.ed.ac.uk/software/ figtree/). to create phylogenies including representative snake and mammalian arenavirus sequences, we first downloaded all sequences from the ncbi nucleotide database w/ query: "txid [organism:exp]", i.e. all sequences annotated as being of arenavirus origin. we removed sequences that were not complete or not coding-complete. we extracted np and l cds from these sequences. we used cd-hit-est to create a set of representative sequences, with sequences sharing > % pairwise nucleotide identity collapsed (-c . ) [ ] . we then created and trimmed multiple alignments and phylogenies as described above. global multiple sequence alignments of all s and all l segments were created using mafft software version . with default parameters [ ] . full genome sequences for described european snake arenavirus isolates were included in these alignments (university of helsinki virus (uhv- ) and boa arenavirus nl; ncbi accessions nc_ . , nc_ . , nc_ . , and nc_ . ). alignments were analyzed with the rdp recombination detection program version . using default parameters except to specify linear molecule topology [ ] . we required that recombination events be detected by at least of the methods implemented in the software. putative recombinant segments were validated by examination of phylogenetic discordance and pairwise sequence alignments. genome segments sequences were divided into nt sliding windows (the approximate size of intergenic regions) offset by nt. centroidfold v . . was used to calculate the minimum free energy of folding for each window using parameters-g -e contrafold [ ] . variant sites were called using samtools version . . , using command mpileup-i. we required that variant sites be supported by at least reads in the context of a minimum coverage level of total reads. the number of variant sites per genome segment was calculated and normalized to the length of each segment. tissue culture ric for inoculation experiments, frozen tissue samples were thawed on ice and homogenized in mem + mm hepes (sf-mem) using a dounce homogenizer. homogenates were clarified by centrifugation at , g for minutes then passed through a . μm filter. filtrates were diluted : in sf-mem then added to cultures of near confluent jk cells. culture medium was replaced periodically and supernatants were stored at - °c until further analysis. we used qrt-pcr to measure viral rna levels in culture supernatant. we extracted rna from μl supernatant using the zymo viral rna kit (zymo research). μl rna ( % of eluate) was used as template in rt reactions as above. resulting cdna was diluted and used in qpcr reactions as above, with primers listed in s table. primer pair efficiencies were calculated as above and used to determine quantities of viral rna relative to the amount of s segment rna present in the first time point sample. jk cells were grown as described above and were plated at a density of cells per well in -well plates. one day later diluted virus stocks were added to cells. cells were incubated for - days and then supernatants were transferred to new well plates. then wells were stained with anti-ggv-np antibody, which cross-reacts with the nps of the viruses used in these experiments. staining and washing was performed as previously described [ ] . stained plates were scanned on an odyssey licor instrument to identify infected wells. supernatant from np-positive wells were transferred to -well plate wells plated the day prior with , jk cells per well. one day later cell culture supernatant was replaced. after an additional days of incubation, culture supernatant was collected and clarified by centrifugation at , g for minute. rna was isolated from these supernatants using the zr viral rna kit according to the manufacturer's protocol (zymo research). rna was used as template in qrt-pcr as described above to measure levels of viral rna of various genotypes. this study did not include experiments involving live animals. in some cases, samples (typically blood) were collected from live animals by attending veterinarians. in other cases, tissues were collected during necropsy. all samples were taken and used with owners consent. some samples were collected in the context of other, related studies: the acquisition of tissue samples at the university of florida was authorized under university of florida institutional animal care and use committee protocol a . the acquisition of samples at the university of california davis was authorized under iacuc protocol . supporting information s table. counts and fractions of viral reads in datasets. (xlsx) s fig. cartoons depicting genome segment organization, coverage levels, and predicted secondary structure. genome cartoons and features are drawn to scale. vertical lines at the end of genome segments indicate that the putative terminal sequences are included in the assembly for that segment. where applicable, partial coding sequences and the approximate location of recombination junctions are indicated. note that it was not possible to confidently identify the recombination breakpoint for the l genome segments so it is not depicted. below each cartoon are plotted coverage levels (the number of sequencing reads supporting each base in the assembly) and predicted free energy of folding (i.e. predicted rna secondary structure; -Δg) of nt sliding windows. displayed are fractional abundances of indicated s or l segment genotypes in individual samples as measured by qrt-pcr using a panel of genotype-discriminating primers (q) and sequencing (s). fractional abundance was measured for qpcr using standard curve-based quantitation and for sequencing using read mapping as in fig . neg snake is a sample from an uninfected snake and hela is total hela cell rna. asterisks ( à ) indicate the following issues related to template/primer compatibility: snake # l contains mismatches in the primer binding regions so doesn't amplify; primers targeting the l genotype also amplify recombinant genotype l in snake # and # ; primers targeting the l genotype also amplify recombinant genotype l in snake # . in these latter two cases, qpcr-measured abundance was split evenly between the two amplified genotypes. (pdf) virus evolution rapid evolution of rna genomes basic concepts in rna virus evolution self organization of matter and the evolution of biological macromolecules viral quasispecies evolution quasispecies theory and the behavior of rna viruses rna virus populations as quasispecies linkage among genes controlling inhibition of lysis in a bacterial virus genetic recombinations leading to production of active bacteriophage from ultraviolet inactivated bacteriophage particles the mechanism of rna recombination in poliovirus virus evolution isolation and preliminary genetic and biochemical characterization of temperature-sensitive mutants of reovirus evolution and ecology of influenza a viruses hiv drug resistance drug resistance, virus fitness and hiv- mutagenesis vaccine-derived polioviruses and the endgame strategy for global polio eradication arenaviridae: the viruses and their replication virus taxonomy: classification and nomenclature of viruses: ninth report of the international committee on taxonomy of viruses arenavirus genetic diversity and its biological implications arenaviruses and hantaviruses: from epidemiology and genomics to antivirals past, present, and future of arenavirus taxonomy identification, characterization, and in vitro culture of highly divergent arenaviruses from boa constrictors and annulated tree boas: candidate etiological agents for snake inclusion body disease inclusion body disease, a worldwide infectious disease of boid snakes: a review detection of novel divergent arenaviruses in boid snakes with inclusion body disease in the netherlands isolation, identification, and characterization of novel arenaviruses, the etiological agents of boid inclusion body disease replication of boid inclusion body disease-associated arenaviruses is temperature sensitive in both boid and mammalian cells structural characterization of the glycoprotein gp core domain from the cas virus, a novel arenavirus-like species new insights into the evolutionary relationships between arenaviruses provided by comparative analysis of small and large segment sequences arenavirus variations due to host-specific adaptation phylogeny and evolution of old world arenaviruses arenavirus diversity and evolution: quasispecies in vivo high genetic divergence and recombination in arenaviruses from the phylogeny of the genus arenavirus phylogeny of new world arenaviruses based on the complete coding sequences of the small genomic segment identified an evolutionary lineage produced by intrasegmental recombination genetic reassortants of lymphocytic choriomeningitis virus: unexpected disease and mechanism of pathogenesis generation of reassortants between african arenaviruses recombination between temperature-sensitive mutants of the arenavirus pichinde updated phylogenetic analysis of arenaviruses detected in boid snakes updated phylogenetic analysis of arenaviruses detected in boid snakes immunohistochemical detection of a unique protein within cells of snakes having inclusion body disease, a world-wide disease seen in members of the families boidae and pythonidae standards for sequencing viral genomes in the era of high-throughput sequencing fast gapped-read alignment with bowtie cd-hit: a fast program for clustering and comparing large sets of protein or nucleotide sequences rdp : a flexible and fast computer program for analyzing recombination coinfection of wild ducks by influenza a viruses: distribution patterns and biological significance mixed infection and the genesis of influenza virus diversity multiple infection oftt virus(ttv) with different genotypes in japanese hemophiliacs coinfection with multiple tt virus strains belonging to different genotypes is a common event in healthy brazilian adults abundance of multiple high-risk human papillomavirus (hpv) infections found in cervical cells analyzed by use of an ultrasensitive hpv genotyping assay persistent infection and promiscuous recombination of multiple genotypes of an rna virus within a single host generate extensive diversity a carrier state of lymphocytic choriomeningitis virus in l cell cultures viral superinfection in cells carrying an arenavirus and/or a togavirus response of cells persistently infected with arenaviruses to superinfection with homotypic and heterotypic viruses studies on the mechanism of lymphocytic choriomeningitis virus homologous interference resistance to superinfection of vero cells persistently infected with junin virus determinants of lymphocytic choriomeningitis interference cross-protection between tacaribe complex viruses. presence of neutralizing antibodies against junin virus (argentine hemorrhagic fever) in guinea pigs infected with tacaribe virus cross-protection in nonhuman primates against argentine hemorrhagic fever engineered luciferase reporter from a deep sea shrimp utilizing a novel imidazopyrazinone substrate generation of recombinant lymphocytic choriomeningitis viruses with trisegmented genomes stably expressing two additional genes of interest a revision of the system of nomenclature for influenza viruses: a who memorandum the modern u.s. reptile industry wet markets-a continuing source of severe acute respiratory syndrome and influenza? the lancet lymphocytic choriomeningitis virus in employees and mice at multipremises feeder-rodent operation, united states a new mathematical model for relative quantification in real-time rt-pcr experimental validation of novel and conventional approaches to quantitative real-time pcr data analysis ball python nidovirus: a candidate etiologic agent for severe respiratory disease in python regius the sequence alignment/map format and samtools price: software for the targeted assembly of components of (meta) genomic sequence data. g genesgenomesgenetics mafft multiple sequence alignment software version : improvements in performance and usability improvement of phylogenies after removing divergent and ambiguously aligned blocks from protein sequence alignments jmodeltest : more models, new heuristics and parallel computing a simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood mrbayes : bayesian phylogenetic inference under mixed models prediction of rna secondary structure using generalized centroid estimators the authors thank jens kuhn and michael buchmeier for thoughtful comments, jennifer mann for logistical assistance, kelly crotty for preliminary analyses, and jessica lund, eric chow and the center for advanced technology at ucsf for assistance with sequencing. key: cord- -j ehiwn authors: verheije, monique h.; raaben, matthijs; mari, muriel; te lintelo, eddie g.; reggiori, fulvio; van kuppeveld, frank j. m.; rottier, peter j. m.; de haan, cornelis a. m. title: mouse hepatitis coronavirus rna replication depends on gbf -mediated arf activation date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: j ehiwn coronaviruses induce in infected cells the formation of double membrane vesicles, which are the sites of rna replication. not much is known about the formation of these vesicles, although recent observations indicate an important role for the endoplasmic reticulum in the formation of the mouse hepatitis coronavirus (mhv) replication complexes (rcs). we now show that mhv replication is sensitive to brefeldin a (bfa). consistently, expression of a dominant-negative mutant of arf , known to mimic the action of the drug, inhibited mhv infection profoundly. immunofluorescence analysis and quantitative electron microscopy demonstrated that bfa did not block the formation of rcs per se, but rather reduced their number. mhv rna replication was not sensitive to bfa in mdck cells, which are known to express the bfa-resistant guanine nucleotide exchange factor gbf . accordingly, individual knockdown of the golgi-resident targets of bfa by transfection of small interfering rnas (sirnas) showed that gbf , but not big or big , was critically involved in mhv rna replication. arf , the cellular effector of gbf , also appeared to be involved in mhv replication, as sirnas targeting this small gtpase inhibited mhv infection significantly. collectively, our results demonstrate that gbf -mediated arf activation is required for efficient mhv rna replication and reveal that the early secretory pathway and mhv replication complex formation are closely connected. viruses rely on cellular host factors for virtually all steps of their infection cycle. however, the cellular proteins required and the cellular pathways hijacked by viruses have hardly been elucidated. all positive-strand rna viruses assemble in infected cells their replication complexes (rcs) in association with intracellular membranes [ , , , , ] . the induction of such local microenvironments is likely advantageous for the virus, as membrane association may facilitate the recruitment of both the viral and cellular components involved in rna replication. alternatively, membrane association may provide a shielded environment that prevents the activation of, or protects against, antiviral host cell responses like those mediated by interferon. coronaviruses belong to a family of enveloped positive-strand rna viruses in the order nidovirales. upon translation of the viral genomic rna, two very large polyproteins (approximately , and , amino acids) are synthesized, the autoproteolytic cleavage products of which collectively form the rcs. these rcs are associated with double membrane vesicles (dmvs [ , , ] ), which appear as cytoplasmic foci when analyzed by fluorescence light microscopy and increase in number during the course of the infection [ , , , ] . it is plausible that the nonstructural viral proteins (nsps) mediate the formation of dmvs by modifying intracellular membranes and by recruiting cellular components to their need. recent studies suggest the endoplasmic reticulum (er) to be the lipid donor compartment of the membrane-bound coronavirus rcs [ , , , ] , although colocalization of nsps with markers for endosomes, golgi and autophagosomes has also been described [ , , , , ] . brefeldin a (bfa) is a well known fungal metabolite that induces the redistribution of golgi proteins into the er [ , ] , effectively resulting in the block of transport though the secretory pathway [ , ] . this drug inhibits the activation of adpribosylation factor (arf) small gtpases by targeting the large guanine nucleotide exchange factors (gefs) gbf (golgi-specific resistance factor ), and big (bfa-inhibited gef) and [ , , ] . more specifically, bfa locks arf*gdp when bound to gef, thereby blocking the gef activity at an early stage of the reaction, prior to guanine nucleotide release [ , ] . the large gefs function in the er to golgi transport pathway [ ] and localize to the cis-(gbf ) and trans-sides (big and big ) of the golgi complex [ ] . the cellular effectors of these gefs, arfs, are divided into three classes: class i (arf - ), class ii (arf and ), and class iii (arf ) [ ] . class i arfs regulate the assembly of coat complexes onto vesicles budding from compartments along the secretory pathway and activate lipid-modifying enzymes (reviewed in [ , ] ). while the function of class ii arfs remains largely unclear, the class iii arf is thought to regulate endosomal membrane traffic [ , ] . gbf and the bigs are likely to activate distinct subclasses of arfs at specific locations in order to regulate different types of transport routes [ ] . in the field of virology, bfa has been used, besides for studying viral protein transport and virus assembly [ , , , , , ] , to investigate the formation of rcs and rna replication of several positive-strand rna viruses [ , , , ] . for example, poliovirus rna replication was shown to be sensitive to bfa. in the presence of this drug, poliovirus replication sites were not formed and rna replication was completely blocked [ , ] . remarkably, other members of the picornavirus family appeared to differ in their sensitivity to bfa. whereas echovirus rna replication was strongly inhibited by bfa, rna replication of encephalomyocarditis virus was not affected at all, while parechovirus exhibited an intermediate sensitivity to it [ ] . relatively little is known about the host pathways involved in coronavirus rna replication and in rc formation. recently, we demonstrated the important role of the er in the generation of the rcs. while mhv nsp was localized to this organelle when expressed alone, it was recruited to the replication complexes in infected cells [ ] . furthermore, coronaviral replication was inhibited when the er export machinery was blocked by use of the kinase inhibitor h or by expression of a dominant active mutant of sar [ ] . other cellular proteins and pathways are likely to contribute to the formation of the coronavirus rcs as well. here, we studied the involvement of bfa-sensitive pathways in mhv replication and rc formation. our results demonstrate that gbf -mediated arf activation is required for efficient mhv rna replication. moreover, together with our recent observation about the relevance of the er in the same process, our data reveal that the early secretory pathway and mhv replication are intimately connected. bfa is known to disturb membrane traffic in most cell types, resulting in a redistribution of golgi proteins into the er [ , ] . we first confirmed the sensitivity of murine lr cells to bfa by immunofluorescence using antibodies directed against the golgi protein marker gm [ ] . indeed, after treatment of the cells with mg/ml bfa for h, the typical golgi staining pattern of gm was lost, concomitant with a reticular redistribution of the protein marker (data not shown). next, we tested whether mhv infection was sensitive to bfa. therefore, lr cells were inoculated with a luciferase-expressing recombinant of mhv-a (mhv-eflm) in the presence or absence of mg/ml bfa. after h, the inoculum was removed and the cells were further incubated either in the presence or in the absence of bfa. at h p.i., the intracellular luciferase expression level was determined relative to untreated cells. luciferase expression was inhibited more than % when bfa was present from - h p.i., whereas bfa treatment during virus inoculation had only a minor effect on reporter gene expression (fig. a) . although this latter decrease might have resulted in part from a reduced entry, the negative effect of bfa on mhv replication and transcription is evident from the profoundly impaired mhv reporter gene expression when bfa was added post inoculation ( - h p.i.). in a control experiment, the effect of bfa on sindbis virus replication in lr cells was assayed by using sindbis pseudovirus particles containing luciferase-expressing replicons. as described previously [ ] , sindbis virus replication was not affected by the bfa treatment (fig. a) . this result indicates that the observed effect of bfa on mhv-driven luciferase expression was not due to non-specific drug-induced toxicity. although we have demonstrated in previous studies that reporter gene expression by mhv is a reliable measure for coronavirus replication [ ] , we wanted to confirm that the reduction in luciferase expression resulted from a corresponding decrease in viral rna synthesis rather than from inhibition of viral protein translation. to this end, a similar experiment as shown in fig. a was performed, in which the amount of intracellular genomic viral rna was determined by real-time taqman pcr. as for the luciferase expression levels, the amount of genomic rna was found to be severely reduced when bfa was added directly after the virus inoculation (fig. b) , whereas a less profound effect was observed when cells were treated during virus inoculation. very similar results were obtained when targeting the taqman pcr to a different region of the viral genome (data not shown). to more directly check for an effect of bfa on the translation of viral mrnas, we performed an additional experiment. lr cells were infected at high multiplicity with the recombinant virus mhv- afls, which expresses the firefly luciferase, and subsequently transfected with a synthetic mrna encoding renilla luciferase. this synthetic mrna mimics viral mrnas as it contains ' and ' untranslated regions identical to those found in the viral genome. the cells were incubated in the presence or absence of bfa ( - h p.i.) after which the intracellular renilla and firefly luciferase expression levels were determined. the results show that bfa treatment did not inhibit the synthesis of renilla luciferase from the synthetic mrna, while firefly luciferase expression driven by the recombinant virus was severely affected (fig. c) . renilla luciferase expression was also not affected in the absence of a viral infection (data not shown). all together, these results indicate that bfa inhibits mhv rna replication while translation of viral mrnas is not affected. next, we determined the post inoculation period during which mhv replication was most sensitive to bfa, by analyzing the luciferase expression levels as they are a reliable measure for rna replication. thus lr cells infected with mhv-eflm were treated with bfa for overlapping h periods. at the end of each incubation period the intracellular luciferase expression levels were coronaviruses are the causative agents of many respiratory and enteric infections in humans and animals. as with all viruses, virtually all of the steps of their infection cycle depend on host cellular factors. as the first and most crucial step after their entry into cells, coronaviruses assemble their replication complexes (rcs) in association with characteristic, newly induced membranous structures. the cellular pathways hijacked by these plus-strand rna viruses to create these ''factories'' have not been elucidated. here, we study the involvement of the secretory pathway in mouse hepatitis coronavirus (mhv) replication by using the drug brefeldin a (bfa), which is known to interfere with er-golgi membrane traffic by inhibiting the activation of adp-ribosylation factor (arf) small gtpases. our observations show that mhv rna replication is sensitive to bfa. in agreement herewith we demonstrate, by using various techniques, that the bfasensitive guanidine nucleotide exchange factor gbf and its downstream effector arf are of critical importance for coronavirus replication. from our results we conclude that mhv rna replication depends on gbf -mediated arf activation. our study provides new insights into the close connection between mhv replication and the early secretory pathway. determined and compared to those in mock-treated cells. the results showed that replication was affected throughout the course of the infection (fig. d) ; however, the effects were most pronounced during the early phases of infection. to confirm our observation that bfa inhibits mhv replication but also to prove that the effects of this drug are due to the inhibition of gef activities, we next analyzed to what extent the expression of a dominant-negative mutant of arf (t n) would affect mhv infection. this arf mutant has a decreased affinity for gtp and, following gdp displacement, it remains 'nucleotidefree' for a longer period than wt arf [ ] . as a consequence, expression of arf -t n mirrors the effects of bfa [ ] . in addition to this protein, we included a constitutive-active arf mutant (arf -q l), which persists in the gtp-bound state longer than wild-type arf, resulting in a prolonged arf activation. expression of this latter mutant is known to inhibit transport at later steps in the secretory pathway, e.g. from vesicular tubular clusters (vtc) to the golgi complex and between golgi stacks [ ] . lr cells were transfected with plasmids expressing yfp fusions of either wild type arf , arf -t n or arf -q l. after transfection, the cells were inoculated with an rfpexpressing mhv-a recombinant (mhv-rfp) that allows flow cytometric analysis of mhv replication [ ] . the percentage of rfp-positive cells in the yfp-expressing population was determined relative to that of the wild type arf expressing cells (fig. e) . overexpression of the wt arf fusion protein itself did not significantly affect mhv infection when compared to nontransfected cells (data not shown). the results indicate that overexpression of the dominant-negative arf mutant inhibited mhv infection profoundly, thereby confirming the results obtained with bfa. in contrast, expression of the constitutiveactive mutant of arf did not influence mhv replication. as bfa is known to affect intracellular vesicle formation and transport, and because mhv replicates its genome in association with dmvs, we next investigated the effect of bfa on the assembly of the mhv rcs. first, we checked whether the morphological integrity of the rcs was affected in the presence of bfa. therefore, lr cells infected with mhv-a were treated with bfa for minutes starting . h p.i. they were subsequently fixed and processed for immunofluorescence using antibodies both against nsp , which served as a protein marker for the mhv replication sites [ , ] , and against the viral structural protein m, known to reside in the golgi [ ] . the nsp antibody revealed the typical perinuclear staining pattern in both treated and non treated infected cells ( fig. a) . in contrast, a dispersed distribution of m protein was observed in bfa-treated cells reflecting the collapse of the golgi, whereas in non-treated cells the m protein showed a clear golgi-like staining ( fig. a) . these results indicate that, once formed, the replication sites are not disrupted by bfa. subsequently, we investigated whether bfa inhibited rc formation early in the infection. bfa was therefore added to lr cells directly after inoculation with mhv-a and staining was performed at h p.i using the nsp antibody. although some perinuclear staining of nsp could be detected in bfa-treated cells, the number and intensity of the nsp containing foci were clearly reduced when compared to non-treated cells (fig. b ). we next investigated whether these nsp puncta represented mhv replication sites. therefore, we studied the ability of the nsp foci to recruit the nucleocapsid protein n, a protein previously shown to localize to the rcs [ , ] . three parallel cultures of lr cells were transfected with a plasmid coding for a mhv n-gfp fusion protein and h post transfection two of them were infected with mhv-a . bfa ( mg/ml) was added to one of these latter cultures directly after inoculation (t = h p.i.). at h p.i., the cells were fixed and subsequently processed for immunofluorescence using the anti-nsp antibody (fig. c ). as expected, n-gfp was diffusely localized to the cytosol in non-infected cells (indicated by an arrowhead in fig. c ). in contrast, when cells were infected with mhv, this fusion protein also appeared in foci that co-localized with nsp (indicated by arrows in fig. c ). this co-localization was observed both in mock-and in bfa-treated cells, indicating that the nsp foci that had been formed in the presence of bfa, though decreased in number and intensity, correspond with the replication sites. in complete agreement with the luciferase expression data shown above, this result demonstrates that bfa inhibits, but does not completely block, the formation of rcs. to study the effects of bfa on the dmvs at an ultrastructural level, mhv-infected lr cells were fixed at h p.i. and embedded in epon resin in order to be analyzed by electron microscopy. dmvs (indicated by the asterisks in fig. a ) were always seen organized in clusters often located in the perinuclear area. the morphology and dimensions of these vesicles were similar to those previously described for the dmvs harboring the rcs [ , , , , , ] . importantly, these vesicles were not observed in mock-infected cells (data not shown). fig. b shows a close view of these dmvs, in which the translucent interior is surrounded by a double membrane. the presence of an inner web-like structure is most likely artificial [ ] . treatment of cells with bfa ( - h) led to the expected disappearance of an apparent golgi complex with the concomitant expansion of the er volume (not shown). in these cells, vesicles with a morphology almost identical to those present in non bfatreated cells were observed (fig. a) . however, the number of these dmvs was significantly decreased (p, . ) in bfa-treated cells as compared to non-treated cells ( . vs. . on average per section, fig. c ). the reduction in the number of dmvs is likely to be an underestimation as only em sections were included in the analyses in which at least one replication vesicle could be detected. strikingly, the double membrane of the replication vesicles was visually more pronounced in bfa-treated cells than in untreated cells (fig. b) , which might relate to the swelling of the er observed after bfa addition. the dmvs were slightly bigger in the bfa-treated cells ( . nm +/ . compared to . nm +/ . in non-treated cells; p, . ; fig. d ), although the significance of this latter observation is not clear at present. overall, our ultrastructural analysis of mhv-infected cells confirms that treatment of cells with bfa decreased the number of replication vesicles, consistent with the reduced viral rna replication in the presence of bfa. to address which arf gefs contribute to mhv replication, we next focused on the bfa-sensitive gefs localized in the secretory pathway, i.e. gbf , big and big . first, we studied whether coronavirus replication was affected by bfa in mdck cells. these cells have a bfa-resistant golgi-apparatus due to a point mutation in gbf (m l; f. van kuppeveld, unpublished results). however, the trans-golgi network (tgn) and the endocytic organelles in mdck cells are still sensitive to bfa [ , , ] . mdck cells stably expressing the ceacam a receptor (mdck(mhvr); [ ] ) were inoculated with mhv-eflm and bfa was added either during ( - h p.i.) or after ( - h p.i.) the inoculation. the results show that mhv replication was not affected by bfa treatment of the cells during either time period (fig. a) , pointing toward a possible involvement of the bfa-sensitive gbf protein in mhv replication. to confirm that gbf , rather than big or big , is required for mhv replication, each one of these gefs was specifically and singularly depleted by rna interference before assaying mhv replication. for each target gene, three sirna oligos were transfected into hela-ceacam a cells. at h post transfection, the cells were infected with the luciferase-expressing mhv- afls. six h later, the number of viable cells and the luciferase expression levels were determined ( fig. s a and s b) as described in the materials and methods. in fig. b the results are presented as relative luciferase expression (rii) levels, i.e. the luciferase activity expressed relative to mock-treated cells after correction for the number of viable cells. transfection of control sirnas targeting the housekeeping protein glyceraldehyde -phosphate dehydrogenase (gapdh) did not change the rii, whereas sirnas targeting firefly luciferase reduced the rii up to % (p, . ) demonstrating the efficiency of the sirna transfection. importantly, down-regulation of gbf resulted in a drastic inhibition of rii (p, . ) whereas sirnas targeting big and big did not have a significant effect (fig. b) . almost identical results were obtained when the three sirna oligos for each gene were singly transfected (data not shown). in a parallel experiment, we demonstrated that the down-regulation of the major target of gbf , arf , had a similar phenotypic effect on mhv replication as seen for gbf (fig. b) . to prove the specificity of our results, we performed a series of controls. first, the specific knockdown of the respective mrnas after sirna transfection was confirmed by quantitative rt-pcr analysis. at h after transfection of the sirnas, the corresponding mrna levels for big , big , gbf and arf were found to be reduced by %, %, %, and %, respectively. the mrna levels were not affected after transfection of non-corresponding sirnas, demonstrating the specificity of the mrna depletion (data not shown). second, the functional knock-down of gbf and arf at the protein level was demonstrated by cotransfection of plasmids encoding gbf -yfp and arf -yfp together with either the gbf -or arf -specific sirnas, respectively. this approach was chosen because of the unavailability of specific anti-antibodies. twenty-four h after transfection, the cells were fixed and yfp-positive cells were counted. fig. c demonstrates that gbf and arf expression are prohibited in the presence of their specific sirnas. next, we analyzed whether inhibition of mhv replication after depletion of arf coincided with a collapse of the golgi complex as observed after bfa treatment. again, hela-ceacam a cells were transfected with sirnas targeting arf and subsequently processed for immunofluorescence at h post transfection using the gm antibody. in the arf sirna-transfected cells, the gm staining was indistinguishable from that in mock-treated cells (fig. d) indicating that loss of arf did not lead to the collapse of the golgi into the er. this is in complete accordance with the results of volpicelli-daley et al. [ ] , who demonstrated that arf depletion alone is not sufficient to mimic the bfa effect on the golgi complex, but rather requires a simultaneous depletion of arf and arf [ ] . having established that depletion of gbf or arf affects mhv replication profoundly, we studied whether the formation of the mhv rcs was similarly affected. to this end, we performed a similar knock down experiment in which we transfected sirnas targeting either arf or gbf and subsequently infected the cells with a recombinant mhv, which expressed an additional copy of nsp , now fused to gfp. the nsp -gfp fusion protein co-localizes with nsp and provides an additional marker for the rcs (data not shown). six hours after infection the cells were fixed and processed for immunofluorescence with the nsp antibody. in mock transfected cells, many gfp and nsp positive foci were observed, which largely co-localized (fig. e ). in agreement with the relative luciferase expression values shown in fig. b , both in arf -and gbf depleted cells, the number and intensity of the nsp positive foci was reduced, similar to what had been observed in bfa-treated cells (fig b) . apparently, the number of mhv rcs is reduced in these cells. strikingly, however, it appeared that the nsp -gfp expression was much more affected than that of nsp by the depletion of either arf or gbf , as hardly any gfp fluorescence could be detected. while nsp is expressed directly from the viral genome, the nsp -gfp fusion protein is expressed from a subgenomic mrna and hence replication and transcription is required for its expression. these results therefore indicate that not only fewer rcs are formed in the absence of either gbf or arf , but that these rcs are also impaired in their rna synthesis. in conclusion, our results demonstrate that depletion of gbf and arf reduces mhv replication as well as the number of rcs. furthermore, our results indicate that the rcs formed in the absence of either gbf or arf are less active. in addition, inhibition of mhv replication is not caused by the collapse of the golgi apparatus per se, as in arf -depleted cells virus replication is severely affected whereas the overall morphology of the golgi complex is unaltered. we next addressed the question whether arf is recruited to the replication sites. to this end, lr cells expressing wild type arf fused to yfp were infected with mhv-a and either fixed at an early ( h) or a late ( h) time point p.i. before identifying the replication sites by immunostaining the cells with nsp antibodies. figure a shows that arf -yfp was predominantly localized to the golgi apparatus (indicated by the arrowhead on the left panel of fig. a ) both at h p.i. and h p.i. at h p.i., only in a minority of the cells co-localization between arf and nsp was observed (indicated by the arrows in fig. a ). no co-localization could be observed in infected cells at h p.i. similar results were obtained for gbf (data not shown). many downstream effectors of arf have been described, and the list is still growing. one of the best known functions of arf involves the regulation of copi-mediated vesicular transport. for the bfa-sensitive poliovirus, copi has been found to localize at the replication vesicles [ ] . to study whether a similar recruitment of copi to the replication vesicles occurs during mhv replication, we determined its localization in mhv-infected cells. thus, hela-ceacam a cells were infected with mhv-nsp gfp. this recombinant virus allowed us to directly visualize the replication vesicles without having to perform an immunostaining with the anti-nsp antibodies. this was desirable as both the antibody against accop (two subunits of the copi coat) and the nsp antibody had been raised in rabbits. at h p.i. the cells were fixed and processed for immunofluorescence analysis using the accop antibody. the results show that, in addition to a diffuse staining throughout the cell, copi was primarily localized in a golgi-like pattern (fig. b) . copi did not co-localize with the nsp -gfp positive sites, indicating that copi was not recruited to the replication sites of mhv. another well known effector of arf is phospholipase d (pld), a lipid-metabolizing enzyme involved in membrane dynamics and vesicular transport [ , ] . to analyze whether rcs recruit pld, lr cells were transfected with a construct expressing pld b fused to gfp and subsequently infected with mhv-a . the cells were fixed at h p.i. before identifying the replication sites by immunostaining the cells with nsp antibodies. no co-localization between the rcs and pld b could be observed (fig. s a) . furthermore, specific inhibition of pld by -butanol [ ] did not affect mhv luciferase expression compared to controls (fig. s b) . further studies will be required to examine the role of other arf effectors. finally, we studied whether normal vesicular trafficking is affected in mhv-infected cells. to investigate this, we made use of a gaussia reporter gene, the protein product of which is secreted upon expression [ , ] . cells were transfected with a plasmid encoding this gene under the control of a cmv promoter and subsequently infected with either mhv-a , mock-infected, or treated with bfa. at . h p.i. the intracellular and extracellular levels of gaussia luciferase were measured. thus, the ratio of the luciferase activity in the cell lysate and in the culture supernatant was determined for each condition. while in mock-infected cells almost % of the total amount of gaussia luciferase was found in the culture supernatant, in mhv-infected cells, the amount of secreted gaussia luciferase was decreased about -fold to % (fig. ) . bfa treatment inhibited, as expected, gaussia protein secretion almost completely. from this we conclude that although mhv rna replication depends on gbf mediated arf activation, mhv infection does not drastically impair the secretory pathway. this result is not unexpected, as coronaviruses require a functional secretory pathway for the release of their progeny virions. rna viruses use and manipulate cellular membranes for the assembly of their replication and transcription structures. we and others have shown that coronaviruses exploit the early secretory pathway, but the way in which they do so is not understood. in this report we have demonstrated using several different approaches that mhv requires a functional gbf -arf pathway for efficient rna replication. first, we showed that mhv, but not sindbis virus replication is sensitive to bfa in murine lr cells. second, we observed that mhv replication is not sensitive to bfa in mdck cells, which contain a bfa-resistant gbf . third, we showed that the specific sirna-based knockdown of the bfasensitive gef gbf , but not big and big , strongly affects mhv infection. fourth, also arf , a downstream effector of gbf , appeared to be required for efficient mhv replication, as shown by the inhibition of mhv-driven reporter gene expression during sirna-mediated down regulation of arf as well as during expression of an inactive arf mutant. the inhibition of coronavirus rna replication in the presence of bfa is either caused by direct inhibition of rc formation, resulting in reduced rna replication, or by inhibition of rna replication via another mechanism, resulting in reduced de novo formation of rcs. though it is difficult to distinguish between these two scenarios, our results indicate the latter option to be most plausible. although bfa reduced the number of rcs, their formation was not completely blocked as demonstrated by immunofluorescence staining of the rcs using the nsp antibody and by quantitative electron microscopy. apparently, bfa did not prevent the formation of rcs after translation of the incoming genomic rna. in addition, mhv replication was inhibited by bfa throughout the infection. early in infection the inhibition was more profound than at later time points, when many transcriptionally active rcs have already been formed. furthermore, while the inhibition of reporter gene expression in the presence of bfa, or after depletion of either gbf or arf , is in complete agreement with the reduced numbers of rcs, our results also indicate that the few rcs that are formed in the absence of gbf or arf are less active. therefore, we hypothesize that bfa inhibits mhv rna replication by affecting rc maturation or functioning rather than rc formation per se (fig. ) . replication of several viruses has now been shown to be sensitive to bfa. these viruses, which include poliovirus [ , , ] , grapevine fanleaf nepovirus [ ] and mhv (this study), all appear to use er-derived membranes for the formation of their rcs ( [ ] , [ ] and [ , , ] , respectively). strikingly, picornaviruses belonging to different genera were found to differ in their sensitivity to bfa, which was suggested to correspond with differences in the assembly of their rcs [ ] . replication of equine arterivirus, a distant relative of coronaviruses, was observed not to be sensitive to bfa [ ] , while other nidoviruses have not been studied to date. unlike for poliovirus [ ] , arf is hardly recruited to coronavirus rcs. we therefore hypothesize that downstream effectors of gbf -arf are involved in mhv replication. to date, more than downstream effectors of arf have been identified [ , , , ] , and each one of these might thus be somehow implicated in the functioning of the mhv rcs. the most well known effector of arf is copi. for picornaviruses, bfa sensitivity was suggested to correlate with the recruitment of copi to these sites [ ] . however, no co-localization between copi and the mhv rcs could be observed. this is in agreement with the almost complete absence of arf at these sites. in addition, coronavirus rcs did not co-localize with pld nor was coronavirus replication affected by inhibition of phospholipase d, a lipid-metabolizing enzyme involved in membrane dynamics and vesicular transport [ , ] . it might be that the gbf -arf pathway simply functions to deliver lipids to the rcs. in agreement herewith, cerulenin, an inhibitor of phospholipid biosynthesis, severely inhibits mhv replication (c.a.m. de haan, unpublished results). nonetheless, the observed inhibition of mhv infection after bfa treatment is probably not an indirect consequence of the collapse of the golgi complex as, unlike bfa treatment, arf depletion did not affect the morphology of the golgi complex (fig. d) . consistent herewith, another recent study showed that arf depletion did not affect the golgi morphology or protein transport [ ] . several studies have indicated that coronavirus replication and the er are closely connected. electron microscopical analyses of infected cells showed the partial co-localization of coronavirus replicase proteins with the soluble er resident protein disulfide isomerise [ ] , while the dmvs were often found in close proximity to the er and occasionally in continuous association with it [ , ] . furthermore, when expressed in the absence of a coronavirus infection, the nsp and nsp proteins were inserted into the er and became modified by the addition of n-linked sugars [ , , ] , whereas expression of tagged mhv nsp in mhv-infected cells resulted in the recruitment of the protein to the replication complexes [ ] . in addition, coronavirus replication was inhibited when the er export machinery was blocked by the use of the kinase inhibitor h or by expression of dominantactive mutant of the small gtpase sar [ ] . we now show by using several approaches that mhv rna replication also depends on gbf -mediated arf activation. apparently, an intimate association exists between the early secretory pathway and mhv replication. interestingly, whereas h blocked rc formation completely [ ] , this was not the case when the gbf -mediated activation of arf was impaired by bfa. rather it appears that the rcs formed in the absence of gbf or arf are less active, suggesting a role for these proteins in rc maturation or functioning (fig. ) . clearly, further investigations are needed to unravel the precise mechanism by which the secretory pathway contributes to the biogenesis of functional coronavirus rcs and to rna replication. hela-ceacam a cells were generated by transfecting hela cells (obtained from the mpi-cbg high-throughput technology development studio [ ] ) with the expression plasmid pmhvr [ ] as described before [ ] . murine lr [ ] , hela-ceacam a, and madin-darby canine kidney-ceacam a [mdck(mhvr); [ ] cells, which all stably express the mhv receptor mceacam a, were maintained as monolayer cultures in dulbecco modified eagle medium (dmem; cambrex) containing % fetal calf serum (fcs), iu of penicillin/ml, mg of streptomycin/ml (all from life technologies), and . mg/ml g (life technologies, paisley, uk). split cells, i.e. bhk- cells stably expressing sindbis virus structural proteins [ ] , were maintained in glasgow mem (invitrogen) containing % fcs, iu of penicillin/ml, mg of streptomycin/ml, mg/ml g and mg/ml hygromycine b (boehringer gmbh) and used to generate sindbis pseudovirus particles containing a replicon expressing firefly luciferase. to this end, the firefly luciferase gene was cloned into the psinrep vector (invitrogen) using conventional cloning procedures. the resulting vector was subsequently processed further according to polo et al. [ ] to produce the pseudovirus particles. lr cells were used to propagate the wild type and recombinant mhvs (based on strain a ). the recombinant viruses expressing the firefly luciferase gene (mhv-eflm and mhv- afls) or the red fluorescent protein (rfp) gene have been described before [ , ] . the recombinant virus mhv-nsp gfp, which expresses a nsp -green fluorescent protein (gfp) fusion protein, was generated in a similar way as described previously for mhv-nsp gfp [ ] . briefly, an nsp -gfp fusion construct was cloned behind an additional transcription regulation sequence into a derivative of the rna transcription vector pmh [ ] . targeted recombination to obtain the recombinant mhv-nsp gfp was performed as described before [ ] . antibodies directed against the mhv nsp (anti-p , kindly provided by m. denison, vanderbilt university medical center, nashville, usa [ ] ), the amino terminus of the mhv m protein (j . , kindly provided by j. fleming, university of wisconsin, madison, usa [ ] ), against accopi (anti-accopi, kindly provided by f. wieland, university of heidelberg, germany), against gbf (anti-gbf ) and against the golgi marker gm (anti-gm ) (the latter two from bd transduction laboratories, figure . model of mhv rcs and their links to the early secretory pathway. two major steps in the anterograde protein secretion route (reviewed in [ ] ) are linked to mhv rc formation and/or rna replication. first, transport of proteins out of the er requires er exit site formation controlled by sar p [ , , ] . blocking this early step by using the drug h [ ] or by expressing of a dominant mutant of sar p [ ] blocks mhv replication profoundly [ ] . next, er exit sites develop into, or form de novo, vesicular-tubular clusters (vtcs) (also called ergic), for which gbf and arf are required. this step, which can be blocked by bfa, by expressing a dominant-negative mutant of arf or by down-regulating arf using sirnas [ ] , is also involved in mhv rc formation (this manuscript). however, a fully functional secretory pathway is not essential, as a dominantactive mutant of arf , which blocks transport between vtcs and cis-golgi [ ] , does not impair mhv replication. doi: . /journal.ppat. .g san jose, usa) were used. the conjugated secondary antibodies were purchased from jackson immunoresearch laboratories. plasmids containing the different arf and gbf genes in frame with either a gfp or a yellow fluorescent protein (yfp) tag were obtained from g. romero [ ] and c. jackson [ ] , respectively. pgbf -yfp and parf -yfp encode the wild type proteins fused to yfp. parf t n-yfp and parf q l-gfp encode a dominant-negative and a dominant-active mutant of arf fused to yfp and gfp, respectively [ ] . the pn-egfp plasmid, which encodes the mhv nucleocapsid (n) protein extended at its c-terminus with gfp was constructed by cloning a pcr fragment, specifying the n gene without its stop codon, into pegfp-n (clontech), using conventional cloning procedures. the plasmid encoding the gaussia reporter gene behind a cmv promoter was generated by replacing the egfp gene in pegfp-c (clontech) with the gaussia luciferase gene from pgluc-basic (new england biolabs) using conventional cloning methods. the viral expression plasmid pm f-rl-m was generated by cloning a synthetic dna segment (genscript ß ) corresponding to the extreme ' nt and the extreme ' nt of the mhv-a genome, separated by a nhei restriction site and flanked by a t promoter and a poly(a) sequence, upstream and downstream, respectively, into puc . subsequently, the coding region for renilla luciferase, obtained from prlnull (promega), was cloned into the nhei-digested vector. subconfluent monolayers of lr cells grown on coverslips in cm tissue culture dishes were overlaid with transfection medium consisting of . ml of optimem (invitrogen) that contained ml lipofectamine (invitrogen) and mg of dna. after hours, the medium was replaced with dmem containing % fcs. at h after transfection the cells were processed further as indicated. the plasmid pm f-rl-m was linearized using a paci restriction site directly downstream of the poly(a) sequence, and subsequently rna transcripts were produced using the t messagemachine kit (ambion) according to the manufacturer's instructions. of the transcripts, . pmol of rna was transfected into mock-or mhv- afls-inoculated lr cells at h p.i. using lipofectamine (invitrogen). next, the cells were treated with or without mg/ml bfa from h until h p.i., after which the cells were lysed and intracellular renilla and firefly luciferase activity was measured with the dual-luciferase assay kit (promega) according to the manufacturer's protocol. cells were fixed using a % paraformaldehyde solution in phosphate buffered saline (pbs), and subsequently permeabilized with . % triton-x in pbs. next, the cells were incubated for h with the first antibody diluted in pbs containing % normal goat serum. after several washing steps, the cells were incubated with an appropriate dilution of secondary antibody in the same buffer for h. after three subsequent washing steps, the coverslips were mounted in fluosave (calbiochem). the immunofluorescence staining was analyzed using a confocal laser-scanning microscope (leica). gfp/yfp and fitc were excited at nm and cy at nm. virus replication was quantified by determining either the virusdriven luciferase expression levels or the amount of genomic rna. to this end, lr or mdck(mhvr) cells were inoculated at a multiplicity of infection (moi) of with mhv-eflm, mhv- afls or sindbis pseudovirus particles in the presence or absence of mg/ ml bfa in dmem. after h, the culture medium was replaced by dmem containing % fcs and antibiotics, again in the presence or absence of mg/ml bfa. at the indicated time points, the luciferase expression in the cells was determined using the firefly luciferase assay system (promega) according to manufacturer's instructions and using a single-tube luminometer (turner designs, td- / ). alternatively, rna was isolated from the cells using the qiagen rneasy kit (qiagen) according to the manufacturer's protocol. taqman single-tube reverse transcription-pcr (rt-pcr) assay (pe biosystems, foster city, california, usa) was performed essentially as described by de haan et al. [ ] . the reactions were performed in triplicate according to the manufacturer's instructions by using the taqman rt-pcr kit (pe biosystems) and an abi prism sequence detector. small interfering (si) rna-mediated knockdown experiments sirna duplexes targeting different sites within the coding sequences of gbf , big , big , and arf were designed by and obtained from ambion inc. (three sirnas per gene; nucleotide sequences available on request). sirnas targeting gapdh, luciferase gl +gl , and kif (all from ambion) were taken along as controls in each experiment. one day after seeding the hela-ceacam a cells, they were transfected with a final concentration of nm sirna using oligofectamine (invitrogen). seventy-two h after transfection, the cells were inoculated with mhv- afls at such a moi that approximately % of the mocktreated cells became infected. at h post infection (p.i.), the cell number and viability was measured by wst- assay according to the manufacturer's protocol (roche diagnostics gmbh). subsequently, the medium was replaced by dmem lacking phenol red (cambrex) and steadylite hts firefly luciferase substrate (perkin elmer) was added. luciferase expression was determined using a luminescence plate reader (berthold centro lb ). each sirna experiment was performed in triplicate. for each well, luciferase values were corrected for the cell number and viability as determined by the wst assay relative to the mock-treated cells. to validate the functional knockdown of the targeted genes, mrna levels of each gene were determined after sirna transfection using taqman gene expression assays (applied biosystems, ca, usa), according to the manufacturer's protocol. to determine whether sirnas targeting the arf and gbf genes effectively depleted hela-ceacam a cells from the corresponding proteins, a sirna transfection experiment was performed in which ng of the plasmids encoding either arf -yfp or gbf -yfp were added to the transfection mixture containing the corresponding sirnas. twenty-four h after transfection, the cells were fixed and representative images were taken by an automated cellworxtm microscope (applied precision) with a objective. lr cells transfected with parf -yfp, parf t n-yfp, or parf q l-gfp were inoculated with mhv-rfp (moi of ) at h post transfection. two h p.i. mm hr peptide [ ] was added to inhibit syncytia formation. at h p.i., the cells were collected and fixed using a % paraformaldehyde solution. after two washes with pbs, the samples were analyzed employing a facscalibur tm flow cytometer (becton dickinson) gating for yfp/gfp-positive cells in the forward and side scatter, such that a limited cell population with similar arf expression levels was selected. from the yfp/gfp-positive population, the fraction of cells expressing rfp was determined. lr cells infected with mhv-a and treated from to h p.i. with or without mg/ml bfa were resuspended in % glutaraldehyde in . m cacodylate buffer (ph . ) for at least h at room temperature (rt). this buffer was then replaced with fresh one and the fixation was continued overnight. cells were then centrifuged, washed times with the . m cacodylate buffer before being post-fixed in % oso , . % ferrocyanide at uc for min. next, the cell pellet was washed times with distilled water and left sit in the last wash for min before being centrifuged and resuspended in warm % low melting point agar (roche, basel, switzerland) and immediately spun down. after solidification of the agar on ice, the tip containing the cells was cut into small mm blocks. these blocks were then dehydrated by immerging them into increasing amounts of ethanol ( %, %, %, %, % and times %) by incubation on a rotatory wheel for at least min at rt for each step. these amalgamations were followed by others in , -propylene oxide (merck, haarlem, netherlands)-epon resin ( : ) for min, , propylene oxide -epon resin ( : ) for min, , -propylene oxide-epon ( : ) for min and epon resin overnight. the epon solution was prepared by mixing g of glycid ether , g of dodecenylsuccinic acid anhydride, g of methylnadic anhydride and ml of benzyldimethylamine (all from serva, heidelberg, germany). the epon resin was then replaced the following day with freshly made resin and the incubation continued for h at rt. after centrifugation at rpm for min, the epon resin was polymerized by heating the sample at uc for days. - nm sections were then cut using an ultracut e ultramicrotome (leica microsystems) and transferred on formvar carbon-coated copper grids. sections were stained first with % uranyl acetate for min at rt and then with a lead-citrate solution ( mm lead nitrate, mm sodium citrate, ph ) for min before being viewed. analysis of em sections was performed by using a jeol electron microscope. dmvs were defined based on the two following morphological criteria: the typical double membrane and the presence of the previously described web-like structure in their proximity [ ] . the size and the number of the dmvs in control and bfa-treated cells were determined by analyzing randomly selected cell profiles. the results were statistically analyzed with the student's t-test. host factors in positive-strand rna virus genome replication wrapping things up about virus rna replication virus factories: associations of cell organelles for viral replication and morphogenesis viral rna replication in association with cellular membranes comparison of the replication of positive-stranded rna viruses of plants and animals localization of mouse hepatitis virus open reading frame a derived proteins rna replication of mouse hepatitis virus takes place at double-membrane vesicles colocalization and membrane association of murine hepatitis virus gene products and de novo-synthesized viral rna in infected cells the putative helicase of the coronavirus mouse hepatitis virus is processed from the replicase gene polyprotein and localizes in complexes that are active in viral rna synthesis ultrastructure and origin of membrane vesicles associated with the severe acute respiratory syndrome coronavirus replication complex localization and membrane topology of the coronavirus nonstructural protein : involvement of the early secretory pathway in replication the intracellular sites of early replication and budding of sars-coronavirus open reading frame a-encoded subunits of the arterivirus replicase induce endoplasmic reticulumderived double-membrane vesicles which carry the viral replication complex localization of mouse hepatitis virus nonstructural proteins and rna synthesis indicates a role for late endosomes in viral replication coronavirus replication complex formation utilizes components of cellular autophagy mouse hepatitis virus replicase proteins associate with two distinct populations of intracellular membranes rapid redistribution of golgi proteins into the er in cells treated with brefeldin a: evidence for membrane cycling from golgi to er novel blockade by brefeldin a of intracellular transport of secretory proteins in cultured rat hepatocytes adp-ribosylation factor/ copi-dependent events at the endoplasmic reticulum-golgi interface are regulated by the guanine nucleotide exchange factor gbf gbf , a guanine nucleotide exchange factor for adp-ribosylation factors, is localized to the cis-golgi and involved in membrane association of the copi coat turning on arf: the sec family of guaninenucleotide-exchange factors the sec family of arf guanine nucleotide exchange factors proteins and cell regulation brefeldin a inhibits golgi membrane-catalysed exchange of guanine nucleotide onto arf protein inhibition by brefeldin a of a golgi membrane enzyme that catalyses exchange of guanine nucleotide bound to arf regulators and effectors of the arf gtpases localization of large adp-ribosylation factor-guanine nucleotide exchange factors to different golgi compartments: evidence for distinct functions in protein traffic characterization of class ii and class iii adp-ribosylation factor genes and proteins in drosophila melanogaster the mechanisms of vesicle budding and fusion bi-directional protein transport between the er and golgi a regulatory role for arf in receptor-mediated endocytosis overexpression of wild-type and mutant arf and arf : distinct perturbations of nonoverlapping membrane compartments evaluation of the primary effect of brefeldin a treatment upon herpes simplex virus assembly brefeldin a blocks protein glycosylation and rna replication of vesicular stomatitis virus requirement of the vesicular system for membrane permeabilization by sindbis virus effect of brefeldin a on rotavirus assembly and oligosaccharide processing envelope glycoprotein interactions in coronavirus assembly release of canine parvovirus from endocytic vesicles foot-and-mouth disease virus replication sites form next to the nucleus and close to the golgi apparatus, but exclude marker proteins associated with host membrane compartments markers for trans-golgi membranes and the intermediate compartment localize to induced membranes with distinct replication functions in flavivirus-infected cells inhibition of poliovirus rna synthesis by brefeldin a grapevine fanleaf virus replication occurs on endoplasmic reticulum-derived membranes involvement of membrane traffic in the replication of poliovirus genomes: effects of brefeldin a differential requirements for copi coats in formation of replication complexes among three genera of picornaviridae the vesicle docking protein p binds gm , a cis-golgi matrix protein, in a mitotically regulated manner differential inhibition of cellular and sindbis virus translation by brefeldin a coronaviruses as vectors: position dependence of foreign gene expression dissection of membrane dynamics of the arf-guanine nucleotide exchange factor gbf dominant inhibitory mutants of arf block endoplasmic reticulum to golgi transport and trigger disassembly of the golgi apparatus mouse hepatitis virus replicase protein complexes are translocated to sites of m protein accumulation in the ergic at late times of infection mouse hepatitis virus c-like protease cleaves a -kilodalton protein from the open reading frame a polyprotein in virus-infected cells and in vitro coronavirus m proteins accumulate in the golgi complex beyond the site of virion budding an electron microscope study of the development of a mouse hepatitis virus in tissue culture cells selective inhibition of transcytosis by brefeldin a in mdck cells brefeldin a causes structural and functional alterations of the trans-golgi network of mdck cells brefeldin a rapidly disrupts plasma membrane polarity by blocking polar sorting in common endosomes of mdck cells mouse hepatitis virus strain a is released from opposite sides of different epithelial cell types isoform-selective effects of the depletion of adp-ribosylation factors - on membrane traffic phospholipase d: a lipid centric review phospholipase d as an effector for adp-ribosylation factor in the regulation of vesicular traffic -butanol interferes with phospholipase d and protein kinase calpha association and inhibits phospholipase d basal activity a highly sensitive assay for monitoring the secretory pathway and er stress codon-optimized gaussia luciferase cdna for mammalian gene expression in culture and in vivo cellular copii proteins are involved in production of the vesicles that form the poliovirus replication complex poliovirus proteins induce membrane association of gtpase adp-ribosylation factor arf proteins: roles in membrane traffic and beyond membrane recruitment of effector proteins by arf and rab gtpases arf and its many interactors identification of severe acute respiratory syndrome coronavirus replicase products and characterization of papain-like protease activity membrane topology of murine coronavirus replicase nonstructural protein genome-wide analysis of human kinases in clathrin-and caveolae/raft-mediated endocytosis cloning of the mouse hepatitis virus (mhv) receptor: expression in human and hamster cell lines confers susceptibility to mhv targeting non-human coronaviruses to human cancer cells using a bispecific single-chain antibody retargeting of coronavirus by substitution of the spike glycoprotein ectodomain: crossing the host cell species barrier stable alphavirus packaging cell lines for sindbis virus and semliki forest virus-derived vectors comparison of six different murine coronavirus jhm variants by monoclonal antibodies against the e glycoprotein the distribution and translocation of the g protein adp-ribosylation factor in live cells is determined by its gtpase activity dynamics of gbf , a brefeldin a-sensitive arf exchange factor at the golgi cleavage inhibition of the murine coronavirus spike protein by a furin-like enzyme affects cell-cell but not virus-cell fusion the coronavirus spike protein is a class i virus fusion protein: structural and functional characterization of the fusion core complex secretory protein trafficking and organelle dynamics in living cells bfa bodies'': a subcompartment of the endoplasmic reticulum coat proteins and vesicle budding maintenance of golgi structure and function depends on the integrity of er export potential role for protein kinases in regulation of bidirectional endoplasmic reticulum-to-golgi transport revealed by protein kinase inhibitor h three distinct steps in transport of vesicular stomatitis virus glycoprotein from the er to the cell surface in vivo with differential sensitivities to gtp gamma s we would like to thank d. duijsings for helpful discussions, m. denison and j. fleming for providing us with the antibodies directed against the mhv nsp and m protein, respectively, and g. romero and c. jackson for providing the plasmids containing arf and gbf , respectively. key: cord- -vg bwed authors: haagmans, bart l.; andeweg, arno c.; osterhaus, albert d. m. e. title: the application of genomics to emerging zoonotic viral diseases date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: vg bwed interspecies transmission of pathogens may result in the emergence of new infectious diseases in humans as well as in domestic and wild animals. genomics tools such as high-throughput sequencing, mrna expression profiling, and microarray-based analysis of single nucleotide polymorphisms are providing unprecedented ways to analyze the diversity of the genomes of emerging pathogens as well as the molecular basis of the host response to them. by comparing and contrasting the outcomes of an emerging infection with those of closely related pathogens in different but related host species, we can further delineate the various host pathways determining the outcome of zoonotic transmission and adaptation to the newly invaded species. the ultimate challenge is to link pathogen and host genomics data with biological outcomes of zoonotic transmission and to translate the integrated data into novel intervention strategies that eventually will allow the effective control of newly emerging infectious diseases. result in the emergence of new infectious diseases in humans as well as in domestic and wild animals. genomics tools such as high-throughput sequencing, mrna expression profiling, and microarray-based analysis of single nucleotide polymorphisms are providing unprecedented ways to analyze the diversity of the genomes of emerging pathogens as well as the molecular basis of the host response to them. by comparing and contrasting the outcomes of an emerging infection with those of closely related pathogens in different but related host species, we can further delineate the various host pathways determining the outcome of zoonotic transmission and adaptation to the newly invaded species. the ultimate challenge is to link pathogen and host genomics data with biological outcomes of zoonotic transmission and to translate the integrated data into novel intervention strategies that eventually will allow the effective control of newly emerging infectious diseases. most of the well-known human viruses persist in the population for a relatively long time, and coevolution of the virus and its human host has resulted in an equilibrium characterized by coexistence, often in the absence of a measurable disease burden. when pathogens cross a species barrier, however, the infection can be devastating, causing a high disease burden and mortality. in recent years, several outbreaks of infectious diseases in humans linked to such an initial zoonotic transmission (from animal to human host) have highlighted this problem. factors related to our increasingly globalized society have contributed to the apparently increased transmission of pathogens from animals to humans over the past decades; these include changes in human factors such as increased mobility, demographic changes, and exploitation of the environment (for a review see osterhaus [ ] and kuiken et al. [ ] ). environmental factors also play a direct role, and many examples exist. the recently increased distribution of the arthropod (mosquito) vector aedes aegypti, for example, has led to massive outbreaks of dengue fever in south america and southeast asia. intense pig farming in areas where frugivorous bats are common is probably the direct cause of the introduction of nipah virus into pig populations in malaysia, with subsequent transmission to humans. bats are an important reservoir for a plethora of zoonotic pathogens: two closely related paramyxoviruses-hendra virus and nipah virus-cause persistent infections in frugivorous bats and have spread to horses and pigs, respectively [ ] . the similarity between human and nonhuman primates permits many viruses to cross the species barrier between different primate species. the introduction into humans of hiv- and hiv- (the lentiviruses that cause aids), as well as other primate viruses, such as monkeypox virus and herpesvirus simiae, provide dramatic examples of this type of transmission. other viruses, such as influenza a viruses and severe acute respiratory syndrome coronavirus (sars-cov), may need multiple genetic changes to adapt successfully to humans as a new host species; these changes might include differential receptor usage, enhanced replication, evasion of innate and adaptive host immune defenses, and/or increased efficiency of transmission. understanding the complex interactions between the invading pathogen on the one hand and the new host on the other as they progress toward a new hostpathogen equilibrium is a major challenge that differs substantially for each successful interspecies transmission and subsequent spread of the virus. new molecular techniques such as high-throughput sequencing, mrna expression profiling, and array-based single nucleotide polymorphism (snp) analysis provide ways to rapidly identify emerging pathogens (nipah virus and sars-cov, for example) and to analyze the diversity of their genomes as well as the host responses against them. essential to the process of identification and characterization of genome sequences is the exploitation of extensive databases that allow the alignment of viral genome sequences and the linkage of these genomics data to those obtained by classical viral culture and serological techniques, and epidemiological, clinical, and pathological studies [ ] . extensive genetic analysis of hiv- , for example, has provided clues to the geography and time scale of the early diversification of hiv- strains when the virus emerged in humans. hiv- strains are divided into multiple clades, each of which has independently evolved from a simian immunodeficiency virus (siv) that naturally infects chimpanzees in west and central africa. current estimates date the common ancestor of hiv- to the beginning of the twentieth century [ ] . because zoonotic pathogens typically may cause variable clinical outcomes in human hosts that differ in age, nutritional status, genetic background, and immunological condition, deciphering the complex interactions between evolving pathogens and their hosts is a great challenge. the genome sequences of many host species have become available the last decade, and with them a range of novel tools are available to study virus-host interactions at the molecular level. this progress, together with advances in high-throughput sequencing technology and, not least, in (bio)informatics and statistics, allows us to analyze the ''genomewide'' networks of gene interactions that control the host response to pathogens. by comparing and contrasting the outcomes of infection with closely related pathogens in different but related host species, we can further delineate the various host pathways involved in the different outcomes. the power of this approach was nicely demonstrated for siv infection of various primate host species. natural reservoir hosts of siv do not develop aids upon infection, whereas non-natural hosts, such as rhesus macaques and pig-tailed macaques, when infected experimentally with siv, develop aids in a similar manner to hiv-infected humans. transcriptional profiling indicates that siv infection of these species produces a distinctive host response [ ] . siv-infected primates with symptoms of aids have a high viral load, immune activation, and loss of certain types of t cells, whereas sivinfected sooty mangabeys (the species from which hiv- is thought to have originated) have substantially lower levels of innate immune activation than the symptomatic primates, partly due to the production of less interferon-a by plasmacytoid dendritic cells in response to siv and other toll-like receptor ligands [ ] . identification of host factors that restrict hiv infection may aid the development of effective intervention strategies. below, we elaborate on two other examples of recent important zoonotic events that led to sustained virus transmission in the human host, and the role that genomics has played in the elucidation of their pathogenesis thus far. influenza is caused by rna viruses of the orthomyxoviridae family. whereas fever and coughs are the most frequent symptoms, in more serious cases a fatal pneumonia can develop, particularly in the young and the elderly. typically, influenza is transmitted through the air by coughs or sneezes, creating aerosols containing the virus; but influenza can also be transmitted by bird droppings, saliva, feces, and blood. birds and pigs play an important role in the emergence of new influenza viruses in humans. fecal sampling of migratory birds has revealed that they harbor a large range of different subtypes of influenza a viruses [ ] . some wild duck species, particularly mallards, are potential long-distance vectors of highly pathogenic avian influenza virus (h n ), whereas others, including diving ducks, are more likely to act as ''sentinel'' species that die upon infection [ ] . following the introduction of a new pandemic influenza a virus subtype from an avian reservoir, either directly or via another mammalian species such as the pig, the virus may continue to circulate in humans in subsequent years as a seasonal influenza virus. in the past century, three major influenza epidemics resulted in the loss of many millions of lives. spanish flu alone caused the deaths of more than million people by the end of world war i in . the outbreak of a new h n virus (causing ''swine flu'') that started in mexico further illustrates the pandemic potential of influenza a viruses. after introduction of a new influenza a virus from an avian or porcine reservoir into the human species, viral genomics studies are essential to identify critical mutations that enable the circulating virus to spread efficiently, interact with different receptors, and cause disease in the new host. for example, the importance of residue of the pb protein of the viral polymerase in determining species restriction has been demonstrated through these kinds of approaches [ ] . furthermore, changes in the hemagglutinin molecules may allow influenza a viruses to switch receptor specificity. the hemagglutinin of avian h n influenza viruses preferentially binds to oligosaccharides that terminate with a sialic acid-a- , -gal disaccharide, whereas the hemagglutinins of mammalian influenza a viruses prefer oligosaccharides that terminate with sialic acid-a- , -gal (figure ) . fatal viral pneumonia in humans infected with avian h n viruses is partly due to the ability of these viruses to attach to and replicate in the cells of the lower respiratory tract, which have oligosaccharides that terminate in sialic acid-a- , -gal disaccharide [ , ] . the sequence of the hemagglutinin protein may also affect its binding affinity for neutralizing antibodies. understanding the relationship between genetic diversity and antigenic properties of these viruses [ ] may help to predict the emergence of influenza viruses and to develop effective vaccines. microarray-assisted mrna expression profiling of emerging zoonotic viral infections, including influenza a virus, is used to phenotype the host response in great detail. by comparing mrna expression in individuals infected with an emerging virus to expression in individuals infected with a related established virus, researchers can generate a ''molecular fingerprint'' of the host response genes or pathways specifically involved in the oftenexuberant host responses to the emerging virus. by using genetically engineered influenza a viruses, a role for the nonstructural ns viral protein in evasion of the innate host response has been demonstrated [ ] . interestingly, the ns protein derived from the spanish h n pandemic influenza virus blocked expression of interferon-regulated genes more efficiently than did the ns protein from established seasonal influenza viruses [ ] . other genomics studies of genetically engineered influenza a viruses containing some or all of the gene segments from either the h n virus or the highly pathogenic avian influenza a virus (h n ), suggest that these highly pathogenic influenza viruses induce severe disease in mice and macaques through aberrant and persistent activation of proinflammatory cytokine and chemokine responses [ ] [ ] [ ] [ ] . application of genomics tools not only supports the elucidation of mechanisms underlying pathogenesis but may also help to identify leads for therapeutic intervention. in ferrets, h n infection induced severe disease that was associated with strong expression of interferon response genes including the interferon-cinduced cytokine cxcl . treatment of h n -infected ferrets with an antagonist of the cxcl receptor (cxcr ) reduced the severity of the flu symptoms and the viral titers compared to the controls [ ] , clearly demonstrating the potential of biological response modifiers for the clinical management of viral infections. the host evasion and evolution of influenza virus is further discussed in [ ] . coronaviruses (covs) primarily infect the upper respiratory and gastrointestinal tract of mammals and birds. five different currently known covs infect humans and are believed to cause a significant percentage of all common colds in human adults. surprisingly, recent studies revealed that approximately % of bats sampled in china were positive for covs [ ] . subsequent phylogenetic studies revealed that bat covs that resembled human sars-cov clustered in a putative group comprising one subgroup of bat covs and another of sars-covs from humans and other mammalian hosts. according to the current hypothesis sars-cov has arisen by recombination between two bat viruses. phylogenetic analysis of sars-cov isolates from animals indicate that the resulting bat virus was transmitted first to palm civets (paguma larvata), a wild cat-like animal hunted for its meat, and subsequently to humans at live animal markets in southern china [ ] . genome analyses have provided evidence that genetic variation in the spike gene of these viruses from civets is associated with increased transmission of the virus [ ] . in addition, species-tospecies variation in the sequence of the gene angiotensin-converting enzyme (ace ), which encodes the sars-cov receptor, also affects the efficiency by which the virus can enter cells [ ] . by a combination of phylogenetic and bioinformatics analyses, chimeric gene design, and reverse genetics-aided generation of viruses that encode spike proteins of diverse isolates, researchers have reconstructed the events that led to the emergence of a virus able to spread efficiently in humans [ ] . structural modeling predicted that the sars-cov that caused the epidemic had an increased affinity for both civet and human ace receptors due to adaptation (figure ) . subsequent functional genomics studies of these viruses in diverse species provided further insight into the role of specific host genes involved in the pathogenic response [ , ] . the pathological changes observed in the lungs are initiated by a disproportionate innate immune response, illustrated by elevated levels of inflammatory cytokines and chemokines, such the hemagglutinin of avian influenza a viruses (blue) preferentially bind to oligosaccharides that terminate in sialic acid-a- , -gal (red), whereas the hemagglutinin on human influenza a viruses (green) prefer oligosaccharides that terminate in sialic acid-a- , -gal (orange). fatal viral pneumonia in humans infected with the h n subtype of avian influenza a viruses is likely due to the ability of these viruses to attach to and replicate in the lower respiratory tract cells, which have sialic acid-a- , -gal terminated saccharides. the horizontal arrows indicate interspecies transmission, including the transmission from an avian or porcine reservoir into the human species. image credit: bart as cxcl (ip- ), ccl (mcp- ), interleukin (il)- , il- , il- , il- b, and interferon-c [ ] . these clinical data were confirmed experimentally by demonstrating that sars-cov infection of diverse cell types induces a range of cytokines and chemokines, thus providing a conceptual framework for sars-cov pathogenesis. host genome expression analyses of various animal hosts and humans with different outcomes of infection indicated differential activation of innate immune genes in, for example, aged subjects compared to young subjects. importantly, treatment of aged macaques with pegylated interferon-a (i.e. interferon-a covalently modified with polyethylene glycol polymer chains, to enhance its bioavailability) reduced sars-cov replication and pathogenic responses [ ] . thus, host genomics analysis may provide markers of pathogenesis and leads for therapeutic intervention, as in this example of sars-cov infection. rapid identification of newly emerging viruses through the use of genomics tools is one of the major challenges for the near future. in addition, the identification of critical mutations that enable viruses to spread efficiently, interact with different receptors, and cause disease in diverse hosts through, for instance, enhanced viral replication or circumvention of the innate and adaptive immune responses, needs to be further expanded. although microarrayassisted transcriptional profiling can provide us with a wealth of information regarding host genes and gene-interacting networks in figure . zoonotic transmission of sars-cov. genomic analyses provided evidence that genetic changes in the spike gene of sars-cov from bats (left) and civet cats (center) are essential for the animal-to-human transmission (horizontal arrows). species-to-species genetic variation in the (thus far unidentified) viral receptor in bats and in the angiotensin converting enzyme (ace ) gene, encoding the sars-cov receptor in civet cats and humans also affects the efficiency with which the virus can enter cells (vertical arrows). the sars-cov that caused the epidemic evolved a high affinity for both civet (center) and human (right) ace receptors (indicated by the single diagonal and the right side vertical arrow). virus-host interactions, future research should focus on combining data obtained in different experimental settings. therefore, the careful design of complementary sets of experiments using different formats of virus-host interactions is absolutely needed for successful genomics studies [ ] . special attention should be addressed to the comparative analysis of the host response in diverse animal species. thus far a limited number of laboratory animal species has been studied, but the recent elucidation of the genome of several other animal species will provide tools to decipher the virus-host interactions in the more relevant natural host. recent developments in the sequencing of the rna transcriptome may aid this development. ultimately, microarray technology may also extend to genotyping of the human host by snp analysis, to identify markers of host susceptibility and severity of disease, that can be used in tailor-made clinical management of disease caused by emerging infections. comparative analysis of host responses to emerging viruses may also point toward a similar dysregulated host response to a range of emerging virus infections, enabling the rational design of multipotent biological response modifiers to combat a variety of emerging viral infections. by focusing on broad-acting intervention strategies rather than on the discovery of a newly emerging pathogen that is not characterized yet, we may be able to protect ourselves from several unexpectedly emerging infections with the same clinical manifestations. this approach may readily reduce the burden of disease and time will be gained to design preventive pathogen specific intervention strategies such as antiviral therapy or vaccination. clearly, for all stages of combating emerging infections, from the early identification of the pathogen to the development and design of vaccines, application of sophisticated genomics tools is fundamental to success. catastrophes after crossing species barriers public health. pathogen surveillance in animals henipaviruses: emerging paramyxoviruses associated with fruit bats viruses and koch's postulates direct evidence of extensive diversity of hiv- in kinshasa by transcriptional profiling in pathogenic and non-pathogenic siv infections reveals significant distinctions in kinetics and tissue compartmentalization divergent tlr and tlr signaling and type i interferon production distinguish pathogenic and nonpathogenic aids virus infections spatial, temporal, and species variation in prevalence of influenza a viruses in wild migratory birds wild ducks as long-distance vectors of highly pathogenic avian influenza virus (h n ) molecular basis for high virulence of hong kong h n influenza a viruses h n virus attachment to lower respiratory tract haemagglutinin mutations responsible for the binding of h n influenza a viruses to human-type receptors mapping the antigenic and genetic evolution of influenza virus cellular transcriptional profiling in influenza a virus-infected lung epithelial cells: the role of the nonstructural ns protein in the evasion of the host innate defense and its potential contribution to pandemic influenza aberrant innate immune response in lethal infection of macaques with the influenza virus early and sustained innate immune response defines pathology and death in nonhuman primates infected by highly pathogenic influenza virus genomic analysis of increased host immune and cell death responses induced by influenza virus global host immune response: pathogenesis and transcriptional profiling of type a influenza viruses expressing the hemagglutinin and neuraminidase genes from the pandemic virus gene expression analysis of host innate immune responses during lethal h n infection in ferrets the role of genomics in tracking the evolution of influenza a virus prevalence and genetic diversity of coronaviruses in bats from china cross-host evolution of severe acute respiratory syndrome coronavirus in palm civet and human receptor and viral determinants of sars-coronavirus adaptation to human ace mechanisms of zoonotic severe acute respiratory syndrome coronavirus host range expansion in human airway epithelium early upregulation of acute respiratory distress syndrome-associated cytokines promotes lethal disease in an aged-mouse model of severe acute respiratory syndrome coronavirus infection functional genomics highlights differential induction of antiviral pathways in the lungs of sars-cov-infected macaques genomic analysis reveals age-dependent innate immune responses to severe acute respiratory syndrome coronavirus pegylated interferon-alpha protects type pneumocytes against sars coronavirus infection in macaques virogenomics: the virus -host interaction revisited key: cord- -daj zcm authors: shang, jian; wan, yushun; liu, chang; yount, boyd; gully, kendra; yang, yang; auerbach, ashley; peng, guiqing; baric, ralph; li, fang title: structure of mouse coronavirus spike protein complexed with receptor reveals mechanism for viral entry date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: daj zcm coronaviruses recognize a variety of receptors using different domains of their envelope-anchored spike protein. how these diverse receptor recognition patterns affect viral entry is unknown. mouse hepatitis coronavirus (mhv) is the only known coronavirus that uses the n-terminal domain (ntd) of its spike to recognize a protein receptor, ceacam a. here we determined the cryo-em structure of mhv spike complexed with mouse ceacam a. the trimeric spike contains three receptor-binding s heads sitting on top of a trimeric membrane-fusion s stalk. three receptor molecules bind to the sides of the spike trimer, where three ntds are located. receptor binding induces structural changes in the spike, weakening the interactions between s and s . using protease sensitivity and negative-stain em analyses, we further showed that after protease treatment of the spike, receptor binding facilitated the dissociation of s from s , allowing s to transition from pre-fusion to post-fusion conformation. together these results reveal a new role of receptor binding in mhv entry: in addition to its well-characterized role in viral attachment to host cells, receptor binding also induces the conformational change of the spike and hence the fusion of viral and host membranes. our study provides new mechanistic insight into coronavirus entry and highlights the diverse entry mechanisms used by different viruses. undergo any dynamic conformational changes. therefore, it is a mystery how s -ntd would play any role in activation of the membrane fusion process, other than its established role in viral attachment. mhv from the β-genus is an extensively studied prototypic coronavirus. mhv is the only known coronavirus that uses the s -ntd to recognize a protein receptor, ceacam a [ , , ] . ceacam a is a cell adhesion protein. due to alternative mrna splicing, ceacam a contains either two (d -d ) or four (d -d -d -d ) ig-like domains [ ] . previously, we determined the crystal structure of mhv s -ntd complexed with ceacam a (d -d ) [ ] . the structure showed that mhv s -ntd has the same fold as human galectins (galactosebinding lectin), but it does not bind any sugar; instead, it binds to d of ceacam a through protein-protein interactions. the cryo-em structures of mhv spike in pre-fusion and postfusion have been determined [ , ] . however, the structure of mhv spike in complex with ceacam a is still not available. as a result, although previous biochemical studies have shown that ceacam a binding triggers the conformational changes of mhv spike [ , ] , the molecular mechanism for the role of ceacam a in the mhv-spike-mediated membrane fusion is unknown. in this study, we determined the cryo-electron microscopic (cryo-em) structure of pre-fusion mhv spike in complex with ceacam a (d -d ), which reveals the structural change of mhv spike associated with receptor binding. using proteolysis and negative-stain em assays, we further investigated the impact of receptor binding on proteases sensitivity and the final structural transitions of mhv spike. our results provide insight into the molecular mechanism for mhv entry and demonstrate the diversity of entry mechanisms for different coronaviruses. we prepared both mhv spike ectodomain (s-e) and mouse ceacam a ectodomain (d -d ) for cryo-em studies. to prepare mhv s-e in the pre-fusion state, we removed the cterminal transmembrane anchor and intracellular tail of mhv spike and replaced them with a gcn trimerization tag and a his tag. ceacam a was also engineered to contain a c-terminal his tag. both mhv s-e and ceacam a were expressed in insect cells, secreted into cell culture medium, and purified to homogeneity using affinity column and size exclusion columns. recombinant mhv s-e molecules were mostly intact and had not been cleaved by proteases. subsequently recombinant mhv s-e and ceacam a were mixed together in solution and the complex was purified using a size exclusion column. we collected cryo-em data on the complex and calculated a density map at . Å (fig a, s fig) . the density of the complex revealed that each mhv s-e trimer binds three ceacam a molecules (fig a) . we built a structural model and refined it (fig b) . the final structural model contained all of the residues of the mhv s-e trimer (except residues - , - , and - in each monomer) as well as six n-linked glycans (two on each monomer). although both the d and d domains of ceacam a could be seen in the cryo-em density, only the density for the d domain was sufficiently robust for building the atomic model ( fig a, fig b) . data collection and model statistics are shown in s table. the overall structure of the receptor-bound mhv s-e is similar to that of the unliganded se in the pre-fusion state. like the unliganded s-e, the receptor-bound s-e contains three monomeric units, with three s heads sitting on top of the trimeric s stalk (fig a, fig b) . three copies of s -ctd are located on the top of the trimer, all of which are in the lying down state. there are significant differences in the structural models of s -ctd in the receptor-bound s-e and unliganded s-e; however, we believe that these differences are due to the improved cryo-em density in the current study, which helped correct the misbuilt structural model in the unliganded s-e from an earlier study [ ] . the revised atomic structures of s -ctd and a second region of s were listed in s fig. in both the receptor-bound and unliganded s-e trimer molecules, three copies of s -ntd are located on each side of s ( fig b, fig b) . the structures of receptor-bound s -ntd and unliganded s -ntd are highly similar to each other (s fig) . each s subunit contains a central helix (ch) (which mediates trimerization of the s stalk), a fusion peptide (fp) (which consists of three α-helices and several connecting loops), and a heptad repeat n region (hr-n) (which consist of three α-helices and several connecting loops). all of these structural elements in s are in the pre-fusion state and would need to undergo dramatic structural changes in order to transition to the post-fusion state. as in the unliganded s-e, the heptad repeat c region (hr-c) was not observed in the receptor-bound se structure probably due to its disorderness. it is worth noting that compared with the unliganded s-e, the proteolysis sites (at the s /s region and s ' site) do not become more exposed in the receptor-bound s-e (s fig). overall, receptor binding does not trigger dramatic structural changes in mhv s-e, which still stays in the pre-fusion conformation. receptor binding by mhv s-e reveals several unique features of a coronavirus spike using its s -ntd as the rbd, as compared with sars-cov spike that uses its s -ctd as the rbd [ ] . first, almost all of the trimeric s-e molecules bind three ceacam a molecules each, while almost all of the sars-cov s-e molecules only bind one or two ace molecules each [ , ] . this is probably due to the fact that the three copies of mhv s -ntd are located on different sides of the spike trimer, are far from each other, and hence the three bound receptor molecules do not have steric clashes. in contrast, the three copies of sars-cov s -ctd are all located on the top of the spike trimer and are near each other, leading to steric clashes between bound ace molecules. depending on the number of receptor molecules on host cell membranes, the high stoichiometry of receptor binding by mhv spike potentially allows efficient viral attachment to target cells. second, in both the receptor-bound and unliganded mhv s-e molecules, all of the three copies of the s -ntd are fully exposed and completely accessible for receptor binding (fig b, fig b) . we compared the receptor-binding affinities of recombinant s -ntd and recombinant s-e using alphascreen assay. the result showed that there is no significant difference in the ceacam a-binding affinities between recombinant s -ntd and recombinant s-e ( fig c) , consistent with our structural observation. therefore, mhv s -ntd is primed to recognize and engage the receptor. in contrast, the s -ctd on sars-cov spike is not accessible in the lying down state and only becomes available to recognize ace in the standing up state. this difference between the receptor-binding modes of mhv s -ntd and sars-cov s -ctd is probably attributed to the different locations and orientations of the two rbds. in this case, s -ctd is the most protruding region on the entire spike molecule (and also on the live virus particle) and is directly exposed to the host immune system. thus, the lying down state of sars-cov s -ctd is likely an immune evasion strategy for the virus, which would counter the neutralization by rbd-targeting antibodies. compared with s -ctd, s -ntd is less exposed and hence is under reduced immune pressure. the readily accessible receptorbinding sites in mhv spike also potentially allow efficient viral attachment to target cells. -e. s : receptor-binding subunit. s : membrane-fusion subunit. gcn -his : gcn trimerization tag followed by his tag. s -ntd: n-terminal domain of s . s -ctd: c-terminal domain of s . ch: central helix. fp: fusion peptide. hr-n and hr-c: heptad repeats n and c, respectively. (b) structure of a monomeric subunit of mhv s-e/ceacam a complex. the structural elements of mhv s-e are colored in the same way as in panel (a). ceacam a is colored in blue. (c) binding interactions between recombinant ceacam a (with a c-terminal fc tag) and recombinant mhv s -ntd or recombinant mhv s-e (with a c-terminal his tag) were measured using alphascreen assay. pbs and mers-cov s -ctd, neither of which binds ceacam a, served as negative controls for mhv s-e and mhv s -ntd. the error bars indicate standard deviation (sd) (n = ). n.s.: statistically not significant (p > . in two tailed t-test). https://doi.org/ . /journal.ppat. .g molecular mechanism for mouse coronavirus entry third, ceacam a binding triggers structural changes in mhv s-e. compared with the unliganded s-e, s in the receptor-bound mhv s-e moves up and away from the s subunit ( fig a) . specifically, there is~ Å movement of the edge of s -ntd away from s . consequently, the interface between s and s in the receptor-bound s-e is significantly smaller than that in the unliganded s-e ( fig b) . specifically, before and after receptor binding, the buried interfaces of s and s decreased from Å to Å and from Å to Å , respectively. thus, ceacam a binding by mhv s -ntd significantly reduces the interactions between s and s . it is worth noting that despite containing misbuilt local regions in s , the global structure of the unliganded mvh s-e was reliable [ ] . furthermore, we compared our structure of the receptor-bound mhv s-e with the unliganded s-e structures from other β-coronaviruses including hku , sars-cov, and mers-cov ( fig c, fig d, fig e) . the results confirm our finding that compared with unliganded coronavirus s-e, the s -ntd in the receptor-bound mhv s-e is farther away from the rest of the s structure, leading to more loosely packing of the spike trimer. as an interesting comparison, for sars-cov s-e, ace binding also reduces the interactions between s and s , but this is achieved through stabilization of the s -ctd in the standing up position by ace [ , ] . nevertheless, as in the case of sars-cov, the reduced interactions between s and s through receptor binding by mhv spike potentially facilitate the dissociation of s from s in the later membrane-fusion process (which we have verified below using biochemical studies). lastly, because s -ntd is located on the side of spike trimer, the orientation of the spikebound ceacam a is perpendicular to mhv spike (s fig). it is worth noting that in the current cryo-em study, recombinant ectodomain of ceacam a was used. in vivo, however, cellsurface-anchored ceacam a would not be able to approach mhv spike from the angle that is perpendicular to the spike. in other words, cell-surface-anchored ceacam a would need to bend in order to bind mhv spike. indeed, previous studies have shown that ceacam a and other cell adhesion molecules have flexible domain hinges and are prone to molecular bending [ , ] . in contrast, for sars-cov, the spike-bound ace aligns with the spike in vitro [ , ] ; hence, cell-surface-anchored ace can simply approach viral-envelopeanchored spike head-on in vivo. although hypothetical, the bending of ceacam a molecule in vivo potentially produces tension in the spike-receptor complex, which may also facilitate the dissociation of s from s in the later membrane-fusion process. in summary, the unique features of receptor binding by mhv spike include the following: all of the three ceacam a-binding sites in mhv spike are readily accessible for the receptor and are fully occupied by ceacam a; receptor binding induces structural changes in the spike that weaken the interactions between s and s ; the orientation of bound receptor, which is perpendicular the spike in vitro, indicates potential bending of the receptor molecule in vivo. these results guided us to further investigate the molecular mechanism of mhvspike-mediated cell entry as follows. to examine the role of receptor binding in protease sensitivity of mhv spike, we performed proteolysis analysis of mhv spike in the presence or absence of ceacam a ( fig a) . we packaged mhv spike into retrovirus particles (which lack their own envelope protein), producing mhv pseudoviruses. subsequently, these mhv pseudovirus particles were incubated with different concentrations of trypsin in the presence or absence of ceacam a. then the proteolysis fragments of mhv spike were examined using western blot. the result showed that even without trypsin treatment, significant amounts of virus-surface mhv spike molecules had been cleaved to s fragment during the virus packaging process in human cells. this result is different from the uncleaved recombinant mhv s-e secreted from insect cells (fig b) , likely reflecting different protease activities in human and insect cells. at low trypsin concentrations, virussurface mhv spike did not demonstrate additional proteolytic cleavage in the presence or absence of ceacam a (fig a) . at intermediate trypsin concentrations, virus-surface mhv spike was not further cleaved in the absence of ceacam a; however, a significant amount of virus-surface spike molecules were further cleaved to s ' fragment in the presence of cea-cam a (fig a) . at high trypsin concentrations, a small percentage of virus-surface spike molecules were further cleaved to s ' fragment in the absence of ceacam a (fig a) . in contrast, a significant amount of virus-surface spike molecules were further cleaved to s ' fragment in the presence of ceacam a ( fig a) . as previous studies showed, the presence of the s ' fragment is strongly associated with the final conformational change of coronavirus spikes [ , , ] . furthermore, it was previously demonstrated that mhv entry depends on the endosome pathway where lysosomal proteases play a critical role [ ] . we recently showed that lysosomal extracts provide a good extracellular system for studying coronavirus entry through the endosome pathway [ ] . thus, to better mimic in vivo conditions, we repeated the above proteolysis assay, using cell-surface-expressed ceacam a (instead of recombinant ceacam a) and lysosomal extracts (instead of trypsin), and obtained similar results (s fig) . therefore, although high concentrations of proteases inefficiently trigger the final conformational change of mhv spike, ceacam a binding significantly facilitates this process. to further understand the role of receptor binding in protease sensitivity of mhv spike, we performed a two-step proteolysis experiment on mhv spike ( fig b) . specifically, recombinant mhv s-e was first cleaved into s and s fragments using trypsin. after stopping the trypsin reaction, the sample was split into two halves: one half was incubated with cea-cam a, and the other was not. then both halves were treated with protease k. the result showed that receptor treatment of the cleaved mhv s-e led to a protease k-resistant s ' fragment. as shown below, mhv s-e that had been cleaved into s and s fragments remained in the pre-fusion conformation. moreover, as discussed earlier, the protease k-resistant s ' here only protein fragments containing the c-terminal c tag (i.e., mhv spike, s and s ', but not s ) could be detected since an antibody targeting the c-terminal c tag of mhv spike was used for the western blot analysis. the result showed that receptor binding enhanced the protease sensitivity of mhv spike and produced more cleaved fragments (particularly s '). (b) silver staining analyses of recombinant mhv s-e that had been subjected to a double proteolysis assay. specifically, recombinant mhv s-e molecules were first treated with low concentration of trypsin. then half of the trypsin-cleaved products were incubated with ceacam a, while the other half were not. subsequently both halves were treated with protease k. here all protein fragments (i.e., mhv s-e, s , s and s ') could be detected as silver staining was used for the detection. the result showed that receptor treatment of the trypsin-cleaved mhv s-e led to a protease k-resistant s ' fragment, suggesting that ceacam a binding facilitated the already cleaved mhv s-e to transition from pre-fusion to post-fusion conformation. see text for more discussion. https://doi.org/ . /journal.ppat. .g fragment likely represents the post-fusion conformation of coronavirus spikes [ , , ] . thus, ceacam a binding facilitated the already cleaved mhv s-e to transition from prefusion to post-fusion conformation, likely due to the removal of the structural restrain of s on s (in other words, dissociation of s from s ). we confirmed this result using virus-surface mhv spike (s fig). these results are consistent with our structural observation showing that ceacam a binding to mhv spike weakens the interactions between s and s . to directly view the final conformational change of mhv spike, we collected negative-stain em images of recombinant mhv s-e that had been treated with trypsin. the results showed that without any treatment, mhv s-e stayed in the pre-fusion conformation (fig a) , which is consistent with our cryo-em structure. low concentration of trypsin did not trigger the final conformational change of mhv s-e (fig b) . however, high concentration of trypsin triggered . % of the mhv s-e molecules to transition to the post-fusion conformation ( fig c) . as previous studies showed, coronavirus spikes in the post-fusion conformation are rod-like structures containing s only; these rod-like structures represent the coiled-coil structures formed by the two heptad-repeat regions (i.e., hr-n and hr-c) of s [ , ] . moreover, because the hydrophobic fusion peptides become exposed in the post-fusion molecular mechanism for mouse coronavirus entry conformation, the post-fusion structures of coronavirus s tend to associate with each other on one end to form rosette-like structures. these negative-stain em results are consistent with the proteolysis sensitivity results, both showing that high concentration of trypsin, but not low concentration of trypsin, can cleave a small percentage of mhv spike molecules to s ' fragments and trigger them to transition to the post-fusion conformation. to investigate the role of ceacam a in the final conformational change of mhv spike, we collected negative-stain em images of recombinant mhv s-e in the presence of recombinant ceacam a. the result showed that after being treated with low concentration of trypsin, all of the receptor-bound mhv s-e molecules remained in the pre-fusion conformation (fig d) . however, also after being treated with low concentration of trypsin, all of these receptor-bound mhv s-e molecules were triggered by urea to transition to the post-fusion conformation ( fig e) . as shown by previous studies, urea (which is a denaturant) can facilitate the dissociation of coronavirus s from s , leading to the final conformational change of coronavirus s [ ] . finally, after being treated with high concentration of trypsin, . % of the receptor-bound mhv s-e molecules underwent the final conformational change and transitioned to the post-fusion conformation (fig f) . these negative-stain em results are also consistent with the proteolysis sensitivity results, showing that ceacam a facilitates protease-cleaved mhv spike to undergo the final conformational change. to analyze the role of ceacam a binding in mhv entry into host cells, we performed both mhv pseudovirus entry assay and live mhv infection assay (s fig, s fig). in both of these assays, virus particles were pretreated with both recombinant ceacam a and trypsin, and then subjected to entry into ceacam a-expressing cells. as a comparison, virus particles were pretreated with either recombinant ceacam a or trypsin before cell entry. the results showed that for both mhv pseudoviruses and live mhv, pretreatment with either recombinant ceacam a or trypsin reduced mhv entry into ceacam a cells. however, pretreatment with both recombinant ceacam a and trypsin further reduced mhv pseudovirus entry and even inactivated live mhv infection. as control experiments, mhv pseudoviruses did not enter cells not expressing ceacam a (except for the trypsin only condition where viral entry slightly increased). these results suggest that recombinant ceacam a alone could competitively inhibit mhv entry into ceacam a-expressing cells, trypsin alone could partially inactivate mhv spikes, and ceacam a and trypsin together drastically inactivate mhv spikes. therefore, in addition to biochemical data, mhv cell entry data are also consistent with our structural observation showing that ceacam a binding to mhv spike weakens the interactions between s and s and facilitates the spike to be proteolysed. recent studies on coronavirus entry have been focused on those coronaviruses that use their s -ctd as the receptor-binding domain. these studies have shown that s -ctds in those coronaviruses undergo a dynamic conformational change: lying down to evade immune surveillance and standing up for receptor binding [ , ] . receptor binding stabilizes s -ctd in the standing up position, reducing the interface between s and s . the weakened interactions between s and s , plus two sequential protease cleavages (one at the s /s boundary and the other at the s ' site), allow s to dissociate from s . subsequently s undergoes the final conformational change and transitions to the post-fusion conformation. mhv differs from the above coronaviruses because it is the only coronavirus that uses its spike s -ntd to bind a protein receptor. as a result of its unique receptor recognition pattern, the molecular mechanism for mhv entry is still elusive. in this study, we investigated the role of receptor binding by s -ntd in the conformational changes of mhv spike, providing insight into the molecular mechanism for mhv entry. we performed a combination of structural and biochemical studies on the receptor-associated activities of mhv spike. these studies included determination of cryo-em structure of receptor-bound mhv spike ectodomain, receptor-dependent protease sensitivity analysis of virus-surface mhv spike, negative-stain em analysis of the receptor-facilitated conformational changes of mhv spike, and receptor-facilitated mhv cell entry. based on our results, we propose the following molecular mechanism for mhv entry (fig ) . during mhv entry into host cells, mhv spike binds to ceacam a on host cell surface for viral attachment. one spike is capable of binding three ceacam a molecules. receptor binding triggers conformational changes in mhv spike, weakening the s /s interactions and positioning mhv spike for two sequential proteolyses (one at the s /s boundary and the other at the s ' site). cea-cam a, which has flexible domain hinges [ , ] , bends in order to approach s -ntd on the side of the spike trimer. the receptor-induced conformational changes, receptor-facilitated proteolysis, and the potential bending of the receptor all contribute to the dissociation of s from s . after s dissociates, s transitions to the post-fusion conformation through a hypothetical elongated intermediate state [ , ] . the molecular mechanism for virus entry is one of the most fundamental questions in virology. our study reveals the unique features of mhv entry, highlighting how receptor binding programs atomic level reorganization of mhv spike to promote membrane fusion. hence mhv has adapted to its special need in receptor recognition and turns this need to its molecular mechanism for mouse coronavirus entry evolutionary advantage in cell entry. our study demonstrates the diversity of cell entry by different coronaviruses and reveals new knowledge about this critical step in viral infection cycles. mhv spike gene (strain a ) was kindly provided by dr. zhaohui qian from chinese academy of medical sciences and peking union medical college, beijing, china. mhv spike ectodomain (s-e) (residues - ) was cloned into pfastbac vector (life technologies inc.); the construct contained an n-terminal honeybee melittin signal peptide and c-terminal gcn and his tags. it was expressed in sf insect cells using the bac-to-bac system (life technologies inc.) and purified as previously described [ ] . briefly, the protein was harvested from cell culture medium, and purified sequentially on ni-nta column and superdex size exclusion column (ge healthcare). mouse ceacam a ectodomain (residues - ) was expressed and purified as previously described [ , ] ; the construct contained a c-terminal his tag. purified mhv s-e and cea-cam a were mixed and incubate at ˚c for hours. the mhv s-e/ceacam a complex was purified on superdex size exclusion column (ge healthcare). for sample preparation, aliquots of the mhv s-e/ceacam a complex ( μl, . mg/ml, in buffer containing mm tris ph . and mm nacl) were applied to glow-discharged cf- / - c c-flat grids (protochips). the grids were then plunge-frozen in liquid ethane using a vitrobot system (fei company). for data collection, images were recorded using a gatan k summit direct electron detector in super resolution mode, attached to a fei titan-krios tem. the automated software serialem [ ] was used to collect , total movies at , x magnification and at a defocus range between and μm. each movie had a total accumulated exposure of e/Å fractionated in frames of -second exposure. data collection statistics are summarized in s table. for data processing, whole frames in each movie were corrected for motion and dose compensation using motioncor [ ] .~ , best images were manually selected. the final images were bin-averaged to reach a pixel size of . Å. the parameters of the microscope contrast transfer function were estimated for each micrograph using gctf [ ] . particles were automatically picked and extracted using relion [ ] with a box size of pixels. initially, , particles were extracted and subjected to d alignment and clustering using relion. the best classes were then selected for an additional d alignment.~ , best particles were selected for creating the initial d model using relion. , particles selected from d alignment were then subjected to d classification. the best class with , particles was subjected to d refinement to generate the final density map. the final density map was sharpened with modulation transfer function of k operated at kev using relion. reported resolutions were based on the gold standard fourier shell correlation (fsc) = . criterion. fourier shell correction curves were corrected for the effects of soft masking by high-resolution noise substitution [ ] . data processing was concluded in s a fig. the initial model of the mhv s-e/ceacam a complex was obtained by fitting the cryo-em structure of unliganded mhv s-e (pdb id: jcl) and the crystal structure of mhv s -ntd/ ceacam a complex (pdb id: r d) into our cryo-em density map using ucsf chimera [ ] and coot [ ] . manual model rebuilding was performed using coot based on the welldefined continuous density of the main chain. side chain assignments were guided through the densities of n-linked glycans and bulky amino acid residues. the structural model of mhv s-e/ceacam a complex was refined using phenix [ ] with geometry restrains and three-fold noncrystallographic symmetry constraints. refinement and model rebuilding were carried out iteratively until no further improvements were achieved in geometry parameters and model-map correlation coefficient. the quality of the final model was analyzed with mol-probity [ ] and emringer [ ] . the validation statistics of the structural models are summarized in s table. alphascreen protein-protein binding assay alphascreen protein-protein binding assay was carried out between recombinant mhv s -ntd and recombinant ceacam a and between recombinant mhv s-e and recombinant ceacam a as described previously [ , ] . briefly, fc-tagged ceacam a (at nm final concentration) was incubated with either his -tagged mhv s -ntd or his -tagged mhv s-e (at nm final concentration) in ½ areaplate (perkinelmer, waltham, ma) at room temperature for hour. alphascreen nickel chelate donor beads and alphascreen protein a acceptor beads (perkinelmer) were then added to one of the mixtures at final concentrations of μg/ml each. the mixtures were then incubated at room temperature for hour away from light. the alphascreen signals were measured using an enspire plate reader (perkinelmer). mhv pseudoviruses were packaged as previously described [ , ] . briefly, full-length mhv spike gene (which contained a c-terminal c tag) was inserted into pcdna . (+) plasmid. retroviruses pseudotyped with mhv spike and expressing a luciferase reporter gene were prepared through co-transfecting hek t cells with a plasmid carrying env-defective, luciferase-expressing hiv- genome (pnl - .luc.re) and the plasmid encoding mhv spike. the produced mhv pseudoviruses were harvested hours post transfection. mhv pseudoviruses (strain a ) were generated as described above. the produced pseudoviruses with indicated treatment were then used to enter hek t cells expressing cea-cam a. after incubation at ˚c for hours, medium was changed and cells were incubated for an additional hours. cells were then washed with pbs and lysed. aliquots of cell lysates were transferred to optiplate- (perkinelmer), followed by addition of luciferase substrate. relative light unites (rlus) were measured using enspire plate reader (perkinelmer). all the measurements were carried out in triplicates. mhv pseudoviruses were purified using a - % sucrose gradient ultracentrifugation at , ×g at ˚c for hours. purified mhv pseudoviruses were incubated alone or with recombinant ceacam a (which is in excess) at ˚c for minutes. then mhv pseudoviruses were incubated with different concentrations of trypsin at ˚c for minutes. subsequently soybean trypsin inhibitor (which is in excess) was added to stop the reaction. samples molecular mechanism for mouse coronavirus entry were then applied for western blot analysis using an antibody targeting the c-terminal c tag of mhv spike. recombinant mhv s-e molecules ( μg) were first treated with low concentration of trypsin at room temperature for min. the reactions were stopped using soybean trypsin inhibitor. the products from this first proteolysis assay were analyzed using silver staining. they were then divided into two halves: one half was incubated with ceacam a at ˚c for hours, and the other half was not incubated with ceacam a. subsequently both halves were treated with proteinase k (final concentration for the assay: μm) on ice for min. the products from the second proteolysis assay were analyzed using silver staining. purified mhv pseudoviruses were also subjected to the same double proteolysis assay, except that western blot (using an antibody targeting the c-terminal c tag of mhv spike) replaced silver staining in analyzing the proteolysis products from both proteolysis assays. lysosomal extracts from hek t cells were prepared according to the lysosome isolation kit procedure (sigma-aldrich) as previously described [ ] . briefly, hek t cells were harvested and washed with pbs buffer and then resuspended in . packed cell volumes (pcv) of extraction buffer. the cells were then broken in a -ml dounce homogenizer using a loose pestle (i.e., pestle b) until % to % of the cells were broken (protease inhibitors from the kit were omitted in our procedure). the samples were centrifuged at , × g for min, and the supernatants were transferred to a new tube and centrifuged at , × g for another min. the supernatants were removed, and the pellets were resuspended in extraction buffer as the crude lysosomal fraction (clf). the clf was diluted in buffer containing % optiprep density gradient medium solution and further purified using density gradient centrifugation at , × g for hours to produce lysosomal extracts. for cleavage of mhv spike using lysosomal extracts, purified mhv pseudoviruses were incubated with membrane-bound cea-cam a (i.e., hek t cells expressing ceacam a on the surface) for hour and then were treated with lysosomal extracts at ˚c for min. subsequently, samples were denatured and analyzes using sds-page gel. cleaved mhv spike molecules were detected by western blot using an anti-c tag antibody. mhv live virus particles (strain a ) were generated from an infectious clone, which is comprised of seven fragments maintained in psmart (lucigen) or pcr-xl-topoa (invitrogen) vectors and was amplified according to previously published protocols [ ] . viral stock was propagated in delayed brain tumor (dbt) cells and viral titers were determined using plaque titration. for live mhv infection, viruses with indicated treatment were used to infect dbt cells with a multiplicity of infection (moi) of . pfu/cell for a one-hour adsorption period, followed by three washes with phosphate-buffered saline (pbs). fresh medium was then added to each culture, and the infection was maintained at ˚c. each condition was performed in triplicate. microscope images were obtained hours post infection. the mhv s-e/ceacam a complex treated under different conditions was diluted to a final concentration of . mg/ml in mm tris-hcl ph . and then loaded onto glow-discharged -mesh carbon grids (electron microscopy sciences). subsequently the grids were stained with . % uranyl formate. all micrographs were collected at the university of minnesota using a tecnai g spirit biotwin at kev (fei company) and an eagle . mega pixel ccd camera at , × nominal magnification. for d image averaging, particles were picked and extracted using relion. the total surface area and buried surface area of pre-fusion mhv s-e and mhv s-e/cea-cam a complex were calculated using the pisa server at the european bioinformatics institute (http://www.ebi.ac.uk/pdbe/prot_int/pistart.html) [ ] . for each trimeric s-e (unliganded or receptor-bound), a pdb file containing both s subunits and s subunits was submitted to the pisa server, and the total surface area and buried surface area on s and s were individually calculated. all data discussed in the paper will be made available to readers. [ ] . two regions are shown: s -ctd (a and b) and another region in s (d and e). also shown are the comparisons of the chain traces of the two models (c and f). in panels c and f, receptor-bound s-e is colored in orange and unliganded s-e is colored in cyan. the protease sites are colored in red. in the unliganded mhv s-e (pdb id: jcl), the previously misbuilt s /s site has been rebuilt based on the deposited cryo-em density (see s fig for more details) . the s ' site in the unliganded mhv s-e as well as the two protease cleavage sites in unliganded hku s-e (pdb id: i ) were not entirely built. nevertheless, the result showed that the cleavages sites in all of these spike molecules are exposed. fig b, except that mhv pseudoviruses were used instead of recombinant mhv s-e. accordingly, western blot analysis of virus-surface mhv spike fragments instead of silver staining of recombinant mhv spike fragments was used for detection of the proteolysis products. as a result, only protein fragments containing the c-terminal c tag (i.e., mhv spike, s and s ', but not s ) could be detected. the result is consistent with that from fig b. (tif) mhv pseudoviruses were pretreated with (i) only trypsin at ˚c for min (the reaction was stopped by trypsin soybean inhibitor), (ii) only ceacam a, or (iii) ceacam a at ˚c for hour followed by trypsin treatment at ˚c for min (the reaction was stopped by trypsin soybean inhibitor). subsequently the above mhv pseudoviruses were used to enter ceacam a-expressing cells, and the entry efficiency was characterized through luciferase signals accompanying entry. cells not expressing ceacam a were used as negative controls. the final concentrations of the proteins in the assay are indicated in the figure. receptor recognition mechanisms of coronaviruses: a decade of structural studies structure, function, and evolution of coronavirus spike proteins. annual review of virology coronaviruses post-sars: update on replication and pathogenesis coronaviruses: structure and genome expression a comparative sequence analysis to revise the current taxonomy of the family coronaviridae cryo-electron microscopy structure of a coronavirus spike glycoprotein trimer pre-fusion structure of a human coronavirus spike protein cryo-em structures of mers-cov and sars-cov spike glycoproteins reveal the dynamic receptor binding domains glycan shield and epitope masking of a coronavirus spike protein observed by cryo-electron microscopy cryo-em structure of infectious bronchitis coronavirus spike protein reveals structural and functional evolution of coronavirus spike proteins cryo-electron microscopy structure of porcine deltacoronavirus spike protein in the prefusion state cryo-em structure of the sars coronavirus spike glycoprotein in complex with its host cell receptor ace tectonic conformational changes of a coronavirus spike glycoprotein promote membrane fusion conformational states of the severe acute respiratory syndrome coronavirus spike protein ectodomain host cell entry of middle east respiratory syndrome coronavirus after two-step, furin-mediated activation of the spike protein host cell proteases: critical determinants of coronavirus tropism and pathogenesis ready, set, fuse! the coronavirus spike protein and acquisition of fusion competence cryo-electron microscopy structures of the sars-cov spike glycoprotein reveal a prerequisite conformational state for receptor binding receptor for mouse hepatitis-virus is a member of the carcinoembryonic antigen family of glycoproteins cloning of the mouse hepatitis-virus (mhv) receptor-expression in human and hamster-cell lines confers susceptibility to mhv redefined nomenclature for members of the carcinoembryonic antigen family crystal structure of mouse coronavirus receptor-binding domain complexed with its murine receptor receptor-induced conformational changes of murine coronavirus spike protein conformational changes in the spike glycoprotein of murine coronavirus are induced at degrees c either by soluble murine ceacam receptors or by ph structure of sars coronavirus spike receptor-binding domain complexed with receptor stabilized coronavirus spikes are resistant to conformational changes induced by receptor recognition or proteolysis the ceacam nterminal ig domain mediates cis-and trans-binding and is essential for allosteric rearrangements of ceacam microclusters regulation of adhesion by flexible ectodomains of igcams. neurochemical research activation of the sars coronavirus spike protein via sequential proteolytic cleavage at two distinct sites coronavirus cell entry occurs through the endo-/lysosomal pathway in a proteolysis-dependent manner lysosomal proteases are a determinant of coronavirus tropism structures and mechanisms of viral membrane fusion proteins: multiple variations on a common theme receptor binding and membrane fusion in virus entry: the influenza hemagglutinin structural and molecular evidence suggesting coronavirus-driven evolution of mouse receptor automated electron microscope tomography using robust prediction of specimen movements electron counting and beaminduced motion correction enable near-atomic-resolution single-particle cryo-em real-time ctf determination and correction relion: implementation of a bayesian approach to cryo-em structure determination high-resolution noise substitution to measure overfitting and validate resolution in d structure determination by single particle electron cryomicroscopy visualizing density maps with ucsf chimera features and development of coot phenix: a comprehensive python-based system for macromolecular structure solution molprobity: allatom structure validation for macromolecular crystallography emringer: side chain-directed model and map validation for d cryo-electron microscopy development of a novel nonradiometric assay for nucleic acid binding to tdp- suitable for high-throughput screening using alphascreen technology a unified mechanism for aminopeptidase n-based tumor cell motility and tumor-homing therapy receptor usage and cell entry of bat coronavirus hku provide insight into bat-to-human transmission of mers coronavirus systematic assembly of a full-length infectious cdna of mouse hepatitis virus strain a inference of macromolecular assemblies from crystalline state negative-stain em images were collected at the characterization facility of the university of minnesota. cryo-em data were collected at the john m. cowley center for high resolution electron microscopy of arizona state university. we thank dr. dewight williams for helping us with data collection. key: cord- -s oxq authors: montelongo-jauregui, daniel; vila, taissa; sultan, ahmed s.; jabra-rizk, mary ann title: convalescent serum therapy for covid- : a th century remedy for a st century disease date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: s oxq nan global recognition and revolutionized the way infectious diseases were treated, and in , emil von behring was awarded the nobel prize for medicine for his work, which served as a basis for treatment of multiple diseases in the s as well as the development of vaccines [ ] . in fact, there are numerous examples throughout history in which convalescent serum was used with some degree of success to treat an array of diseases, including rheumatic fever [ ] , scarlet fever [ ] , mumps [ ] , measles [ , ] , chickenpox [ ] , and pneumococcal and meningococcal infections [ ] (fig ) . most notable use was during the spanish flu pandemic ( to ) , where meta-analysis studies showed a significantly reduced mortality risk in patients treated with convalescent serum [ , ] . however, with the advent of antimicrobials, by the middle of the th century, the use of serum therapy had declined. nevertheless, the interest in passive antibody therapy has been renewed periodically when new epidemics or pandemics have emerged. one example is during the ebola virus (ebov) outbreak in in the democratic republic of congo, where an infected laboratory worker recovered after transfusion with convalescent plasma containing anti-ebov antibodies. similarly, in , patients with argentine hemorrhagic fever virus treated with convalescent plasma had a lower mortality rate compared with subjects treated with normal plasma, and similar results were reported for subsequent epidemics of the disease [ ] . over the following decades, convalescent plasma therapy was successfully employed during the h n swine influenza pandemic ( ), the h n avian flu epidemic ( ), as well as during the ebov outbreak in west africa in . most relevant and encouraging is the use of convalescent plasma during previous coronavirus epidemics: severe acute respiratory syndrome (sars) in , and middle east respiratory syndrome (mers) in [ ] . the high degree of success in achieving favorable clinical outcomes during these coronaviruses outbreaks establishes a strong precedent and supports the notion that convalescent plasma could be a viable option for treatment of covid- patients, particularly upon early administration [ , , , [ ] [ ] [ ] . the convalescent plasma therapeutic approach is based on the principle of passive antibody therapy, a short-term strategy whereby antibodies from the blood of someone who recovered from an infection can be administered to protect or treat another person [ , ] . effectively, the end goal is the same as vaccines, making antibodies against a specific infectious agent readily available. for instance, a vaccine relies on the host immune cells (b lymphocytes specifically) to produce antibodies after antigen recognition and signal amplification by the immune system, a process that may take weeks [ ] ; on the other hand, in the case of passive antibody therapy, the process is expedited by providing a patient with immediate immunity when the premade antibodies are given. therefore, for covid- patients, the expedited approach could prove lifesaving. nevertheless, this advantage does not come without caveats, as immunization with passive antibody therapy is typically of shorter-term protection, in part because of the half-life of antibodies in circulation [ ] and lack of new production by b lymphocytes. today, passive antibody therapy relies primarily on pooled immunoglobulin preparations that contain high concentrations of antibodies. in contrast, plasma has been used emergently in epidemics in which there is insufficient time or resources to generate immunoglobulin preparations [ ] . despite the high rate of sars-cov- infection, the relatively low mortality rate provides a rich pool of donors [ ] . however, potential covid- donors must meet several eligibility criteria that ensure the donor has antibodies against sars-cov- and lacks the presence of other types of infections [ , ] . additionally, only plasma with high anti-sars-cov- titers of immunoglobulins g and m (igg and igm) are used. once collected, plasma can be tested and administered within hours, following conventional donor-patient blood compatibility typing [ ] . in addition to rapid mobilization, this therapeutic approach is also versatile in applicability as it can be used for prophylaxis or treatment, as illustrated in fig . in the case of prophylaxis, a subject considered at high risk for infection (because of age or underlying medical conditions or who is likely to be in contact with people with covid- ) could be administered convalescent plasma or neutralizing antibodies for protection against infection. alternatively, plasma can be administered to treat subjects who have contracted the infection but have not made sufficient antibodies against the virus yet in order to augment their immune response, improve disease course, and enhance recovery [ ] . however, it is important to note that passive antibody therapy is most effective when administered prophylactically or implemented early after the onset of symptoms [ ] . convalescent plasma with neutralizing antibodies is currently being used for investigational purposes in the covid- pandemic, and preliminary results from small studies performed in china are encouraging. a pilot study exploring the feasibility of convalescent plasma transfusion to rescue a group of patients with severe disease showed that dose ( ml) of convalescent plasma with high neutralizing antibody titers was well tolerated, resulted in disappearance of viremia, and improved clinical symptoms in all patients within days of administration [ ] . similar results were reported from another study with critically ill patients on mechanical ventilation [ ] . although these small, nonrandomized studies had limitations, these findings indicate that convalescent plasma could be a promising rescue option for severe covid- [ ] . although convalescent plasma therapy is considered a relatively safe therapeutic modality, there are some potential risks [ ] . one theoretical complication that may arise is an antibodymediated proinflammatory disease enhancement known as antibody-dependent enhancement (ade), whereby antibodies that developed during a prior infection exacerbate severity of the disease [ , ] . the transfer of these antibodies may aberrantly activate fragment crystallizable (fc) or complement receptors, increasing recruitment of proinflammatory cytokines and chemokines to the site of infection and causing severe tissue damage [ ] [ ] [ ] . additionally, the presence of non-neutralizing antibodies may exacerbate viral endocytosis or phagocytosis into host cells via fc receptors, potentializing viral replication [ , ] . however, although this phenomenon is well known with dengue and other viral diseases, there have not been any reported ade cases with the use of convalescent plasma for sars, mers, or covid- [ , , [ ] [ ] [ ] [ ] . nevertheless, it is crucial to have a clear understanding of the role of the recipients' immune response [ ] . transmission of the virus through transfusion is another concern; however, the risk is relatively low because of strict transfusion protocols, and when used for treatment purposes, the recipient is already infected [ , ] . the success of convalescent plasma therapy hinges on the availability of plasma with high concentrations of antibodies, which may not be a major limitation for covid- because sars-cov- has been shown to elicit high neutralizing antibody titers in recently convalesced individuals [ ] [ ] [ ] . apheresis is an automated technology that allows for selective collection of a blood fraction while other components can be transfused back to the donor. therefore, for donation of convalescent plasma, plasmapheresis is recommended because it is highly efficient and approximately - ml of plasma can be collected in a single donation, providing - units of convalescent plasma for transfusion [ ] . there are some practical and logistical limitations for the implementation of a large-scale convalescent plasma transfusion program such as training of study personnel, recruitment of donors, and transport of plasma to hospitals, as well considerations for plasma shelf-life and half-life of antibodies in the plasma [ , ] . although large-scale randomized clinical trials will ultimately confirm the safety and efficacy of convalescent plasma therapy for covid- , a recent safety study by joyner and colleagues [ ] provided encouraging data. by analyzing key safety metrics following transfusion of convalescent plasma in , hospitalized adults with severe or life-threatening covid- , the incidence of serious adverse events was found to be < %, and the -day mortality incidence was . %. these early indicators are encouraging and highly supportive of the use of convalescent plasma as a rescue therapy in hospitalized patients, given that the reported fatality rate of covid- among patients admitted to the intensive care units (icus) is % [ ] . in fact, the low risk indicated by the study support expanding the use of convalescent plasma therapy in less ill individuals [ ] . the global reach of the covid- pandemic and the desperate need for effective treatments have provided an impetus to develop convalescent plasma therapy into a viable (albeit shortterm) treatment option particularly for the critically ill. although its efficacy and safety have not yet been fully proven, convalescent plasma therapy for covid- patients is projected to be a safe and potentially effective therapy for prophylaxis and treatment. however, it is critically important to perform rigorous randomized controlled trials to confirm efficacy and safety and to provide evidence for improved meaningful clinical outcomes. nevertheless, despite the nuanced challenges, the substantial evidence of benefit with use for prior viral infections offers strong precedent for convalescent plasma as a therapeutic approach. importantly, efforts should be focused not only on evaluation of the feasibility of plasma treatment for infectious diseases but also ensure that use of convalescent plasma therapy takes place according to ethical and controlled conditions. genomic characterisation and epidemiology of novel coronavirus: implications for virus origins and receptor binding the sars, mers and novel coronavirus (covid- ) epidemics, the newest and biggest global health threats: what lessons have we learned? origin and evolution of pathogenic coronaviruses bat coronaviruses in china remdesivir for the treatment of covid- -preliminary report the convalescent sera option for containing covid- treatment of critically ill patients with covid- with convalescent plasma effectiveness of convalescent plasma therapy in severe covid- patients anti-sars-cov- virus antibody levels in convalescent plasma of six donors who have recovered from covid- treatment with convalescent plasma for covid- patients in wuhan convalescent serum lines up as first-choice treatment for coronavirus early safety indicators of covid- convalescent plasma in , patients ueber das zustandekommen der diphtherie-immunitä t und der tetanus-immunitä t bei thieren untersuchungen über das zustandekommen der diphtherie-immunitä t bei thiere. philipps-universitä t marburg remembering emil von behring: from tetanus treatment to antibody cooperation with phagocytes the action of vaccines and of concentrated antistreptococcus serum in experimental streptococcal arthritis serum treatment of scarlet fever university of nebraska the use of convalescent serum in the treatment of measles, chickenpox, mumps and whooping cough, including the prophylactic value of parental blood treatment of preeruptive measles with convalescent serum serum therapy revisited: animal models of infection and development of passive antibody therapy deployment of convalescent plasma for the prevention and treatment of covid- convalescent plasma treatment reduced mortality in patients with severe pandemic influenza a (h n ) virus infection hyperimmune iv immunoglobulin treatment: a multicenter double-blind randomized controlled trial for patients with severe influenza a (h n ) infection principles of vaccination public health foundation. th therapeutic antibodies: successes, limitations and hopes for the future severe acute respiratory syndrome coronavirus (sars-cov- ) and coronavirus disease- (covid- ): the epidemic and the challenges points to consider in the preparation and transfusion of covid- convalescent plasma covid- : immunopathology and its implications for therapy impact of immune enhancement on covid- polyclonal hyperimmune globulin therapy and vaccine development how immune complexes from certain igg nabs and any f(ab')( ) can mediate excessive complement activation immune complex-mediated tissue injury: a multistep paradigm antibody-dependent enhancement of sars coronavirus infection and its role in the pathogenesis of sars participants in the summit on dengue immune correlates of p. immune correlates of protection for dengue: state of the art and research agenda is covid- receiving ade from other coronaviruses? microbes infect dengue viruses and mononuclear phagocytes. i. infection enhancement by non-neutralizing antibody convalescent plasma: new evidence for an old therapeutic tool? clinical course and outcomes of critically ill patients with sars-cov- pneumonia in wuhan, china: a single-centered, retrospective, observational study. lancet we would like to thank dr. arturo casadevall for critical review of the manuscript. key: cord- -rb vwolt authors: reuven, eliran moshe; ali, mohammad; rotem, etai; schwarzter, roland; gramatica, andrea; futerman, anthony h.; shai, yechiel title: the hiv- envelope transmembrane domain binds tlr through a distinct dimerization motif and inhibits tlr -mediated responses date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: rb vwolt hiv- uses a number of means to manipulate the immune system, to avoid recognition and to highjack signaling pathways. hiv- infected cells show limited toll-like receptor (tlr) responsiveness via as yet unknown mechanisms. using biochemical and biophysical approaches, we demonstrate that the trans-membrane domain (tmd) of the hiv- envelope (env) directly interacts with tlr tmd within the membrane milieu. this interaction attenuates tnfα, il- and mcp- secretion in macrophages, induced by natural ligands of tlr both in in vitro and in vivo models. this was associated with decreased levels of erk phosphorylation. furthermore, mutagenesis demonstrated the importance of a conserved gxxxg motif in driving this interaction within the membrane milieu. the administration of the env tmd in vivo to lipotechoic acid (lta)/galactosamine-mediated septic mice resulted in a significant decrease in mortality and in tissue damage, due to the weakening of systemic macrophage activation. our findings suggest that the tmd of env is involved in modulation of the innate immune response during hiv infection. furthermore, due to the high functional homology of viral env proteins this function may be a general character of viral-induced immune modulation. the ongoing race between pathogens and their hosts' responses to eliminate them led to the development of many mechanisms driven by the invading pathogen to impair immune responses. the cell populations that are mainly targeted by the virus include mononuclear phagocytes (e.g. macrophages and monocytes) and t cells [ ] . several mechanisms of immune evasion and suppression have been described for the pathology of the human immunodeficiency virus type (hiv- ) [ , ] . regarding mononuclear phagocytes, studies implicated the importance of early genes, expressed by hiv- in advanced phases of infection, for immune manipulation [ , ] . however, as these cells are hallmarks of innate immunity, there is also a requirement for immune manipulation at stages of viral entry and latency. little is known about the ability of hiv- to modulate innate immune responses of these cells during its entry and latent stages, particularly against members of the toll-like receptor (tlr) family. tlrs are critical in the immediate innate immune response against bacterial and viral pathogens [ , ] . tlrs are conserved membrane receptors that recognize a wide variety of pathogenassociated molecular patterns (pamps), such as lipopolysaccharide (lps) from gram-negative bacteria, lipoteichoic acid (lta) from gram-positive bacteria, flagellin, in addition to intracellular molecules such as single-stranded dna and rna [ , ] . to induce ligand recognition and subsequent signaling, the heterodimerization of tlr with tlr or tlr is required. this is coordinated through ligand binding to the extracellular regions of the proteins and conformational changes throughout the proteins [ , , ] . the significance of the tlr and tlr tmds in the regulation and activation of formation of the receptor complex and in downstream signaling has been recently described [ ] , revealing that activation of tlr increases resistance of macrophages to hiv- infection [ ] . interestingly, dendritic cells (dcs) infected with hiv- were reported to be less responsive via tlr upon expression of env on the membrane [ ] . these emerging studies link the manipulation of tlr responses and hiv- pathogenesis through as yet unknown mechanisms. hiv- infects cells via the hiv- env protein which mediates viral entry to host cells that express cd together with an additional co-receptor such as monocytes and dendritic cells, through membrane fusion. in addition to its fusogenic activity, the env protein binds proteins localized to membrane microdomains on macrophages and dcs. in addition, tlrs expressed on the cell membrane are recruited to cholesterol-enriched membrane microdomains upon their ligand recognition, initiating signal transduction [ , ] . env is also targeted to cholesterol-enriched membrane microdomains through a well-defined localization signal located adjacent to its tmd. the tmd of env and its adjacent regions are extremely conserved among all clades currently reported of hiv- , and are similar to the tmd of hiv- gp [ ] . interestingly, recent studies showed that tlr recognition of several viral related glycoproteins (gp) induces activation of an antiviral response [ ] . taken together, we hypothesized that the env plays a role in impairment of tlrinduced responses, contributing both to its evasion from recognition during the fusion process and to expropriate native tlr responses at latent stages, thus assisting in viral replication upon its initiation. here, we report that the gp tmd associates with tlr tmd in the membrane. as a result, cultured macrophages treated with peptides derived from the gp tmd displayed reduced tlr -mediated signaling and decreased pro-inflammatory cytokine secretion. moreover, the ectopic expression of the intact gp and gp inhibited lta mediated cytokine secretion with gp having the highest potency. to demonstrate the tlr inhibitory effect in vivo, we show that the gp tmd protected mice against lta and d-galactosamine (lta/gln)-mediated acute sepsis, concomitant with a decrease in tissue damage. we synthesized a peptide derived from the n terminus of the env tmd region (gp tmd) previously reported to be involved in cellular responses to viral infection [ ] . since this region harbors the well-defined gxxxg motif known to drive the assembly of tmds [ ] , we also synthesized a peptide lacking this motif (gp mutant) (table s in text s ). we then utilized the macrophage cell line raw . to study the functionality of tlr . we activated tlr by purified lta that was recently shown to be regulated, at least in part, through interactions between the tmds of tlr and tlr [ ] . we measured the level of phosphorylated erk , upon treatment with lta, as these are key regulators of the tlr signaling pathway. figure a and b show that minutes after addition of lta to raw cells that were pre-incubated with the gp tmd peptide, there was a significant decrease in erk , phosphorylation compared to nontreated cells, indicating less receptor activation. as a negative control, pre-incubation with the gp mutant peptide did not change the levels of phosphorylated erk , . noteworthy, since the cells in culture that respond through the tlrs also respond to endogenous signals, the basal level of phosphorylated erk , was also decreased by the gp tmd peptide but not by the mutant peptide ( figure a ). we next measured the expression levels of nfkb downstream genes; tumor necrosis factor a (tnfa), a hallmark cytokine of tlr activation, and monocyte chemotactic protein (mcp- ), a major chemokine mainly secreted by macrophages [ ] , in order to ensure that the inhibitive effect on erk , phosphorylation is a result of inhibition of tlr signaling. treatment of the cells with gp tmd peptide resulted in a % decrease in mcp mrna expression levels compared to non-treated cells ( figure c ). the treatment also resulted in a % decrease in the mrna expression levels of tnfa ( figure d ). when cells were treated with the mutant peptide, only a % decrease in mrna levels was detected for tnfa ( figure c ), and no significant change was detected for the expression levels of mcp- ( figure d ). altogether, it is clear that the association between the tmds of gp and tlr functionally impacts receptor activity. in order to confirm that the effects observed at the transcription levels influence the protein expression levels we measured tnfa, mcp- and il- secretion levels. il- is an additional target gene of tlr but it is transcribed via a different transcription factor complex than tnfa. cells were pre-incubated with env tmd peptides for two hours prior to lta activation. cytokine expression was measured in accordance to the expected time of expression of the various targets. the results show that the envtmd peptide attenuates the secretion of tnfa (figure a ), il- ( figure b ) and mcp- ( figure c ). however, and in line with the mrna expression levels and phosphorylation inhibition, the gp mutant peptide did not decrease cytokine secretion. in order to validate that the effect observed is not limited to murine cells we evaluated the effect of the peptides on human thp- cells. we followed the secretion of tnfa under the same conditions as for the mice cell line. the data show that the wt peptide had similar effect on human cells as in mice cells and that the mutant peptide did not affect the secretion levels of tnfa ( figure d ). these results suggest that incubation of gp tmd peptides with cells leads to inhibition of tlr / signaling. together, these results suggest a potential potent immunosuppressive effect of gp hiv- env protein via inhibition of tlr mediated signaling. in order to confirm this hypothesis we utilized the ectopic expression of hiv- env-yfp chimeric protein in raw . cells, which mimics the hiv- infection of macrophages, and tested their responsiveness to lta by measuring tnfa secretion. we expressed two forms of the env protein, the full length gp and the transmembrane protein gp . lta responsiveness was compared to mock transfected cells (see materials and methods). lta treatment resulted in elevated tnfa secretion in mock cells, whereas gp expressing cells showed a reduction in tnfa secretion. furthermore, gp expressing cells showed even more reduction of tnfa secretion almost to the basal level of the nontreated cells (figure ). the better efficiency of gp in inhibiting tnfa secretion compared to gp can be in part due to the smaller size of its ectodomain compared to that of gp . this should result in less hindrance upon its interaction with tlr . we confirmed similar expression levels of hiv- gp and gp , as they showed similar extracellular expression levels (see example in to understand viral pathology and the tools needed to eliminate infection, it is important to understand how viral immune evasion occurs. one such mode of inhibition is the decreased responsiveness of toll-like receptors (tlrs). to date, the exact mechanism inducing this inhibition is not clear. in this study, we utilized a multidisciplinary approach and report on direct modulation of tlr activity by the envelope trans-membrane protein of hiv- through trans-membrane domain interactions. this interaction resulted in a decreased response in vitro of tlr to its natural ligand lta. through mutagenesis analysis we show that the gxxxg motif is the driving force of this interaction. interestingly, the inhibitory effect was also highly effective in protecting mice from lethal effects in a sepsis-like model. our findings implicate that env participates in innate immune impairment, which may occur during viral entry and at latent stages. furthermore, due to the high functional homology between viral env proteins, this function may exhibit a general character of viral-induced immune modulation. supplementary figure s in text s ). these data reveal that the fully processed env protein, once expressed on the cellular membrane, results in inhibition of tlr activation. we studied the ability of the env tmd to target the tlr tmd as it naturally resides in the membrane, and the tmds of tlr / were shown to be important for their regulation and function. the gallex system [ , ] was utilized to evaluate the hetero-association of gp and tlr tmds in biological membranes. b-gal activity was measured after the expression of lexa-tmd-mbp chimera proteins in which the tmds are those of env (wild type and mutant) and of tlr . the gpa tmd sequence and its non-assembling mutant g i (table s in text s ) served as positive and negative controls, respectively. strong dimerization results in low b-gal activity. the level of association between the tmds of gp wt and tlr was significantly higher than that of the tmds of the gp mutant and tlr . moreover, there was no detectable association between the tmds of tlr and gpa (containing the gxxxg motif) ( figure a ). therefore, we conclude that the gxxxg motif contributes but is not sufficient for the assembly of the tmds of gp and tlr . fret analysis was used to explore the direct interaction between the tmd peptides within phospholipid membranes (pc:chol : ). the decrease in the emission of nbd conjugated peptides (gp tmd/mutant), after the addition of successive amounts of rho-labeled tlr tmd, served as an indication of the interaction between a pair of peptides. the gp tmd peptide showed strong interaction with the tlr tmd peptide ( figure b ). this is evident from the large decrease in the emission signal at nm after the first addition of the rho-tlr tmd peptide and by the dose dependent extent of association resulting in a decrease of over % at a : ratio. in comparison, the interaction between the gp mutant and tlr tmds peptides was significantly lower (less than %) (figure c ). to determine the degree of interaction between the tmds of env and tlr within macrophages we performed immunoprecipitation assays using the gp tmd and mutant peptides, and the antimicrobial peptide ll as a negative control. ll was used due to its strong membrane affinity properties and lack of specific protein targets in macrophages. figure d shows the strong precipitation of gp tmd with tlr compared to almost no precipitation of the gp mutant peptide. in order to validate that the inhibitory effect of the gp env peptide is due to inhibition of the interaction between the tmds of tlr and tlr , we performed a fret competition assay between tlr and in the absence ( figure s a in text s ) or presence ( figure s b in text s ) of the gp wt peptide. the data show that when the env peptide is present, the degree of association between the tlr peptides decreased from % fret to %. this inhibition was further corroborated by immunoprecipitation of tlr -tmd peptides with the tlr protein in the absence ( figure s c, left lane in text s ) or the presence (figure s c, right lane in text s ) of the gp wt peptide. in order to verify that the tmd peptides preserved helical structures, we used circular dichorism (cd) spectroscopy in a membrane mimicking solution ( % lpc). all three peptides exhibited a-helical secondary structures, typical of a tmd peptide ( figure s in text s ). it is clear from the cd results that the gp mutant peptide (loss of the gxxxg motif) preserves a helical structure suggesting that any functional discrepancies are not as a result of major structural deformation. in order to corroborate the effect of the env tmd on tlr activation in vivo we utilized a murine model for acute sepsis caused by hyper-activation of tlr [ ] . mice were injected intraperitoneally (i.p) with mg of lta and mg/kg dgalactosamine (gln), and were either treated intravenous (i.v.) with gp tmd peptide ( mg/kg), mutated peptide ( mg/kg) or pbs. upon injection of lta/gln, / pbs-treated mice and / of the gp mutant treated mice died while only / of the gp tmd treated mice died ( figure a ). histological analysis of the liver indicated that lta/gln treatment resulted in massive parenchyma damage associated with hemorrhage in both pbs and gp mutant treated mice ( figure b ). however in the gp tmd treated samples, the tissue resembled that of control mice ( figure b ) with no observable tissue damage or hemorrhage. this was also evident from the external feature of the tissues themselves, showing that mice not treated with the env tmd peptides had enlarged and inflamed livers and spleens ( figure s in text s ). we analyzed serum levels of tnfa and il- as these cytokines are secreted by resident macrophages and blood monocytes in the lta/gln model [ ] . in accordance with the survival rates, our results indicate that the blood serum levels of tnfa and il- were significantly lower for the gp tmd peptide-treated group compared to the untreated group (figure a and b, respectively). we therefore examined spleen, in order to test activation of the immune system. the effect of an uncontrolled inflammatory response may lead to secretion of high levels of steroids, leading to massive cell death in the lymphocyte zones [ ] . lta/gln injection resulted in a large number of apoptotic cell foci in the white pulp and congestion throughout the tissue in comparison to control mice spleens ( figure c middle and top panels, respectively). comparably, gp tmd treatment decreased apoptosis to levels similar to the naïve samples ( figure c lower panel) . cell death was also detected in the red pulp of untreated mice and was absent from gp tmd treated mice. this result indicates that gp tmd decreased monocyte activation, resulting in less apoptosis in the spleen. in order to test the specific attenuating effect of gp tmd on lta/gln mediated sepsis, we tested whether the treatment resulted in an inhibitive effect on macrophages, as macrophages are the main immune cells that orchestrate this type of inflammation. we investigated whether the inhibitory effect of gp tmd peptide was tissue specific or systemic by staining both liver (tissue specific) and spleen (systemic) for the monocyte activation marker, mac [ ] . liver staining showed that the administration of lta/gln resulted in increase in mac positive cells, in addition to the appearance of round-shaped mac positive cells that could be either activated residential macrophages (kupffer cells) that phagocysed dead hepatocytes, or infiltrated activated monocytes ( figure a, white arrows) . the mutant gp showed minor amount of mac positive cells that resembled the non-treated control mice ( figure b ). these results suggest that the attenuating effect of gp tmd was systemic rather than tissue specific, with high specific effects on monocytes and macrophages. in this study, we report that the responsiveness of tlr is modulated by the env tmd. this inhibitory effect is driven through the direct association of the env tmd with the tlr tmd. functionally, the env tmd inhibits tlr signaling in response to lta and inhibits secretion of pro-inflammatory cytokines both in vitro and in an animal model of lta/gln sepsis. recent studies show that peptides derived from tmds of several membrane proteins interfere with their assembly and function [ , , ] . we utilized this strategy to assess the ability of env to affect tlr activation, and to identify the important amino acids within the tmd that contribute to this interaction. it has been shown that the tmd of tlr regulates its function in response to natural and synthetic ligands. the env tmd possesses a gxxxg motif, which is well defined as a membrane dimerization motif. interestingly, the tlr tmd also possesses such a sequence ( figure s , boxed in text s ). the precise conformational changes of the tlr tmd that induce its dimerization with its partners leading to signal transduction are not known. nevertheless, it is conceivable that the gxxxg motif of the tlr and tlr tmds move in a piston mechanism (for an explanation on the piston motion, see ref. [ ] ). this motion could explain how these motifs are important for the tlr function although they are not located parallel to each other within the membrane. mutating the gxxxg motif significantly decreased the ability of the env tmd to inhibit macrophage responses to tlr ligands. this is expected because the peptides' mode of action is by inhibiting dimerization of tlr and tlr through interactions within the membrane milieu. interestingly, the interaction of tmd sequences is not driven solely by the gxxxg motif, since the gpa tmd did not show any association with the tlr tmd. the specific nature of the interaction is demonstrated by the co-immunoprecipitation of tlr with the env tmd, while no interaction was detected with the mutated tmd. overall, the data suggest that the env tmd interferes with the dimerization of tlr / tmds partially through their gxxxg motif. this subsequently leads to impaired complex signaling and to inhibition of cytokine secretion. the general effect of this inhibition is demonstrated by inhibition of the initial steps of signal transduction (erk phosphorylation), as well as, by inhibition of an array of end point targets of tlr signaling (tnfa, il- and mcp- ) each transcribed in a different complex of nfkb. note that it has been shown that the gxxxg is important, but not sufficient, for helix-helix interactions within the membrane [ ] . as cd and tlrs may reside in similar membrane environments [ , ] , and membrane-bound tlrs recognize viral glycoproteins, it is reasonable to assume that this leads to initiation of a response towards hiv- during fusion. furthermore, activation of tlr signaling prior to hiv infection causes dendritic cells to be less permissive to infection [ ] . therefore, inhibiting the initial response towards the invading pathogen is a powerful tool to allow virus entry into the cell. an additional possible need for this inhibitory activity is at latent stages of viral infection. hiv- env and tlr both possess a membrane localization signal motif adjacent to their tmd ( figure s in text s ). this signal motif determines the precise environment within the membrane in which the proteins reside. although for t cells, env is expressed at low levels during latency, env expression levels on mononuclear phagocytes are high [ ] . several studies indicated the impairment of tlr signaling after viral infection and incorporation [ ] which may assist in the establishment of infection by opportunistic bacteria and fungi, in addition to the loss of cd + t cells [ ] . together with our findings about the modulatory effect of env on tlr responses, this impairment may occur as a result of the interactions of the tmds of env and tlrs, leading to the prevention of tlr activation upon ligand recognition. noteworthy, several studies have linked the activation of tlr cascades to viral replication and burst. these studies focus on cells with established infection. this means that the virus successfully transmitted its genome into the cells and initiated the expression of its early genes [ ] . in view of the small number of proteins expressed by the hiv genome, this immunosuppressive activity might exist with its fusogenic activity to help the virus to escape immune recognition and response towards it, prior to the activity of the viral early genes. in summary, we demonstrate that the env tmd inhibits the activation of tlr signaling by interacting with its tmd. this property could be of great importance during fusion and serves as an additional mean of regulation of the activity of tlr at stages of env expression. our findings further demonstrate the complex strategies used by hiv- to manipulate the immune system. interestingly, the env tmd is also involved in the late steps of the membrane fusion reaction. such dual activity further demonstrates how viruses although expressing a limited number of proteins have evolved to alter so many cellular processes. considering the strong ability of other gp -derived segments to inhibit t-cell activation by various mechanisms, our study may indicate that these effects are also applicable to mononuclear phagocytes and additional targets should be further investigated. apart from hiv pathogenesis, specific gp derived peptides may provide potential tools for treating uncontrolled immune responses, manifested here by sepsis. all in vitro assays were performed on raw . murine macrophages (atcc-tib ). cells were grown at uc in the presence of % co in dmem supplemented with % fbs, lglutamine, sodium pyruvate, non-essential amino acids, and antibiotics (biological industries, beit-haemek, israel). for cytokine secretion measurements cells per well were cultured overnight in a -well plate. at the following day, media were replaced by fresh dmem, including all supplements. gp tmd peptides were dissolved in dmso and added to the cells in different concentrations. the final concentration of dmso was % for all groups. cells were incubated with the peptide for hours, washed and incubated with fresh media containing lta. cells were incubated with lta for hrs (tnfa) or hours (il- , mcp- ) at uc, after which samples of the media were collected and stored at uc. cytokine levels were evaluated using a mouse tnfa/il- enzyme-linked immunosorbent assay kit (elisamax, biolegend) according to the manufacturer's protocol. all experiments were done in triplicates. raw . macrophages were transfected twice, h and again h prior to the experiment, either with p zm gp -cd -opt plasmid for the expression of hiv envelope protein gp , or with gp -yfp expressing plasmid for the expression of gp hiv fusion protein [ ] . to increase the expression rate we utilized turbofect reagent (thermo scientific, waltham, ma, usa) according to the manufacturer protocol. surface expression of untagged gp was analyzed using immunofluorescence. briefly, cells were washed twice with pbs and fixed with % paraformaldehyde for min at room temperature. antibody staining was conducted using a goat polyclonal anti-hiv- gp -biotin conjugated antibody and a atto conjugated, goat igg (h&l) antibody. fluorescence intensities were obtained using an inverted fluoview confocal microscope (olympus, tokyo, japan) with a oil immersion objective (numerical aperture . ) at uc with a frame size of pixels. atto was excited with a laser diode at nm and detected between and nm. large unilamellar vesicles (luvs) composed of phosphatidylcholine (pc) and cholesterol (chol) ( : , w/w) were prepared using the extrusion method as described previously [ ] . the fluorescence experiments were performed with pairs of peptides using -fluoro- -nitrobenzofurazan (nbd, biochemika) -labeled peptides as donors and rhodamine-labeled peptides as acceptors and as described previously [ ] . maximum fret values were obtained by calculating the changes in the emission of the donor peptide at nm. cd measurements were performed by using an applied photophysics spectropolarimeter. the spectra were scanned using a thermostatic quartz cuvette with a path length of mm. wavelength scans were performed at uc; the average recording time was s, in nm steps, through the wavelength range of - nm. peptides were scanned at a concentration of mm in buffer only (hepes mm) or in a membrane mimetic environment of % lysophophatidilcholine (lpc) in buffer. the hetero-interactions of the tmds within a natural membrane environment were studied using the gallex assay [ ] . the expression vectors and e. coli strains required for the assay were kindly provided by dr. dirk schneider. briefly, two plasmids are used in the assay containing either the wild-type lexa sequence (pblm, encoding for resistance to tetracycline) or a mutated form of lexa (palm, encoding for resistance to ampicillin). the desired tm sequences are inserted between these two protein sequences using standard cloning techniques. the resulting plasmids were both transformed into an su e. coli strain, which then expressed the lexa-tm-male fusion proteins (after induction with mm iptg). hetero-association of tm domains leads to the formation of lexa wt /lexa mut heterodimers that bind to a hybrid lexa promoter/operator in the reporter strain su (that recognizes the mutant lexa domain), and repress the expression of b-gal. furthermore, as the hybrid promoter is designed to bind only the wt and mutant heterodi-mers, it will not recognize wild-type lexa homodimers. thus, tm homo-oligomer formation does not interfere with the measurement of hetero-association. the tmds of glycophorina (gpa) and its g i mutant, used as positive and negative controls, respectively, are itliifgvmagvigtilli and itliifgvmai-vigtilli. all the constructs were confirmed by dna sequencing. the dimerization propensity of the two constructs was measured by transforming an e. coli su indicator strain with the relevant palm and pblm plasmids (each harboring a different tm sequence) simultaneously. the bacteria were grown overnight in the presence of mm iptg in lb medium with ampicillin and tetracycline in a -well plate. ml of bacteria were resuspended in ml of z-buffer ( mm na hpo , mm n a h po , mm kcl, mm mgso , ph = . , % chloroform, % b-mercaptoethanol), and the absorbance (od ) was measured. bacteria were lysed with ml of % sds in zbuffer. o-nitrophenyl-galactopyranose (onpg) was added and a kinetic reading of absorbance at od was performed for min to measure the activity of b-gal. vmax was calculated and divided by the absorbance at od to correct for bacterial growth. strong dimerization results in strong inhibition of b-gal expression and less color formation. cell lysates were prepared in radioimmunoprecipitation (ripa) assay buffer ( mm tris hcl ph . , mm nacl, % nonidet p- , . % sodium deoxycholate, . % sds) containing mm naf, mm na vo , protease and phosphatase inhibitors (sigma-aldrich). protein concentration was measured using the bca protein assay kit (pierce chemical co.). fifty mg of protein was loaded and separated on - % sds-page and transferred to a nitrocellulose membrane. the membrane was blocked using % bovine serum albumin (bsa) in phosphate-buffered saline containing . % tween- (pbst) for h at room temperature. the primary antibody was diluted in pbst containing % bsa and incubated with the membrane at uc overnight. after washes with pbst, membranes were incubated with the secondary antibody in pbst containing % bsa at room temperature for h. the following sequences were used for quantitative mrna analysis: tnfa forward: cttgtggcaggggccaccac reverse: ccatgccgttggccaggagg mcp- forward: tcacctgctgctactcattcacca reverse: agcacagacctctctcttgagctt anti phospho-erk , and anti-total erk , were purchased from sigma-aldrich and anti gapdh was purchased from millipore. goat polyclonal anti-hiv- gp -biotin conjugated antibody was purchased from abcam (cambridge, united kingdom). atto conjugated goat igg (h&l) antibody was purchased from rockland (gilbertsville, pa, usa). s. aureus lta was purchased from sigma-aldrich and d-galactosamine was purchased from calbiochem. tnfa and il- elisa kits were purchased from biolegend. the plasmid p zm gp -cd -opt was obtained through the nih aids reagent program, division of aids, niaid, nih: from drs. yingying li, feng gao, and beatrice h. hahn. the plasmid gp -yfp, provided by roland schwarzer encodes a hiv- gp fusion protein with the c-terminal external parts of the protein replaced by a yellow fluorescent protein. animal studies were carried out in strict accordance with the israeli law and the national research council guidelines. all animal experiments were conducted at the weizmann institute of science and approved by the weizmann institutional animal care and use committee (iacuc permit no. - ). week-old c black female mice were injected ip with mg/kg of s.aureus lta and mg/kgof d-galactosamine in pbs, as previously described [ ] , in combination with mg/kg of gp -tmd or mutated gp -tmd or pbs, and monitored for survival for hours. blood samples were collected for tnfa and il- serum levels measurements. serum was extracted by blood coagulation in rt followed by spin down centrifugation. serum samples were analyzed by elisa assays. spleen and liver samples were collected either post mortem or from sacrificed mice hours after the injection of lta/gln and kept in formaldehyde ( %) for hours and embedded in paraffin blocks. mm sections were then prepared and stained with hematoxylin and eosin by standard protocols. peptides were synthesized by a -fluorenylmethoxylcarbonyl (fmoc) solid-phase method on rink amide mbha resin (calbiochem-novabiochem, san diego, california) by using an abi a automatic peptide synthesizer (applied biosystems, foster city, ca). peptide synthesis was followed by cleavage from the resin by incubation for h with % tfa, . % h o, and . % triethylsilane, followed by purification by rp-hplc (. %) on a vydac c column (grace discovery sciences, deerfield, il), and then identification by electro-spray mass spectroscopy. the following fluorophores were used for fluorescent labeling: -fluoro- -nitrobenzofurazan (nbd, biochemika) and ( )-carboxytetramethylrhodamine n-succinimidyl ester (tamra, bio-chemika). peptides used for in vivo experiments were treated twice with % acetic acid in order to replace the trifluoroacetate anion added during hplc purification. text s this file contains table s and figures s -s . figure s . this figure shows that gp tmd disrupts the interaction between tlr and tlr as revealed by fluorescence resonance energy transfer (fret) measurements. in this experiment the nbd-labeled tlr tmd peptide was added first from a stock solution in dmso (final concentration . mm and a maximum of . % (v/v) dmso) to a dispersion of pc:chol luvs ( mm) in pbs. this was followed by the addition of the following: a. rhodamine labeled tlr tmd or in addition with un labeled wt gp (b), in sequential doses ranging from . mm to . mm (stock in dmso), generating a ratio of : , : , : and : rhodamine:nbd presented from top to bottom. in both graphs, the upper spectrum represents the emission of the nbd-labeled peptide alone. (c) ip of tlr from raw . cells in the presence of the indicated rho-labeled peptides. the result of the last lane from the marker shows that in the presence of wt gp there is a decrease in the binding of the tlr -tmd peptide to the tlr protein. figure s . this figure shows the secondary structure of the peptides used in this study as measured by cd spectroscopy. spectra were measured at mm in a % lpc solution. graphs are the mean of measurements. figure s . this figure shows that treatment with gp wt peptide rescues mice organs form inflammatory related damage. in this experiment tissue samples of liver (a) and spleen (b) from mice taken at the experimental end point. in both tissues inflammation is evident by the tissue color. figure s . this figure shows sequence alignment of the tmds of gp (top) and tlr (bottom). arrows indicate the predicted tmd. underline indicates the localization motif to cholesterol enriched regions within the membrane. figure s . monocyte-derived macrophages and myeloid cell lines as targets of hiv- replication and persistence immune evasion and counteraction of restriction factors by hiv- and other primate lentiviruses hiding in plain sight: how hiv evades innate immune responses hiv impairment of immune responses in dendritic cells human immunodeficiency virus type mediates global disruption of innate antiviral signaling and immune defenses within infected cells pathogen recognition receptors: ligands and signaling pathways by toll-like receptors sensing of microbial molecular patterns by toll-like receptors tlr : cellular sensor for microbial and endogenous molecular patterns toll-like receptors linking innate and adaptive immune response toll-like receptor signalling crystal structure of the tlr -tlr heterodimer induced by binding of a tri-acylated lipopeptide activating immunity: lessons from the tlrs and nlrs assembly of the tlr / transmembrane domains is essential for activation and is a target for prevention of sepsis activation of toll-like receptor increases macrophage resistance to hiv- infection dendritic cells from hiv- infected individuals are less responsive to toll-like receptor (tlr) ligands mediators of innate immune recognition of bacteria concentrate in lipid rafts and facilitate lipopolysaccharide-induced cell activation intracellular lipid flux and membrane microdomains as organizing principles in inflammatory cell signaling hiv- broadly neutralizing antibody extracts its epitope from a kinked gp ectodomain region on the viral membrane toll-like receptor on inflammatory monocytes induces type i interferon in response to viral but not bacterial ligands hiv- gp and tcralpha trans-membrane domains share a motif exploited by the hiv virus to modulate t-cell proliferation motifs of two small residues can assist but are not sufficient to mediate transmembrane helix interactions tolllike receptor (tlr) - agonists-induced cytokines and chemokines: i. comparison with t cell receptor-induced responses transmembrane domains interactions within the membrane milieu: principles, advances and challenges analyzing oligomerization of individual transmembrane helices and of entire membrane proteins in e. coli: a hitchhiker's guide to gallex the spleen as a target in severe acute respiratory syndrome neuronal accumulation of glucosylceramide in a mouse model of neuronopathic gaucher disease leads to neurodegeneration specific inhibition of a pathogenic receptor tyrosine kinase by its transmembrane domain computational design of peptides that target transmembrane helices lipid raft distribution of cd depends on its palmitoylation and association with lck, and evidence for cd -induced lipid raft aggregation as an additional mechanism to enhance cd signaling location, location, location: is membrane partitioning everything when it comes to innate immune activation? grampositive bacteria enhance hiv- susceptibility in langerhans cells, but not in dendritic cells, via toll-like receptor activation biosynthesis, cleavage, and degradation of the human immunodeficiency virus envelope glycoprotein gp cd (+) t-cell depletion in hiv infection: mechanisms of immunological failure signaling through toll-like receptors triggers hiv- replication in latently infected mast cells the cholesterol-binding motif of the hiv- glycoprotein gp regulates lateral sorting and oligomerization characterization of the interacting domain of the hiv- fusion peptide with the transmembrane domain of the t-cell receptor gallex, a measurement of heterologous association of transmembrane helices in a biological membrane we thank dr. ori brenner of the weizmann institute of science for his help in analysis of the histological specimens and their interpretation. we also thank dr. avraham ashkenazi and mr. yoel klug for their critical revision of the manuscript and fruitful inputs. key: cord- - mjmttec authors: wodrich, harald; henaff, daniel; jammart, baptist; segura-morales, carolina; seelmeir, sigrid; coux, olivier; ruzsics, zsolt; wiethoff, christopher m.; kremer, eric j. title: a capsid-encoded ppxy-motif facilitates adenovirus entry date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: mjmttec viruses use cellular machinery to enter and infect cells. in this study we address the cell entry mechanisms of nonenveloped adenoviruses (ads). we show that protein vi, an internal capsid protein, is rapidly exposed after cell surface attachment and internalization and remains partially associated with the capsid during intracellular transport. we found that a ppxy motif within protein vi recruits nedd e ubiquitin ligases to bind and ubiquitylate protein vi. we further show that this ppxy motif is involved in rapid, microtubule-dependent intracellular movement of protein vi. ads with a mutated ppxy motif can efficiently escape endosomes but are defective in microtubule-dependent trafficking toward the nucleus. likewise, depletion of nedd ligases attenuates nuclear accumulation of incoming ad particles and infection. our data provide the first evidence that virus-encoded ppxy motifs are required during virus entry, which may be of significance for several other pathogens. many viruses use the microtubule network of the host cell for transport to their site of replication (i.e. the nucleus) [ ] . access to the microtubule network is achieved through recruitment of cytoplasmic dynein motor proteins followed by efficient retrograde transport towards the nucleus [ , ] . virus-induced cellular signaling cascades help stimulate the directionality and efficacy of the transport [ ] . viral interaction with dynein motor proteins occurs either directly through capsid proteins or indirectly via hijacking of adapters from existing transport pathways [ ] . most dna viruses accumulate transiently at the microtubule organizing center (mtoc) prior to nuclear translocation [ , , ] . how they release from the microtubules or the mtoc and transport to nuclear pores is poorly understood. mtoc release may involve a switch from dynein to kinesin mediated transport, the cellular ubiquitin/ proteasome system and/or nuclear transport receptors [ , , [ ] [ ] [ ] [ ] . indirect evidence that the host's ubiquitylation machinery participates in parts of the viral entry process comes from studies using pharmacological inhibitors of the ubiquitin/proteasome system. for example, translocation of a murine coronavirus from the endosome to the cytoplasm is facilitated by the ubiquitinproteasome system [ ] . similarly, influenza viruses appear to be trapped in an endosomal compartment upon pharmacological inhibition of the proteasome [ ] . in contrast, blocking the proteasome increases the transduction efficiency of adenoassociated virus vectors and this correlates with ubiquitylation of capsid proteins [ , ] . the semliki forest and the vesicular stomatitis virus, however, do not seem to be affected by proteasome inhibition during their entry suggesting different host factor requirements [ ] . a role for the ubiquitylation machinery during egress of enveloped viruses is better understood. egress involves the transport of assembled capsids, subviral structures or individual capsid proteins to assembly and budding sites at the cell surface or at intracellular membranes [ ] . budding, and potentially trafficking, to the egress site requires an intact class e vesicular sorting pathway (vsp, [ , ] . the vsp is believed to involve the consecutive activity of three distinct heteromeric complexes termed endosomal sorting complexes required for transport (escrt-i, -ii and -iii, [ ] ). the capsid proteins of several enveloped viruses encode 'late domains' that specifically interact with escrt components and redirect them towards the site of viral egress [ ] . some late domains of the ppxy motif type (where x can be any amino acid) require the binding of ubiquitin ligases of the nedd family of hect-e ubiquitin ligases (homologous to e -ap carboxyl terminus) for efficient escrt recruitment [ ] . nedd . and its close relative nedd . are prototypic members of this family, which is conserved from yeast to mammals [ ] . they encode a n-terminal c domain for ca + -dependent lipid interaction, a catalytical hect domain and three to four wwdomains for protein-protein interactions with proline-rich domains such as the ppxy motif [ , ] . the exact role of ppxyrecruitment of nedd ligases in vsp-mediated viral budding is still unclear. a possible link was recently shown by enhanced escrt ubiquitylation through nedd . overexpression [ , ] . late domains, including the ppxy type, have also been found in some nonenveloped reoviruses but a general function in virus release remains to be shown [ ] . ppxy type late domains where also described for the ad capsid protein penton, which can interact with ubiquitin ligases of the nedd family. however, its role in ad infection is unclear [ ] . at least in vitro, ad infects cells by first attaching to primary receptors, including car, cd and sialic acid, via the fiber protein [ ] . in some cells endocytosis of ad may be triggered by penton base-mediated signaling through alpha(v) integrins [ ] [ ] [ ] . in epithelial cells, ad serotype (ad ) particles undergo stepwise disassembly during entry, starting with detachment of the fiber at or near the cell surface and followed by a passage through endosomal compartments in which acidification serves as additional disassembly trigger for membrane penetration and cytosolic translocation [ , ] . partial disassembly releases the internal capsid protein vi, which can lyse membranes in vitro via its predicted n-terminal amphipathic helix [ ] . in the cytosol, the particle engages in microtubule-directed transport towards the mtoc and is translocated to the nuclear pore complex (npc) for nuclear import of the genome [ ] [ ] [ ] . in this study we address the mechanisms of ad cell entry. we demonstrate that the internal capsid protein vi is rapidly exposed to antibodies during cell entry, possibly at the cell surface or immediately after endocytosis. we further determine that protein vi remains partially associated with ad capsids as they traffic to mtocs and the npc. we identify a functional ppxy motif within protein vi that mediates the association of protein vi with nedd e ubiquitin ligases and facilitates its ubiquitylation. recombinant ad in which the protein vi ppxy motif is mutated have normal capsid morphology, escape from endosomes with similar efficiency as wildtype viruses, but are defective in genome delivery to the nucleus. we show that the ppxy motif in protein vi is involved in its efficient microtubule-mediated transport and mutating it in the virus alters the intracellular targeting of ads towards the mtoc region concomitant with a post-entry block in viral infectivity. furthermore, nedd . and nedd . are involved in ad infection and intracellular targeting of incoming virions to the mtoc. we propose that the ppxy motif, in other viral systems, may also function during entry and interact with novel cellular pathways for efficient viral entry. protein vi is exposed during ad infection and partially remains associated with the viral particle the fate of ad particles immediately after internalization is only partially characterized. in the context of endosome escape of ad , very little is known about how this occurs and which, if any, cellular proteins are involved. from in vitro studies it was proposed that the internal capsid protein vi mediates ad endosome escape [ ] . previous reports showed that protein vi dissociates from the ad capsid very early after attachment [ , ] . to delineate the fate of protein vi during ad entry we performed infection assays and followed the intracellular distribution of the viral capsid and protein vi as a function of time. to this end, fluorescently labeled ad particles were adsorbed to either human retina epithelia pigment cells (htert-rpe , figure ) or human osteosarcoma cells (u os, figure s ) at uc and then transferred to uc to synchronize internalization. cells were fixed at various times and analyzed by confocal microscopy (figure ). protein vi was detected using an affinity purified polyclonalantiserum and the location of the mtoc was marked by detecting the primary cilia, which originates at the mtoc using antibodies against acetylated tubulin [ ] . at uc viral particles accumulated at the cell periphery showing sporadic positive staining with the protein vi antiserum (, %) possibly due to the recognition of protein vi from damaged particles (figure first row). in contrast, min after the temperature shift, ad particles were still localized close to the cell periphery but approximately % of them gave a signal with the protein vi antibody indicating that more protein vi was accessible ( figure , second row). after min, particles had entered the cell with some localized at the mtoc region ( figure , third row) and some at the nuclear rim as described previously [ ] . about % of the particles remained protein vi positive, including particles at the nuclear rim. after min the majority of the particles were concentrated at the mtoc region ( figure , bottom row) as previously reported [ ] . protein vi staining was also concentrated at the mtoc region but most of the signal was not particle associated. similar results were obtained in u os cells ( figure s ). together these data suggested that structural rearrangements leading to protein vi exposure take place at or close to the cell surface during ad entry. in addition, the data showed that protein vi trafficked to the mtoc region and partially remained associated with the capsid. to identify possible trafficking determinants, we analyzed the sequences of protein vi from several ad serotypes and identified a highly conserved ubiquitin-ligase interacting motif present in ppxy-type viral late domains (ppxy, figure s ). to examine the role of this ppxy motif in ad cell entry, we used an e /e -deleted ad that had the protein vi ppsy motif mutated to pgaa (ad -vi-m , detailed in figure s ) [ , ] . this mutation, when viruses exploit cellular functions during entry and exit of cells. to redirect cellular functions for their own purpose, viruses encode high-affinity binding sites for key-cellular factors. one such domain is the ppxy motif, which is present in structural proteins of several, mainly enveloped viruses. this motif binds to ubiquitin ligases of the nedd family and recruits their function to sites of virus budding from cells. here we show that adenoviruses also encode a ppxy motif in the internal structural protein vi and that the ppxy motif has an unprecedented function in virus entry. adenoviruses with mutations in the protein vi ppxy motif undergo normal endosomal uptake and membrane penetration but have reduced infectivity, altered intracellular targeting and lack efficient gene-delivery. we also find that protein vi is ubiquitylated by nedd ligases in a ppxy dependent manner following partial capsid disassembly and displays rapid intracellular movement. depletion of nedd ligases also alters virus movement within cells during entry and reduces viral infectivity. given that ppxy motifs are important for virus exit our findings might have uncovered an additional function for ppxy motifs in virus entry, potentially expanding the significance of ppxy motifs and functionally related domains for viral replication. introduced into mason-pfizer monkey virus, was previously shown to abolish late domain functions with no apparent structural changes, which would impair virus assembly [ , ] . to control for unintended mutations introduced during the cloning, we reverted the pgaa sequence back to ppsy (ad -vi-wt). because the , copies of protein vi appears to be in contact with several proteins in the mature capsid, modifications that disrupt the tertiary structure could also affect the capsid composition. in large-scale preparations, mutant and wt virus banded at identical densities and gave similar yields of particles as determined by genome and protein quantification. a biochemical analysis of the capsid composition of purified viral particles showed no apparent differences between wt and mutant viruses (figure a and data not shown). to confirm that viral capsid integrity between mutant and figure . timecourse of protein vi release during ad entry. ad - was pre-bound to htert-rpe cells at uc (top row) and shifted to uc for min (second row), min (third row) and min (bottom row) as indicated. protein vi was detected using anti-protein vi antibodies (left column), adenoviral particles by detecting the alexa- fluorescent signal (second column) and the mtoc by staining the primary cilia (third column). a composite of all three signals including the nucleus (in grayscale) is shown to the left. the inset shows an inverted magnification of representative virus, protein vi and the primary cilia signals from the small dashed box. in the composite protein vi signals are shown in red, ad is shown in green, the primary cilia in grey and the nucleus in blue. colocalization of protein vi and ad appears as yellow. the scale bar is mm. doi: . /journal.ppat. .g figure b capsid integrity and morphology of the mutant virus was indistinguishable from the wt virus. in contrast, the infectious versus physical particle ratio of ad -vi-m was , -fold lower than ad -vi-wt as assayed by plaque formation on monolayers of cells ( figure c ). because infectious vs. physical particles can vary between preparations, we assayed plaque size, which is more informative measurement of propagation rate. plaques were significantly smaller for ad -vi-m versus ad -vi-wt (see below), suggesting that the altered ppxy domain affects some stages of virus propagation. to determine whether the m mutation influences ad cell entry, we performed a fluorescent focus forming assay and stained cells at , and h post-infection for expression of the e a protein, which marks the appearance of viral replication centers ( figure d and data not shown). compared to ad -vi-wt, the ad -vi-m virus produced approximately -fold fewer fluorescent foci when equivalent numbers of viral particles were used for infections. this suggested that steps prior to replication (i.e. internalization) require an intact ppxy motif in protein vi. to address trafficking using a different approach, we inserted a gfp expression cassette into the frt site in the e -deleted region of the wt and the m mutant virus (see figure s for details). the gfp expressing viruses showed no difference in the quantitative yield and biochemical composition after large-scale purification. we repeated plaque forming assays ( figure e ) and single round infection assays ( figure f ), this time using gfp expression as the quantification method in non-complementing u os cells. we observed a reduction in infectivity in the same order of magnitude as previously (compare figures c with e and d with f). in addition the gfp expression allowed us to follow the plaque formation over time. as shown in figure s the spread of the m mutant virus was significantly slower and led to fewer and much smaller plaques ( figure e and figure s ). next we asked whether ad endosomal lysis efficiency following internalization is affected by the mutation in the ppxy motif of protein vi. we infected a cells with , or particles per cell of either ad -vi-wt or ad -vi-m in the presence of alpha-sarcin, a membrane impermeable toxin that inhibits translation when it enters the cytoplasm. alpha-sarcin enters the cell by virus-mediated endosomal membrane lysis thus providing a quantifiable marker for endsome membrane lysis ( figure g , [ ] ). we measured the incorporation of radiolabeled amino acids over time as a means to determine translational efficiency. as control we used the temperature sensitive ad mutant ad ts that is closely related to ad . ad ts mutants poorly lyse the endosomal membrane when the virus is grown at non-permissive temperatures due to an increased capsid stability that lacks intraendosomal disassembly. ad ts mutants grown at permissive temperatures lyse the endosome with the same efficiency as the wt virus. incorporation of radiolabeled amino acids was compared to alpha-sarcin treated cells. when we added alpha-sarcin and ad -vi-wt, ad -vi-m or the ad ts mutant grown at a permissive temperature translation was diminished in a dosedependent manner over at least two orders of magnitude, showing efficient cytoplasmic delivery of the toxin. ad ts grown at the non-permissive temperature did not inhibit translation in this assay ( figure g ). we observed no difference between the ppxy-mutant virus and the wt controls. this indicated that an attenuating effect of the ppxy-mutation occurs after endosomal lysis and prior to the onset of replication. we next characterized the release of protein vi from fluorescently labeled ad-vi-m and ad -vi-wt following synchronous infections of u os cells using the infection assay as described for figure . ad -vi-wt released protein vi within min ( figure a , left panel). in contrast, we observed a delayed release of protein vi from ad -vi-m ( % of m after min compared to % of wt, figure a ) and an increase in colocalization of viral particles with protein vi at the nucleus ( figure a ). this observation suggests a delayed accessibility of protein vi within the virus or a defect in protein vi dissociation from the virion ( figure a , images to the right). in addition to the increase of protein vi capsid association, ad -vi-m appeared to be more evenly distributed throughout the cell and did not efficiently accumulate at the mtoc region ( figure b , images to the left). quantification revealed that min post-infection approximately % of the wt viral particles in proximity of the mtoc could be found within a mm radius around the mtoc and % within - mm. in contrast, the localization for the m virus was % for each region showing a decreased targeting towards the mtoc ( figure b , right panel). in summary these data suggest that the ppxy motif in protein vi is required for proper uncoating and normal nuclear targeting. to understand the accumulation defect of ad -vi-m at the mtoc, we expressed wt (vi-wt), mutant (vi-m ) and protein vi deleted in the amphipathic helix (vi-dw) fused to mrfp in cells and analyzed protein vi localization in relation to microtubules ( figure ). we found vi-wt in a punctuated distribution throughout the cell suggesting association with a vesicular compartment or tubulo-vesicular structures associated with microtubules ( figure , top row). in contrast, the ppxy motif mutant vi-m localized to a more central compartment and was rarely associated with the microtubules (figure , middle row). deletion of the amphipathic helix, in contrast, abrogated purified ad capsids. electronic microscopy images of purified ad -vi-wt capsids (left panel) and purified ad -vi-m (right panel) are shown. the inset in each figure shows a magnification of individual capsids. the scale bar is nm. c) plaque assay comparison of ad -vi-wt vs. ad -vi-m . quantification of plaques at day for different physical particle per cell ratios for ad -vi-wt and ad -vi-m . shown is the average plaque number per well (+/ standard deviation) of three individual experiments. d) focus forming assay. e complementing cells were infected with ad -viwt or ad -vi-m and replication centers were stained with e a antibodies h post-infection and at different particle per cell ratios. . random fields with . cells were counted, standard deviation represents field-to-field variations. e) plaque forming assay comparison of ad gfp-vi-wt vs. ad gfp-vi-m . quantification of gfp positive plaques at day at different physical particle to cell ratios. the values show the average number of gfp positive plaques per -well (+/ standard deviation) of two independent experiments. f) single round infection assay comparison of ad gfp-viwt vs. ad gfp-vi-m . u os cells were infected with increasing amounts of physical particle to cell ratios as indicated. gfp expression was quantified using facs. values correspond to the average percentage of gfp positive cells of two experiments done in triplicates (+/ standard deviation). note that the , % infection of wt infected cells at physical particles per cell is saturated and not comparable to m . g) analysis of membrane penetration. a cells were infected with ad -vi-wt, ad -vi-m , ad ts grown at uc or at uc at different physical particle per cell ratios as indicated and in the presence of alpha-sarcin and translational efficiency was determined by measuring the incorporation of radiolabeled amino acids over time. values are given in percentage normalized to % translation measured in the presence of the toxin but in absence of virus. conditions are indicated below each bar and are the mean of at least three independent experiments done in triplicates. doi: . /journal.ppat. .g owing to its association with membranes, microtubules and the viral capsid, we next asked whether protein vi displayed intracellular dynamics that could explain virus trafficking. we therefore performed live-cell imaging (lci) using cells expressing mrfp-vi-wt or mrfp-vi-m . we found that vi-wt was fast moving with short-and longrange movements whereas vi-m was essentially motionless ( figure a and video s ). the length of the trajectories and the movement of . particles were plotted ( figure b ). we found that protein vi-m motility was greatly reduced compared to protein vi-wt. we next asked whether vi-wt motility depends on intact microtubules and/or actin filaments. disrupting actin filaments with cytochalasin b had no apparent effect on vi-wt localization or motility ( figure b , right panel) suggesting that actin was not involved in the movement. in contrast, protein vi motility in nocodazole-treated cells was strongly reduced resembling the reduced motion observed for the m mutant ( figure b , right panel, see also video s in the supporting information). we asked whether vi-m showed only plus-end microtubule directed movement. we applied nocodazole to vi-m transfected cells, followed by washout of nocodazole. during treatment and after removal of nocodazole no movement or relocalization towards the cell center was observed for vi-m . in contrast viwt rapidly stopped and restarted bidirectional movement under these conditions (data not shown). together these data suggested that protein vi is a highly mobile protein that moves along microtubules, presumably in association with vesicular structures whose motion depends on the ppxy motif. ppxy motif is essential for protein vi ubiquitylation upon partial ad disassembly ppxy domains are the physiological targets of ubiquitin ligases of the nedd family [ ] . therefore we asked whether protein vi ubiquitylation depends on the ppxy motif. we adapted an in vitro ad disassembly assay mimicking the partial capsid disassembly believed to occur during ad entry by exposing virions to uc. projection. two concentric circles with mm and mm in diameter were positioned around the mtoc as shown in the left panel. virus particles were then counted inside the mm radius and in the region between - mm. the graph to the right shows the relative abundance within the two regions. distribution for the wild type virus is shown on the left of the graph and for the ppxy mutated virus to the right. the analysis shows that mutated particles are less likely to accumulate at the mtoc then wt particles. the error bar represents cell-to-cell variation (n. , p, . ). doi: . /journal.ppat. .g figure . subcellular localization of protein vi. u os cells were transfected with protein vi fused to mrfp, either using wt protein vi (vi-wt, top row), with mutated protein vi (vi-m , middle row) or with deleted amphipathic helix (vi-dw, bottom row). cells were co-stained for microtubules. the protein vi-signal is shown in the left column, the microtubule stain is shown in the second column and an overlay of the signals is shown in the third column. protein vi fusions appear in red, microtubules in green and the nucleus in blue. the right column shows the field of cells from which the insets (small white square) was taken. the scale bar is mm, please note that the lower panel is a higher magnification. association of protein vi with microtubules in tubulo-vesicular structures is indicated by arrows in the top row. further examples of tubulo-vesicular structures can also be seen in video s showing life-cell imaging of vi-wt transfected cells. doi: . /journal.ppat. .g this assay dissociates the vertices including fiber, penton, protein vi and peripentonal hexons, but leaves the remainder of the capsid intact ( figure s ; [ ] ). heat and mock-treated samples were subjected to in vitro ubiquitylation reactions, using free ubiquitin, recombinant e and e enzymes and purified cytosol as source for the e ubiquitin ligase(s), and analyzed by western blot (figure , [ ] ). western blot analysis showed that partial capsid disassembly resulted in the appearance of protein vi reactive signals with discrete size increments suggesting predominant modification with two to three ubiquitin as well as some higher molecular weight bands (lane , figure a ). in contrast, the lack of capsid disassembly (lane ) or cytosol (lane ) showed no additional protein vi reactive bands. we also tested ubiquitylation of the capsid proteins fiber, protein iiia and penton base as internal control. we only detected ubiquitylation of the penton base (which also harbors two ppxy domains at its n-terminus), while the fiber and protein iiia (which lack ppxy motifs) were not modified ( figure s ). the ubiquitylation of protein vi was also confirmed by using gst-ubiquitin in the above assay, followed by gstpulldown to show covalent modification of protein vi with ubiquitin confirming the predominant modification with two to three ubiquitin-moieties (data not shown). to address the role of the ppxy motif in protein vi ubiquitylation, we repeated the in vitro ubiquitylation assay using wt or m mutant protein vi purified from e. coli followed by western blot analysis. we detected protein vi-reactive bands, consistent with protein vi modified with two to three ubiquitin ( figure b , lane ). in contrast, no modification was observed when the ppxy motif was mutated ( figure b , lane ) or in the absence of atp ( figure b, lane and ) . using viral particles in in vitro ubiquitylation reactions (following partial capsid disassembly) protein vi of ad -vi-m was not ubiquitylated ( figure c , figure . subcellular dynamics of protein vi. a) u os cells were transfected with vi-wt (top row) or vi-m (bottom row) fused to mrfp and analyzed by live-cell imaging. frames were taken at sec intervals using a cascade b camera. the left image in all rows shows maximum image projections of the full frame depicting the cell with scale bar of mm. the four inverted images to the right show magnifications from the boxed inset of four consecutive frames (in the case of vi-wt) and every fifths frame (in the case of vi-m ). in the top panel a moving particle is depicted by a grey arrow within each frame using the departure point depicted by a white arrow as reference. the lower panel shows vi-m not moving. b) analysis of trajectory length and relative particle speed of vi-wt or vi-m (left side of both panels) or trajectory length and relative particle speed of vi-wt using drugs as indicated (right side of both panels). movies were processed and all recorded trajectories were analyzed for length and relative speed using the imaris tm software package. individual particles were plotted for length of trajectories (in mm, left panel) and the net movement (in mm/ sec, right panel) under the conditions tested (indicated below each chart). significant differences are indicated by bars. doi: . /journal.ppat. .g lane and ), while protein vi from ad -vi-wt was ( figure c , lane and ). thus, the ppxy motif in protein vi is inaccessible in intact capsids but can recruit ubiquitin ligase activity from cytosol when protein vi is released from the capsid interior. together our results show that protein vi ubiquitylation depends on i) virus disassembly, ii) an intact ppxy domain and iii) the presence of a cytosolic ubiquitylation activity. to identify the ligase responsible for protein vi ubiquitylation, we focused on the nedd -family members nedd . , nedd . , aip /itch, wwp and wwp because they can interact with viral late domains that harbor ppxy motifs [ ] . we first coexpressed the vi-wt or vi-m mrfp fusion protein together with each of the e ligases fused to gfp in u os cells. when expressed alone, most ligases localized primarily to the cytoplasm (data not shown, wwp localized to the plasma membrane and wwp accumulated in an uncharacterized intracellular membrane compartment). in contrast, when vi-wt is coexpressed with nedd . , nedd . or aip /itch, the ligases are recruited to the same membrane compartment as protein vi ( figure a, row - ). wwp appears to sequester protein vi at the plasma membrane ( figure a, row ) . wwp does not colocalize with vi-wt ( figure a , row ). we did not detect significant colocalization between vi-m and the e ligases, consistent with a ppxydependent interaction ( figure s ). to determine whether any of the ligases specifically interact with protein vi, we used purified cytosol from cells overexpressing gfp-tagged ligases and performed pull-downs with beads coated with recombinant protein vi-wt or vi-m . two ligases, nedd . and nedd . , were highly enriched on vi-wt beads while none of the other ligases showed strong binding to vi-wt-or to vi-m beads ( figure b ). taken together, these data suggest a preferential interaction between nedd . and nedd . and the ppxy motif in protein vi, which leads to relocalization of the ligases from the cytoplasm to a membrane compartment. to further characterize the interaction between protein vi and the ligases, we knocked down nedd . , nedd . , aip /itch, wwp and wwp using sirnas ( figure a ). the cells were then incubated with an ad vector harboring a gfp expression cassette (adgfp) at a low multiplicity of infection ( physical particles per cell) for h to achieve approximately % transduced cells and limit the time of virus exposure. the following day the percentage of gfp-positive cells was quantified by flow cytometry. most ligase knockdowns had no significant effects on transduction, but nedd . knockdown diminished transduction by % ( figure a ). because nedd . showed the strongest effect on ad transduction we determined whether bacterially expressed and purified nedd . could ubiquitylate purified protein vi in vitro. a minimal system where cytosol was replaced by recombinant nedd . was sufficient for protein vi ubiquitylation ( figure b, lane ) . the ubiquitylation pattern was similar to that obtained with cytosol anti-protein vi antibodies is shown with ubiquitylated proteins marked with grey arrows (vi-ubi) and unmodified protein vi (vi) is marked with a black arrow. c) reactions as in a) followed by detection of protein vi by western blot using ad -vi-wt (lane and ) and ad -vi-m capsids (lane and ). disassembly of capsids is indicated above each lane and protein vi reactive bands are marked as in b to the right. doi: . /journal.ppat. .g ( figure ) and required an intact ppxy and a catalytically active nedd . ( figure b, lane and ) . nedd . and nedd . both showed strong interaction with protein vi in pulldown assays. to further investigate the role of each ligase we transduced cells with lentiviral vector expressing shrnas against either nedd . or nedd . or luciferase as a control. we transduced cells in a dose-dependent manner to achieve different levels of knockdown. transduction efficiency was monitored using a gfp expressing lentiviral vector in control cells. seven days post-transduction shrna treated cells were infected with adgfp virus as described above and the transduction rate was determined by flow cytometry. we observed a dose-dependent decrease in infectivity for two different shrnas against nedd . , which was similar to what we observed when we used sirnas ( figure c ). the results for shrnas against nedd . were less clear. one shrna also reduced viral infection at very high transduction rates but to a lesser extent than shrnas against nedd . while a second shrna showed no effect on ad transduction ( figure c) . a combined treatment of cells with either sirnas or shrnas against nedd . and nedd . did not further decrease ad-transduction indicating that the effects of nedd -ligase knockdowns on ad transduction may be complex (data not shown). a hallmark of ad infection is its transient accumulation at the mtoc during entry [ ] . the mutation of the ppxy in the ad -vi-m virus seemed to alter this localization (figure ) . similarly, the ppxy motif was required for nedd . dependent ubiquitylation of protein vi. because knockdown of nedd ligases also diminished transduction with adgfp vectors we examined accumulation at the mtoc region in cells depleted with control shrna and nedd . and nedd . specific shrnas. we used the same strategy as in figure by quantifying viral particles in proximity to the mtoc, which was identified by stain for pericentrin. mtoc accumulation for nedd . and nedd . shrna treated cells was reduced when compared to cells treated with control shrna indicating that both ligases might be involved in proper targeting of viruses towards the mtoc ( figure d ). in summary, these data provide evidence that release of protein vi during entry and a possible interaction between the ppxy motif of protein vi and nedd -family ligases are determinants of ad trafficking during infection. in this study we show that the ads internal capsid protein vi harbors a ppxy-motif that is involved in virus entry and infectivity. for ads, reaching the nucleus requires a series of sequential steps: receptor-mediated endocytic uptake, partial capsid disassembly, endosomal rupture, microtubule based transport to the mtoc and nuclear trafficking. the link between these steps has been best exemplified in the case of the thermostable temperature-sensitive mutant ad ts . this mutant enters cells by receptor-mediated endocytosis, but remains in an endosomal compartment due to increased capsid stability. therefore, ad ts particles are directed to lysosomes for destruction and/or recycled back to the surface thus precluding accumulation at the nuclear pore complex [ , , ] . the role of facilitating endosomal escape during ad entry was initially assigned to the penton base [ ] . later, wiethoff and co-workers showed that most membrane lytic activity of ad viral capsids comes from the predicted n-terminal amphipathic helix of the internal capsid protein vi, and that membrane lytic activity required partial capsid disassembly to release protein vi [ ] . here we present several lines of evidence that protein vi plays an additional and previously unidentified role in nuclear targeting of the ad capsid. we show that protein vi exposure from ad capsids occurs within minutes when pre-adsorbed ad is shifted from uc to uc. this is consistent with the loss of the fiber prior to internalization and the rapid accumulation of ad in the cytosol [ , ] . we found that significant amounts of protein vi remains partially associated with the viral capsid after the initial exposure and until the viral particle accumulates at the mtoc or the nuclear rim. we show that protein vi is engaged in rapid intracellular trafficking that depends on intact microtubules and requires the nterminal amphipathic helix for microtubule association and the ppxy motif for motion. to our knowledge protein vi is the first ad capsid protein described that possesses its own microtubuledependent dynamics and future work has to address if other capsid proteins have similar properties. inactivation of the ppxy in the viral context (ad -vi-m ) resulted in a post-entry delay that reduced infectivity and prevented efficient accumulation of the entering virus particles at the mtoc. while we cannot exclude the possibility that the ppxy motif in protein vi also plays a role in adenoviral replication, assembly or egress, our single round infection assays showed that the majority of the titer reduction for the mutant was related to steps prior to the initiation of replication or the delivery and expression of a reporter gene. moreover, our data show that the efficiency with which a toxin is delivered to the cytosol during ad infection is similar for mutant and wt virus. this provides strong evidence that the ppsy motif in protein vi has a function during cell entry, but probably only after endosomal membrane lysis has occurred. current structural data place protein vi inside the assembled capsid, therefore potentially precluding it from functions during egress at least when capsid associated [ ] [ ] [ ] . our observations show that protein vi is exposed after entry and that capsid disassembly is required for its ubiquitylation, which is consistent with our hypothesis that the ppxy motif is accessible only during ad entry following partial capsid disassembly. furthermore, it is currently not clear whether late domains containing ppxy motifs present in other viral systems, which are required for viral egress, have additional functions. mutational inactivation of ppxy motifs in the vp structural protein of ebola virus and matrix protein of rabies virus both showed an attenuation of the virus and a reduction of infectivity [ , ] . interestingly, for ebola virus, the ppxy mutants showed no budding defect, but virus production was reduced, which could indicate a disruption earlier on in the life cycle than previously thought [ ] . for rabies virus, wirblich et al. describe a figure . protein vi interacts with nedd ligases via the ppxy motif. a) protein vi (vi-wt) was n-terminally fused to mrfp and co-transfected with gfp-nedd . (first row), gfp-nedd . (second row), gfp-aip /itch (third row), gfp-wwp (fourth row) and gfp-wwp (last row). confocal images of representative cells are shown and the mrfp signal for protein vi (left column), the gfp signal for the ligases (center column) and the merged signals together with dapi stain of the nucleus (right column) is indicated above each column. transfected plasmids are indicated. colocalization of nedd and protein vi results in a yellow signal. the scale bar is mm. b) diverse gfp-tagged nedd ligases were over-expressed in cells and cytosolic extracts were used for pulldown experiments using recombinant protein vi-wt or vi-m coupled to beads. % of the input material (ip) is shown in the first lane. bound material for protein vi-wt (lane ) and vi-m (lane ) was detected with respective antibodies as indicated to the right. co-eluted protein vi detected with a protein vi specific antibody is shown as loading control in the lower lane. doi: . /journal.ppat. .g budding defect of the late-domain mutant but also noted a reduction of early mrna production [ ] . earlier studies have shown that ppxy type late domains bind to ubiquitin ligases of the nedd family rather then directly to class e vsp proteins [ ] . our study showed that the ppxy motif in protein vi can interact with all nedd ligases tested but preferentially binds to nedd . and nedd . and re-targets them to membranes. owing to topological constrains, recognition of the ppxy domain should require membrane rupture because of the cytosolic localization of nedd ligases, which according to our data does not seem to be affected by the ppxy mutation in the m virus. the ppxy motif in protein vi seems to favor interaction with nedd . and nedd . although we observed interactions with aip /itch and wwp as well. it is possible that an interaction between protein vi and wwp , as we observe in transient transfections, is circumvented by exposure of the ppxy domain after the virus has entered the endosomal compartment. a role for nedd . or nedd . in ad entry is underscored by our observation that nedd . can directly ubiquitylate protein vi via the ppxy motif and its depletion (and to a lesser extent also depletion of nedd . ) reduces ad transduction. in addition this depletion also reduced mtoc accumulation of viral particles following infection, which is similar to the effect of the ppxy mutation in the m virus. however the effects were modest, indicating that additional mechanisms contribute to ad entry. further studies will be needed to identify a specific role for each ligase in ad entry and trafficking towards the mtoc and to determine whether other ligases like aip /itch and wwp are involved. how ubiquitylation of protein vi or interaction with nedd ligases directs accumulation of ads at mtocs remains unknown. ubiquitylated protein vi could be specifically recognized by cellular factors. alternatively, recruitment of nedd . and/or nedd . by protein vi could result in the ubiquitylation of other cellular factors that constitute an efficient transport means used by the virus. recent work has shown that some members of escrt-i become ubiquitylated when nedd . is overexpressed [ , ] . therefore, it is possible that nedd . (or other nedd -ligases) binding to protein vi could activate the escrt pathway via ubiquitylation. whether membrane compartments or the escrt pathway plays a direct role in ad virus transport during entry remains to be addressed. however, escrt components can be found at the endosomal compartments as well as associated with the centromeric region [ ] . it is noteworthy that endosomal escape is also required for interferon induction by ad via yet unknown mechanisms [ ] . this pathway may also be related to protein interaction between protein vi and nedd -family ligases. release and separation of protein vi from the capsid is clearly defective in the ad -vi-m mutant virus. a lack of functional ppxy may therefore block disassembly steps, preclude efficient endosomal escape following the membrane lysis event or fail in the recruitment of subsequent factors required to efficiently link the virus with retrograde transport pathways. penton base also encodes ppxy motifs [ ] . because eliminating the ppxy motif from protein vi had only modest effects on mtoc accumulation, there may be functional overlap between the ppxy in protein vi and penton base. this is further supported by the observation that ubiquitylation of penton could be abolished by depleting the cytosol with protein vi indicating that maybe the same ligases are involved ( figure s ). in conclusion, our study is the first demonstration of a ppxy motif, previously described for viral late domains, present in a non enveloped dna virus that exerts a function during entry. mastadenovirus suggests that protein vi fulfils a key function. we suggest that after endocytosis cell or serotype specific capsid disassembly cues regulate exposure, membrane-interaction and ubiquitylation of protein vi (and possibly other capsid proteins). this could facilitate the recruitment of a common cellular microtubule-dependent pathway for retrograde trafficking. this mechanism could be more important in vivo in highly polarized epithelia or neurons where long-range movement is crucial and might be less important in cell culture models [ ] . given the prevalence of viral late domains, ubiquitylation and trafficking towards the mtoc our results may have uncovered a more general mechanism by which viruses and other cargos achieve intracellular delivery and provide a rational to look for further ''early'' functions of ppxy motif containing late-domains in other viral systems. cytoplasmic extracts for pulldowns were prepared from t cells (human embryonic kidney cells). all cells except htert-rpe were grown in dmem glutamax tm (gibco) supplemented with % of fetal calf serum (fcs) (biowest). htert-rpe cells were a kind gift from m. bonhivers (university of bordeaux ) and grown in dmem/hamsf media supplemented with % fcs according to the suppliers instructions. prior to infection experiments, cells were serum starved for h to induce primary cilia growth [ ] . recombinant ad -vi-wt and ad -vi-m viruses and their gfp expressing counterparts were constructed as described in the supplemental material. amplification of viruses was done in cells and purified using double cscl -banding. virus particle to cell ratios were calculated based on the estimated copy numbers of viral genomes. copy numbers were calculated according to mittereder et al. [ ] . briefly, purified particles were diluted : in virus lysis buffer ( . % sds, mm tris/hcl ph . , mm edta) and incubated for min at uc to release the viral genomes and the od was determined. calculations were based on od = . particles/ml [ ] . lentiviral vector production for shrna encoding vectors was done by the service platform for lentiviral vector production of the indicated (from - %) and transduced with adgfp. gfp expression levels were determined h later using flow cytometry. gfp expression levels within each condition were normalized to transduction controls of cells treated with shrna expressing lentiviral vectors against luciferase (arbitrarily set to ). values are the mean (+/ standard deviation) of at least two experiments done in triplicates d) accumulation of ad at the mtoc region following nedd depletion. fluorescently labeled ad was used to infect cells following control depletions (shluc) or depletion of nedd . or nedd . using (sh . ( ) and sh . ( ) as in c). subcellular localization of viral particles was determined at min. post infections. cells were fixed and stained for the mtoc using a pericentrin antibody. particles were counted and scored for their relative proximity to the mtoc using two concentric circles with and mm diameter around the mtoc (compare also figure ). the graph shows percentage of viral particles within mm or - mm proximity to the mtoc as indicated. note that in nedd . and nedd . depleted cells the relation is inverted compared to control depleted cells showing less viral particles accumulating at the mtoc. the error bar represents cell-to-cell variation (n. , p, . ). doi: . /journal.ppat. .g institute federative de recherche of the bordeaux university. prevalidated lentiviral vectors encoding shrnas in the vector backbone plko. against nedd . and nedd . were purchased from the mission tm shrna collection from sigma. for downregulation of nedd . we used nm_ . - s c (sh . ( ), ccggccggagaattatgggtgtcaactcga-gttgacacccataattctccggttttt) against the coding sequence and nm_ . - s c (sh . ( ), ccggg-cctttctcttgcctgcatatctcgagatatgcaggca-agagaaaggcttttt) against the utr. for downregulation of nedd . we used nm_ .x- s c (sh . ( ), ccgggcgagtacctatgaatggattctcgagaatcca-ttcataggtactcgcttttt) against the coding sequence and nm_ .x- s c (sh . ( ), ccggcctgtt-tgtatgcgtttgctactcgagtagcaaacgcatacaa-acaggttttt) against the utr. control vectors for shrnas encoded for shrna against luciferase (sigma) and control vectors for transduction and estimation of the titer encoded for gfp. all sequences for protein vi were derived from ad serotype (ad ) and cloned into the gateway tm compatible entry vector pdonr . sequence verified donr plasmids were used for recombination into gateway tm compatible destination vectors for n-terminal fusion of mrfp (l -mrfp, kindly provided by e. bertrand). bacterial expression vectors for protein vi are based on pet b. site-directed mutagenesis was used to change amino acids -ppsy- to -pgaa- in protein vi. n-terminal tagged expression vectors for nedd . , and nedd . were provided by e. bertrand [ ] and tagged expression vectors for aip /itch and wwp were a kind gift of paul bieniasz (rockefeller university, new york). bacterial expression vectors for catalytically active murine gst-nedd . and the inactive gst-nedd . -dn was kindly provided by s. kumar [ ] . sirnas were purchased as duplexes from eurogentec (only the reverse strand is shown): scramble ( -cgcaauucgauguccc-gugdtdt), nedd . ( -aaacaacccagccaggcucdtda), nedd . ( -cugugacuuuguguuguggdtda), were previously described by segura-morales et al. ( ) , aip /itch sirnas ( -ucaucauucugagaagcacdtdt, [ ] , and wwp sirna ( -cuucuacgaucaucaacucdtdt) was previously described by chen et al. ( ) . the wwp sirna was a smartpool from dharmacon. depletions were performed in -well dishes using u os cells. cells were transfected after and h with pmol of each sirna duplex per well. forty-eight hours after the first transfection cells were transduced using physical particles per cell of ad -gfp virus for h without prior pre-adsorption. cells were harvested h later and analyzed by flow cytometry for gfp expression and further processed for western blot analysis to verify knock-down efficiency. acquisitions were done with facscali-burh or facscantoiih cytometer (bd biosciences) and the data were processed and analyzed by the cellquesth pro and facsdivah software (bd biosciences). purified ad particles were labeled using the alexa- microscale protein labeling kit (invitrogen) using the manufacturers protocol. infectivity of labeled virus preparations was determined by quantification of gfp-transduction. only preparations with . % activity where used. for time course experiments, u os cells were seeded at semiconfluency on coverslips. pre-binding was done with physical particles per cell in ml at uc on a shaking platform for h. at t coverslips where rinsed in cold dmem and transferred to pre-equilibrated ( uc, % co ) dmem. at indicated time points the cells where fixed and processed for if analysis. cells grown on coverslips were rinsed in pbs and fixed with % pfa in pbs and blocked/permeabilized with if-buffer ( % fcs in pbs and . % saponin). primary and secondary antibodies where applied to the coverslip in if-buffer for h each. cells were mounted in dako mounting media containing dapi and analyzed by confocal-or epifluorescence microscopy. for if involving microtubule staining cells were treated with crosslinkers prior to fixation. the following primary antibodies were used in this study: mouse anti-actubulin (kind gift from c. janke, montpellier), mouse anti e a (kind gift of t. dobner, hamburg), rabbit anti pericentrin (abcam) and rabbit anti-protein vi antibodies raised against recombinant protein vi and affinity purified (see supplemental material). secondary antibodies alexa anti mouse was from affinity research and atto anti rabbit was from sigma. confocal pictures were taken on a zeiss lsm meta confocal microscope or a leica sp confocal microscope and epifluorescence pictures were taken on a zeiss axiolmager z microscope with coolsnap hq photometrics camera both equipped with metamorph tm software. confocal stacks where taken every . mm with a pinhole setting of for all channels to achieve high local resolution. images were processed using imagej and adobe photoshop tm . counting of viral particles was performed using the semi-automated cell counting tool from imagej. colocalization analysis: stacks from confocal images where combined as z-projection using maximum intensity, converted into -bit images for each channel. colocalization between protein vi and ad was then determined using the colocalization finder plugin from imagej. live-cell imaging: u os cells were seeded in . cm glass-bottom dishes and transfected with . mg of protein vi-expressing vectors. twenty-four h later the medium was replaced by pre-warmed opti-mem (gibco). movies were acquired on a nikon te microscope with cascade b camera using metamorph tm software for data acquisition ( frames, frame/sec). in some cases, h before the acquisition, cells were incubated with either nocodazole (sigma) or cytochalasin b (sigma) diluted respectively at . mg/ml and mg/ml in opti-mem. drugs were not removed during acquisition. acquired movies were further processed using imagej to enhance protein vi particle detection by background subtraction and bleach correction. particles were tracked using the spot-tracking tool of imaris tm software to determine the length of their trajectories and the speed of their movement. particle detection size was scaled to . mm and tracks were built with a maximum displacement of . mm between consecutive frames, a maximum gap size of frames and a minimal track length of s. at least cells were analyzed for each condition that equals a minimum of analyzed tracks per condition. three microliter of purified sample virus was adsorbed to a carbon-coated film ( mesh grids). the grids with adsorbed virus were floated onto a solution of the negative stain ( % solution of uranyl acetate). the film was picked up by a copper em grid and then air-dried. specimens were examined under a hitachi h electron microscope operating at kv and images were further processed using imagej software. affinity purified rabbit anti-protein vi antibodies were used at a dilution of : . other antibodies used for the study were: rabbit polyclonal anti-nedd . and anti-nedd . antibodies that were a kind gift of o. staub (lausanne, switzerland) (dilution : ), rabbit polyclonal anti-wwp antibody (sc- , santa cruz biotechnology) (dilution : ), goat polyclonal anti-wwp antibody (sc- , santa cruz biotechnology) (dilution : ), mouse monoclonal anti-aip /itch antibody (sc- , santa cruz biotechnology) (dilution : ) and mouse monoclonal anti-gfp antibody (roche) (dilution : ). sds-page was done using % poly-acrylamide gels and transferred to nitrocellulose membranes. membranes were blocked in tbs containing % of dry-milk and . % of tween (sigma), followed by over-night detection of antigens using primary antibodies diluted in tbs containing % of dry-milk and . % of tween (sigma). primary antibodies were detected using hrp-conjugated secondary antibodies against rabbit, goat or mouse (sigma) at a dilution of : . specific signals were revealed using the enhanced chemiluminescence detection system (ecl) (perkinelmer). data are presented as mean, error bars as std. statistical analysis if not indicated otherwise was done using unpaired students t-test (*:p, . ; **:p, . ; ***:p, . ). figure s protein vi release in u os cells during ad entry. ad -vi-wt- was pre-bound to cells at uc (top row) and shifted to uc for min (second row), min (third row) and min (bottom row). protein vi was detected using affinity purified antiprotein vi antibodies (left column) and ad by detecting the alexa- fluorescent signal (middle column). a composite of both signals including the nucleus (in greyscale) is shown in the left column. the inset shows a magnification of representative virus and protein vi signals in the small white box. protein vi signals are shown in red, ad is shown in green and colocalization of protein vi and ad is shown in yellow. the scale bar is mm. the rabbit polyclonal serum against protein vi was generated against recombinant purified his-tagged protein vi. rabbit serum that reacted positive and specific against protein vi in western blots of purified viruses was used for further affinity purification for use in immunofluorescence applications. affinity purification was done using recombinant purified protein vi coupled to cnbr + -activated sepharose beads. bound antibodies were eluted with . m glycin ph , neutralized with m tris ph . and dialyzed against pbs. affinity purified antibodies were used at : dilutions in immunofluorescence. found at: doi: . /journal.ppat. .s ( . mb tif) figure s alignment of the ppxy motif in the sequence of protein vi from different human and non-human adenovirus serotypes. a partial alignment of protein vi sequences from different human adenoviral serotypes as well as non-human adenoviruses from the genus mastadenovirus is shown. the conserved ubiquitin ligase-recruiting motif is boxed. sequences were retrieved from public databases with the following accession numbers; canine (cav- , ap_ ), bovine (boad , ap_ ), huad (serotype b, abb ), huad (serotype b, ap_ ), huad (serotype e, yp_ ), huad (serotype d, ap_ ), huad (serotype c, ap_ ), huad (serotype c, ap_ ), huad (serotype a, ap ), huad (serotype f, np_ ), murine (muad , ap_ ). found at: doi: . /journal.ppat. .s ( . mb tif) figure s construction of mutant ad with altered ppxy motif in protein vi using bac technology. a) to construct a bacterial artificial chromosome (bac) carrying an infectious ad genome, we cloned an adeasy system (stratagene) based virus genome into pksb vector as described previously (warming et al. [ ] ; ruzsics et al. [ ] ). this recombinant ad lacked the e and e regions and carried an frt site in the place of its e region, which was introduced through an frt containing pshuttle (stratagene) clone. the resulting bac, was termed pad -frt and can be reconstituted to fully infectious recombinant ad viruses after transfection of e complementing cell lines such as cells. to construct protein vi-modified viruses, pad -frt was transformed into the e. coli strain sw , which encodes the l-red recombination system from the bacteriophage under a heatinducible promoter [ ] . we next amplified a kanamycin resistance cassette using primers with nt extensions homologous to protein vi coding regions. the forward primer was flanked with a homology located upstream to the ppsy motif and introduced a clai site into the protein vi orf without affecting its amino acid sequence. the homology region attached to the reverse primers carried the same clai site and overlapped with the ppsy motif. two different reverse primers were generated: one carried an unaltered ppsy motif and another that encoded the amino acids pgaa instead of ppsy and introduced an additional pst i site by the new coding sequence. the pcr products were transformed into the sw bacteria harbouring the ad -bac following heatshock to induce red-recombination. chloramphenicol and kanamycin double resistant clones were selected and bac dna was prepared from individual clones. the isolated bac dna was digested with clai and subsequently religated. this procedure eliminated the kanamycin cassette and reconstituted the protein vi orf concomitant to the recircularisation of the clai treated bacs, because there was no other clai site present in the rest of the pad -frt. if the reverse primer with an intact ppsy motif was used for amplification, the wild type protein vi amino acid sequence was reconstituted with a silent genetic tag introducing a clai site. if the reverse primer with pgaa motif was used for amplification, the protein vi coding sequence was modified at two positions i) a silent genetic tag was inserted introducing a clai site as above and ii) the ppsy motif was replaced by the pgaa motif introducing a new psti site. after the clai treatment and re-ligation the modified genomes were retransformed in e. coli dh b. kanamycin negative colonies were identified by replica plating and the resulting mutants were analysed by restriction digestions and verified by sequencing. the mutant ad genomes were released from the respective bacs by pac i digestion and transfected into cells. following the appearance of cytopathic effects the virus was amplified and purified by double cscl banding, dialyzed into pbs/ % glycerol and snap frozen. b) to verify the identity of the purified virions and analyse whether detectable reversion occurred during reconstitution and propagation of the virus stocks viral dna was extracted from purified virions and was pcr amplified with protein vi specific primers. the pcr products were digested with pst i (left) and cla i (right) to identify the recombinant viruses with or without the altered protein vi sequences. to insert a gfp expression cassette we used bacterial flp-recombination using the frt site in the e -deleted region of the ad -vi-wt and ad -vi-m bacs. we cloned the left end of the ad (nt - ) flanked by a pac i restriction site into the plasmid porir k-ie [ ] ; genbank acc. ay ) upstream of its frt site. we also replaced its zeocin resistance marker with an pgps . (neb) derived kanamycin cassette and cloned an egfp orf from pegfp-n (clontech) in its expression locus and termed this plasmid po -a -gfp. the porir kie derived plasmids can only be maintained in special e. coli strains such as pir (invitrogen) because they are dependent on the presence of r kc phage replicase [ ] . to carry out the recombination, e. coli strain dh b (invitrogen) was co-transformed with pad -frt derived bacs and pcp encoding the flp-recombinase [ ] and cultured at uc. the flp recombinations were carried out as described in bubeck et al. [ ] . briefly, the e. coli cell carrying the target bacs and the flp expression plasmid pcp were transformed with the po -a -gfp and selected for chloramphenicol and kanamycin resistance upon induction of the flp expression by a temperature shift to uc. this treatment induced a recombination between the two frt sites (one in the pad -frt derivative and one on the po -a -gfp) and induced unification of the bac and the po construct. only the recombined construct survived the double selection because po constructs are not maintained in dh b. this approach essentially replaced the old left end of the ad bac by a cmv promoter driven gfp-expression cassette containing fragment, which also possessed all the cis elements needed for virus reconstitution as described above. found at: doi: . /journal.ppat. .s ( . mb tif) figure s the ppxy-mutant ad gfp-vi-m forms smaller and fewer plaques. a) shown is a comparison of the growth of individual plaques starting from a single infected cell. e complementing cells were infected at low multiplicity of infection with ad gfp-vi-wt and the ppxy mutant virus ad gfp-vi-m for h and then washed and overlayed with agarose. virus growth was monitored by the appearance of gfppositive cells and images of representative cells/plaques were taken on days , , and . the images in the left row show the plaque formation of the wild type virus , , and days after the initial infection (top-to-bottom). at day and significant large plaques with lesions of the cell monolayer can be observed. in contrast the mutant virus to the right shows a slow expansion of gfp-positive plaques with less damage to the cell monolayer. images are superimpositions of the gfp signal and the phase contrast image of the monolayer. b) the image shows the damage in the cell monolayer caused by plaque formation on day . the arrow indicates the average size of the plaque. the scale bar is mm. found at: doi: . /journal.ppat. .s ( . mb tif) figure s penton is a target for ubiquitylation following partial disassembly of the virus. a) localization and sequences of conserved ppxy-motifs in protein vi and penton for ad (black box). note that processed protein vi is shown as it is present in the capsid during viral entry. b) schematic representation of the in vitro ubiquitylation assay. virus disassembly was induced by mild heattreatment, in vitro ubiquitylated using ubiquitin or recombinant gst-ubiquitin, recombinant e and e , an energy regenerating system and purified cytosol as source for the e -ligase and analyzed by western blot. controls lack the mild heat-treatment. c) western blot analysis of viral capsid proteins penton, fiber and protein iiia following capsid disassembly and in vitro ubiquitylation. heat-treatment is indicated above each lane. antibodies are indicated to the right of each blot. specific bands are labeled accordingly. grey arrows indicate band shifts due to ubiquitylation, black arrows indicates the unmodified protein. d) western blot analysis of in vitro ubiquitylation reactions of heat-treated viral particles. heat treatment is indicated above each lane. for individual reactions the cytosol was depleted with recombinant fiber beads (control), recombinant vi-wt beads or recombinant vi-m beads as indicated above each lane. reactions were blotted with anti-penton. the assay shows that the ubiquitylation activity can be depleted from cytosol with recombinant wt protein vi but not when the ppsy motif is mutated. the same assay also abolishes protein vi ubiquitylation showing that similar ubiquitylation activities are responsible (data not shown). e) protein vi depleted cytosol renders nedd . active for penton ubiquitylation. in vitro ubiquitylation reactions using catalytically active or inactive nedd . substituted with cytosol depleted by protein vi-wt (as indicated above each lane) and analyzed by western blot with antipenton antiserum. black arrows indicate ubiquitylated proteins, grey arrows the unmodified protein. this assay shows that additional cytosolic factors are required for full nedd . activity for penton ubiquitylation. found at: doi: . /journal.ppat. .s ( . mb tif) figure s protein vi with altered ppxy motif does not colocalize with nedd ligases. protein vi with ppsy motif mutated to pgaa was n-terminally fused to mrfp and cotransfected with gfp-nedd . (top row), gfp-nedd . (second row), gfp-aip /itch (third row), gfp-wwp (forth row) and gfp-wwp (bottom row). confocal images of representative cells are shown and the mrfp signal for vi (left column), the gfp signal for the ligases (centre column) and the merged signals together with dapi stain of the nucleus (right column) is indicated above each column. transfected plasmids are indicated left of each row. note that the cytoplasmic localization of each ligase is similar to that in cells without cotransfection of protein vi (data not shown). found at: doi: . /journal.ppat. .s ( . mb tif) video s intracellular dynamics of protein vi. u os cells were transfected with vi-wt, fused to mrfp. the movie was acquired using a nikon te microscope equipped with cascade b camera at frame per second. the movie shows rapid intracellular movement of vi-wt depicting compartments resembling vesicular, tubulo-vesicular and reticular structures. video s comparison of intracellular dynamics for vi-wt vs. vi-m vs. vi-wt (+ nocodazole). u os cells were transfected with vi-wt (left and right panel) or vi-m (middle panel) fused to mrfp. the cell to the right was treated with nocodazole ( mg/ml) prior to image acquisition. movies were acquired using a nikon te microscope equipped with cascade b camera at frame per second. the movies were labeled and assembled using imagej and converted into quicktime tm movies. all three displayed movies were taking at the same frame rate. the movies show rapid intracellular movement of vi-wt at nearly real-time (left). vi-m shows strongly reduced movement (middle). treatment of vi-wt transfected cells with nocodazole, strongly reduces the vi-wt movement resembling vi-m dynamics (right). found at: doi: . /journal.ppat. .s ( . mb mov) virus trafficking -learning from single-virus tracking a superhighway to virus infection intracellular trafficking of adenovirus: many means to many ends signalling in viral entry viral strategies for intracellular trafficking: motors and microtubules association of adenovirus with the microtubule organizing center microtubule-dependent plus-and minus end-directed motilities are competing processes for nuclear targeting of adenovirus nuclear targeting of adenovirus type requires crm -mediated nuclear export the ubiquitin-proteasome system facilitates the transfer of murine coronavirus from endosome to cytoplasm during virus entry the ubiquitin-vacuolar protein sorting system is selectively required during entry of influenza virus into host cells endosomal processing limits gene transfer to polarized airway epithelia by adeno-associated virus ubiquitination of both adeno-associated virus type and capsid proteins affects the transduction efficiency of recombinant vectors late budding domains and host proteins in enveloped virus release the emerging shape of the escrt machinery functional reconstitution of escrt-iii assembly and disassembly the nedd family of e ubiquitin ligases: functional diversity within a common modular architecture regulation of functional diversity within the nedd family by accessory and adaptor proteins efficient and specific rescue of human immunodeficiency virus type budding defects by a nedd -like ubiquitin ligase nedd l overexpression rescues the release and infectivity of human immunodeficiency virus type constructs lacking ptap and ypxl late domains ppey motif within the rabies virus (rv) matrix protein is essential for efficient virion release and rv pathogenicity adenovirus protein involved in virus internalization recruits ubiquitin-protein ligases adenovirus receptors adenovirus endocytosis hautala t ( ) rab gtpase regulates adenovirus endocytosis integrins alpha v beta and alpha v beta promote adenovirus internalization but not virus attachment stepwise dismantling of adenovirus during entry into cells the role of the adenovirus protease on virus entry into cells adenovirus protein vi mediates membrane disruption following capsid disassembly nuclear import of adenovirus dna in vitro involves the nuclear protein import pathway and hsc import of adenovirus dna involves the nuclear pore complex receptor can/nup and histone h adenovirus core protein pvii is translocated into the nucleus by multiple import receptor pathways a bbsome subunit links ciliogenesis, microtubule stability, and acetylation recombineering: a powerful new tool for mouse functional genomics transposon-assisted cloning and traceless mutagenesis of adenoviruses: development of a novel vector based on species d a proline-rich motif (pppy) in the gag polyprotein of mason-pfizer monkey virus plays a maturation-independent role in virion release the mason-pfizer monkey virus pppy and psap motifs both contribute to virus release switch from capsid protein import to adenovirus assembly by cleavage of nuclear transport signals hect ubiquitin ligases link viral and cellular ppxy motifs to the vacuolar protein-sorting pathway infectious adenovirus type endocytosis of adenovirus and adenovirus capsid proteins role of vesicles during adenovirus internalization into hela cells the first step of adenovirus type disassembly occurs at the cell surface, independently of endocytosis and escape to the cytosol visualization of alphahelices in a -angstrom resolution cryoelectron microscopy structure of adenovirus allows refinement of capsid protein assignments adenoviruses: update on structure and function a quasi-atomic model of human adenovirus type capsid ebola virus vp late domains are not essential for viral replication in cell culture human escrt and alix proteins interact with proteins of the midbody and function in cytokinesis key role of splenic myeloid dcs in the ifn-alphabeta response to adenoviruses in vivo novel partner proteins of adenovirus penton carassociated vesicular transport of an adenovirus in motor neuron axons evaluation of the concentration and bioactivity of adenovirus vectors for gene therapy tsg and alix interact with murine leukemia virus gag and cooperate with nedd ubiquitin ligases during budding regulation of neuronal voltage-gated sodium channels by the ubiquitin-protein ligases nedd and nedd - the e ubiquitin ligase itch controls the protein stability of p comprehensive mutational analysis of a herpesvirus gene in the viral genome context reveals a region essential for virus replication gene disruption in escherichia coli: tcr and kmr cassettes with the option of flp-catalyzed excision of the antibiotic-resistance determinant simple and highly efficient bac recombineering using galk selection we thank e. bertrand, p. bienasz, m. bonhivers, t. dobner, e. everitt, s. kumar, a. lieber, g. nemerow and o. staub for their generous gifts of reagents. we thank j. chroboczek and m. piechazcyk for helpful discussions during the course of this work and michael kann for critical reading of the manuscript. we thank the members of the kremer lab for discussion. we would like to thank k. rogowski and l. doelken for valuable help. we further thank the montpellier rio imaging platform mri. we like to acknowledge the excellent technical support from e. gontier at the electron microscopy platform of the bordeaux imaging center (bic) and the help of n. dugot-senant, v. guyonnet-duperat and v. pitard from the imaging, lentiviral vector production and cytometry platforms at the institute federative de recherché (ifr ) at the university of bordeaux . conceived and designed the experiments: hw csm oc zr cmw ejk. performed the experiments: hw dh bj zr. analyzed the data: hw dh bj zr cmw. contributed reagents/materials/analysis tools: hw csm ss oc zr cmw. wrote the paper: hw zr cmw ejk. key: cord- -kgpvdb authors: sa ribero, margarida; jouvenet, nolwenn; dreux, marlène; nisole, sébastien title: interplay between sars-cov- and the type i interferon response date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: kgpvdb the severe acute respiratory syndrome coronavirus- (sars-cov- ) is responsible for the current covid- pandemic. an unbalanced immune response, characterized by a weak production of type i interferons (ifn-is) and an exacerbated release of proinflammatory cytokines, contributes to the severe forms of the disease. sars-cov- is genetically related to sars-cov and middle east respiratory syndrome-related coronavirus (mers-cov), which caused outbreaks in and , respectively. although ifn treatment gave some encouraging results against sars-cov and mers-cov in animal models, its potential as a therapeutic against covid- awaits validation. here, we describe our current knowledge of the complex interplay between sars-cov- infection and the ifn system, highlighting some of the gaps that need to be filled for a better understanding of the underlying molecular mechanisms. in addition to the conserved ifn evasion strategies that are likely shared with sars-cov and mers-cov, novel counteraction mechanisms are being discovered in sars-cov- –infected cells. since the last coronavirus epidemic, we have made considerable progress in understanding the ifn-i response, including its spatiotemporal regulation and the prominent role of plasmacytoid dendritic cells (pdcs), which are the main ifn-i–producing cells. while awaiting the results of the many clinical trials that are evaluating the efficacy of ifn-i alone or in combination with antiviral molecules, we discuss the potential benefits of a well-timed ifn-i treatment and propose strategies to boost pdc-mediated ifn responses during the early stages of viral infection. the severe acute respiratory syndrome coronavirus (sars-cov- ) is a beta-coronavirus that emerged at the end of in china and rapidly spread around the world, causing a pandemic [ , ] . sars-cov- infection is responsible for covid- , a disease associated with mild symptoms in the majority of cases but that can progress to an acute respiratory distress syndrome [ , ] . so far (july th, ), the virus has infected more than million people and caused more than , deaths worldwide. sars-cov- is genetically related to other betacoronaviruses that have caused epidemics: sars-cov and mers-cov (for middle east respiratory syndrome-related coronavirus), in phosphorylation, irf and/or irf dimerize and translocate into the nucleus, where they induce the expression of ifn-i and a subset of isgs referred to as early isgs (reviewed in [ ] ). secreted ifn-i then bind to the interferon alpha and beta receptor (ifnar, composed of the ifnar and ifnar subunits), leading to the activation of the jak tyrosine kinases tyrosine kinase (tyk ) and janus kinase (jak ), which in turn phosphorylate the signal transducer and activator of transcription (stat) and stat [ , ] . phosphorylated stats heterodimerize and associate with the dna binding protein irf to form a complex known as ifn-stimulated growth factor (isgf ). the isgf complex translocates into the nucleus and binds to interferon-stimulated response elements (isres) in isg promoters, thus inducing the expression of hundreds of isg products that establish the antiviral state at the site of viral infection [ ] . the antiviral response is intensified by various signaling factors, including sensors and transcriptional regulators, which are themselves isgs induced by isgf and/ or directly by the irf /irf transcriptional activators. aside from the ifn-i response, the recognition of double-stranded viral rna elements by the protein kinase receptor (pkr) triggers a translational arrest in infected cells (reviewed in [ , , ] ). this host response is highly connected to the ifn-i response because pkr is also an isg (reviewed in [ , ] ). ifn-i response requires fine-tuning because its overactivation is deleterious to the host. notably, some isgs are involved in the regulation of cell metabolism, intracellular rna degradation, translation arrest, and cell death, for which changes can be potentially detrimental to the host. ifn-i also potentiates the recruitment and activation of various immune cells. thus, although a robust ifn-i response is required as a first line of defense against viral infections, systemic/uncontrolled or prolonged ifn-i production can lead to inflammatory diseases. for example, an exacerbated ifn-i response contributes to the development of autoimmune diseases [ ] . covid- is no exception to the rule, and it is therefore critical to understand the regulation of the ifn-i response upon infection. sars-cov- is a poor inducer of ifn-i response in vitro and in animal models as compared with other respiratory rna viruses [ , ] . ifn-i levels in the serum of infected patients are below the detection levels of commonly used assays, yet isg expression is detected [ , ] , thus suggesting that a limited ifn-i production could be sufficient to induce isgs. alternatively, ifn-i production could be restricted to specific immune cells, such as plasmacytoid dendritic cells (pdcs). despite a more efficient replication in human lung tissues, sars-cov- induced even less ifn-i than sars-cov [ ] , which is itself a weak inducer in human cells [ ] [ ] [ ] ]. an ineffective ifn-i response seems to be a hallmark of other coronavirus infections, as observed with mers-cov in ex vivo respiratory tissue cultures [ ] and with animal coronaviruses such as the porcine epidemic diarrhea virus (pedv) or the mouse hepatitis virus (mhv), which are alpha-and beta-coronaviruses, respectively [ , ] . indeed, coronaviruses have developed multiple strategies to escape and counteract innate sensing and ifn-i production. sars-cov encodes at least proteins that allow the virus to either escape or counteract the induction and antiviral action of ifn (fig and table ) [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . initial observations already suggest that the sars-cov- anti-ifn arsenal is at least as efficient as that of sars-cov [ , , ] , although detailed mechanistic studies are required to determine whether the ifn antagonists identified in other coronaviruses have equivalently competent counterparts in sars-cov- . a virus-cell protein interaction map performed with of the sars-cov- proteins expressed in human embryonic kidney (hek) t identified several innate immune signaling proteins as partners of viral proteins cells (fig ) [ ] . sars-cov- orf b, like sars-cov orf b, interacts with mavs through its association with tom , thus suggesting a conserved mechanism of ifn-i evasion [ , ] (fig ) . furthermore, sars-cov- nsp and nsp were found to interact with tbk and the tbk activator ring finger protein (rnf )/nrdp , respectively [ ] (fig ) . nsp , which is a highly conserved endoribonuclease encoded by various coronaviruses, including sars-cov [ , , ], antagonizes the induction of ifn-i by cleaving the -polyuridines of the negative-sense viral rna, as demonstrated for mhv and pedv in various cellular models [ , , ] (table and fig ) . if further validated, the interaction between sars-cov- nsp and tbk may reveal that the viral endoribonuclease antagonizes ifn induction via at least mechanisms. sars-cov orf b was reported to inhibit ifn induction and to act either on irf or possibly on mavs because it translocates to mitochondria when overexpressed in vero cells [ , ] . despite the fact it encodes a shorter protein than sars-cov, sars-cov- orf b was recently found to suppress ifn induction even more efficiently [ ] . by screening , sars-cov- sequences, the authors identified a natural variant encoding a longer orf b and displaying an even greater inhibitory activity [ ] . finally, sars-cov- nsp was also recently found to bind s ribosomal subunits (fig ) , thus inhibiting host mrna translation, including that of ifn-i on this cartoon are schematically represented the signaling pathways triggered by sars-cov rna recognition by the cytoplasmic rna sensors rig-i and mda , which leads to ifn induction (a) and subsequent ifn signaling in surrounding cells, resulting in the expression of isgs (b). sars-cov proteins that have been reported to interfere with these pathways are indicated. ifn, interferon; ifnar, interferon alpha and beta receptor; iκb, inhibitor of nuclear factor κb; ikkε, iκb kinase-ε; irf, ifn regulatory factor; isg, ifn-stimulated gene; jak, janus kinase; m, membrane; mavs, mitochondrial antiviral signaling protein; mda , melanoma differentiation-associated gene ; n, nucleocapsid; nsp, nonstructural protein; orf, open reading frame; p, phosphate; plp, papain-like protease; rig-i, retinoic acid-inducible gene ; sars-cov, severe acute respiratory syndrome coronavirus; stat, signal transducer and activator of transcription; tank, traf family member associated nf-κb activator; tbk , tank-binding kinase ; traf , tumor necrosis factor receptor-associated factor ; tyk , tyrosine kinase . https://doi.org/ . /journal.ppat. .g [ ], a feature that was previously demonstrated for other coronavirus-encoded nsp , including sars-cov [ , ] (fig and table ) . another viral strategy to inhibit ifn-i signaling is to enhance the host retrocontrol of this pathway. several isgs are themselves repressors of the ifn-i response, and their regulatory functions operate at the viral and host mrna transcription and translation steps, acting via a wide-range of mechanisms (reviewed in [ , ] ). for example, the inducible negative regulators such as the suppressor of cytokine signaling (socs and socs ) act at various levels of the jak-stat pathway or by targeting irf for degradation [ ] . in the context of sars-cov, the s protein induces the expression of socs expression in b cells [ ] . induction of socs / expression is also detected in sars-cov-infected cells, albeit to a lower extent as compared with other respiratory viruses [ ] . recent genomic screen approaches identified a set of repressors of the ifn-i response depending on the cell type and activation pathway involved [ ] [ ] [ ] . hence, one might anticipate that distinct repressors of the ifn-i response are induced depending on the cell type targeted by sars-cov- , the level of replication, and the microenvironment. for example, in the context of coronaviruses, an inefficient detection of mhv infection likely results from an inhibition of the basal levels of sensors mrna expression in several cell types [ ] . it is conceivable that this inhibition might involve negative regulators such as the ifn-inducible rnf , which targets signaling components such as rig-i, mda , and mavs for degradation [ ] . inhibition of protein synthesis is a conserved host response to prevent viral infections. the host translation is dynamically regulated by pkr, activated via recognition of viral rna (reviewed by [ ] ). activated pkr inhibits the eukaryotic initiation factor (eif α), a major regulator of the initiation phase of mrna translation, by phosphorylating its α subunit. the pkr-induced translational arrest shuts down the negative feedback on the ifn-i response, which can thus result in a prolonged and/or amplified ifn-i response [ ] . because pkr is an isg, the translational arrest is, in turn, potentiated by the ifn-i response (reviewed in [ ] ). this highlights a paradoxical situation in which translation arrest prevents viral replication but also set a threshold of viral detection to commensurate the host transcriptional antiviral response to the level of infection [ ] . whether the pkr pathway is modulated by sars-cov is unknown, yet different coronaviruses regulate pkr-eif α axis and host translation. for example, the mers-cov protein a (p a) accessory protein impedes pkr activation [ , ] . future studies are needed to further uncover the relationship between ifn-i response and host translation and their dynamics in the context of sars-cov infection. the ifn-i response varies among different cell types and within different microenvironments. studies at the single-cell level suggest that the amplitude and kinetic of the response is also heterogeneous for a given cell type. mathematic modeling revealed that ifn-i response is, at least in part, stochastic because only a fraction of cells are able to produce ifn-i upon activation by agonists of the sensors and are sensitive to the paracrine stimulation by ifn-i [ ] [ ] [ ] [ ] [ ] . the heterogeneity of the ifn-i response can be imprinted by the state of the cell at the activation time, including its global translation activity, metabolism, expression levels of signaling molecules (sensors, adaptors, and receptors) [ ] [ ] [ ] [ ] . additionally, the distinct onsets of the ifn-i induction depend on the rapidity and amplitude of viral replication. this heterogeneous responsiveness at the individual cell level consequently shapes the dynamics of the host antiviral response at the whole population level [ ] [ ] [ ] . this model of the ifn-i response dynamics yielded in the context of various rna viruses provides a framework likely at play for coronavirus infections. a delayed induction of the isg expression via virus-induced modulation of the basal activity of transcriptional activity of stat and pkr pathways leads to a peak of coronavirus replication preceding the isg response [ ] . additionally, in vivo study of the dynamic of mhv infection showed that a fast and robust ifn-i production by pdcs down-regulate the ifn-i response by other cells [ ] . this suggests that the ifn-i response at play in different cell types might drive the control of coronavirus infection and potentially contribute to the progression of the disease. as mentioned above, coronaviruses possess various mechanisms to defeat the ifn-i response within infected cells, and this inhibition ability is associated with clinical severity (reviewed in [ ] ). clinical studies showed that coronaviruses evade innate immunity during the first days of infection, which corresponds to a period of widespread inflammation and steadily increasing viral load [ , ] . elevated virus replication eventually leads to inflammation and hypercytokinemia, referred to as a "cytokine storm" [ ] [ ] [ ] [ ] (fig ) . the delayed ifn-i response indeed promotes the accumulation of pathogenic monocyte-macrophages [ , ] . this cell infiltrate results in lung immunopathology, vascular leakage, and suboptimal t cell response [ , ] . immune phenotypic profiling in peripheral blood mononuclear cells (pbmcs) of covid- patients similarly revealed that high viremia is associated with an exacerbated ifn-i response, an aggravated cytokine secretion, and inflammation, driving clinical severity [ ] . although the ifnar signaling pathway was up-regulated at an earlier disease stage, down-regulation of isgs, together with exacerbated nf-κb activation, promotes a cytokine storm and hyperinflammation, found in critically ill patients [ ] . collectively, these findings highlight the negative impact of a delayed ifn-i response on viral control and disease severity. however, the underlining mechanisms that drive the temporal control of the ifn-i response in patients are still elusive. in particular, the host and viral determinants driving the on/off switch of the ifn-i response in infected cells, noninfected cells, and/or stimulated immune cells need to be investigated. such studies will certainly benefit from longitudinal studies of immune profiling in sars-cov infected patients at the single-cell level and in combination with the clinical data. by producing , -fold more ifn-i than any other cell types, pdcs are at the heart of the antiviral ifn-i response [ , ] . they also produce other proinflammatory cytokines, which contribute to modulate the function of several immune cells, such as the mobilization of natural killer (nk) cells or the licensing of virus-specific t cell responses [ ] [ ] [ ] [ ] . because pdcs are refractory to most viral replication, their antiviral response cannot be inhibited by viral proteins [ , ] . this unopposed response likely contributes to the exceptional magnitude of pdc ifn-i production [ , [ ] [ ] [ ] . pdcs are circulating immune cells; nonetheless, their response is mostly localized at the infection site because their activation requires physical contact with infected cells [ , , ] . the contact site between pdcs and infected cells, which we named the interferogenic synapse, is a specialized platform for pamp transfer from infected cell to the toll-like receptor (tlr ) sensor in pdc, leading to an antiviral response [ ] . previous studies on sars-and mers-cov demonstrated that the rapid production of ifn-i by pdcs is essential for the control of potentially lethal coronavirus infections in mouse models [ , ] . pdcs migrate into the lungs at the early phase of infection (i.e., pdc number peaks at day ), temporally coinciding with the peak of ifnα production [ , ] . the pdc number was found to be reduced in blood of covid- patients as compared with control patients [ ] , potentially resulting from a prior response followed by a vanished number of circulating pdcs and/or their mobilization to the infected site. future studies are needed to address how pdc responsiveness evolves in the course of sars-cov- infection and how pdcs respond to contact with coronavirus-infected cells. despite the abovementioned viral inhibitory mechanisms of ifn-i response (table ) , exogenous ifn-i in cell cultures efficiently inhibit sars-cov, sars-cov- , and mers-cov spread [ , , , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . consistently, ifn-i was shown to have a protective effect against sars-cov and mers-cov, alone or in combination with other antivirals, in various animal models including mice, marmosets, and macaques [ , , ] . ifn-i and iii interfere with virus infection by inducing the expression of several hundred isgs [ ] . numerous welldescribed isgs exhibit direct antiviral activities by targeting specific stages of the viral life cycle, including entry into host cells, replication, protein translation, and assembly of new virus particles. as mentioned above, many signaling regulators are themselves isgs, thus leading to amplification of the antiviral ifn-i pathway. as a first step towards identifying isgs able to restrict sars-cov- replication, transcriptomic responses to infection have been analyzed in different cellular models, including primary cells, organoids, and clinical samples [ , [ ] [ ] [ ] , as summarized in table . these studies demonstrate that, despite triggering very little to no ifn expression (table ) , sars-cov- replication induces moderate levels of a limited number of isgs. a small subset of infected cells may be refractory to the antagonistic mechanisms of sars-cov- , producing minute but sufficient amounts of ifns to trigger isg induction in larger population of cells. alternatively, isgs may be up-regulated in noninfected cells, which were analyzed together with infected ones. indeed, interpretation of genome-wide investigations of virus-pathogen interactions are often obscured by analyses of mixed populations of infected and uninfected cells [ ] . of note, by contrast to low-multiplicity of infection (moi) infection of a cells expressing angiotensin i converting enzyme (ace ), normal human bronchial epithelial (nhbe) cells, and patient samples, high-moi infections of a -ace and calu- cells led to the high induction of ifns and isgs, including isgs with broad antiviral activities [ ] ( table ). this discrepancy of ifn production/signaling between the levels of viral replication and/or proportion of infected cells might reflect that the counteraction measures employed by sars-cov- are less potent at high moi. alternatively, as suggested by blanco-melo and colleagues, high-moi infections in cell culture may generate more pamps, such as defective noninfectious viral particles, than low-moi infections [ ] . despite being expressed at moderate levels in vitro and in vivo, several up-regulated isgs identified by these transcriptomic studies ( table ) exhibit well-characterized broad-spectrum antiviral activities and could thus have additive restrictive effects on sars-cov- replication. for instance, the members of the interferon-induced protein with tetratricopeptide repeats (ifitm) family, known to inhibit entry of numerous enveloped rna viruses [ ] , similarly restrict entry of sars-cov, mers-cov, and the globally circulating human coronaviruses e and nl in t and a cell lines [ , ] . oas and mycovirus resistance protein (mx)a could also contribute to the ifn-i-mediated inhibitory effect on sars-cov- because a clinical study revealed that single nucleic polymorphisms in the oas -utr and mxa promoter region appear associated with host susceptibility to sars-cov in the chinese han population [ ] . moreover, the fact that mers-cov nonstructural protein b (ns b) is a - - oligoadenylate synthetase (oas)-rnase l antagonist [ ] suggests that the oas pathway contributes to the antiviral effects of ifns on coronavirus replication. isgs positively potentiating ifn signaling, such as ifih /mda , tank, irf , and stat , were also increased in the bronchoalveolar lavage fluid (balf) of covid- patients as compared with healthy controls [ ] and could potentially contribute to the amplification of ifn-i response against sars-cov- replication. zinc finger antiviral protein (zap), which is encoded by an isg, contributes to the anti-sars-cov- effect of ifns in human lung calu- cells [ ] . zap is known for restricting the replication of numerous viruses such as retroviruses and filoviruses [ ] . the protein recruits the cellular mrna degradation machinery to viral rna via -c-phosphate-g- (cpg) dinucleotide recognition [ ] . to further determine which individual isg or combination of isgs mainly restricts sars--cov- replication in vitro, several previously established approaches could be used, such as, for example, screening for single or combined isg activity using a lentiviral vector-based library, as successfully performed by schoggins and colleagues for other viral infections [ ] [ ] [ ] [ ] . indeed, this library of around human isgs was recently screened in human hepatoma cells for antiviral activity against hcov- e [ ] . the screen identified ifn-inducible lymphocyte antigen complex, locus e (ly e) as a potent inhibitor of the replication of multiple coronaviruses, including sars-cov, sars-cov- , and mers-cov, by blocking fusion of viral and cellular membranes [ ] . mice studies revealed that ly e directly protects primary b cells and dendritic cells from murine coronavirus infection [ ] . pursuing the identification and characterization of ifn effectors with potent anti-sars-cov- activities will reveal weakness points in the life cycle of sars-cov- and may lead to the design of drugs that activate antiviral isgs or either mimic or amplify their action. recent advances in systematic screening strategies have revealed the existence of a small subset of isgs exhibiting proviral activities [ , ] . these proviral isgs act either by exhibiting direct proviral activities such as facilitating viral entry [ ] or via their abilities to negatively regulate ifn signaling and facilitate the return to cellular homeostasis. the receptor tyrosine kinase axl is a well-characterized example of an isg that is used by enveloped virus for cellular internalization [ ] [ ] [ ] . alternatively, isgs that possess antiviral activities against a viral family can be hijacked by unrelated viruses to favor infection. this is the case for ifitm and ifitm , which potently block entry of a broad range of enveloped viruses [ ] while promoting entry step of human coronavirus oc (hcov-oc ) in human cells [ ] . sars-cov- uses ace and transmembrane serine protease (tmprss ) to enter cells [ ] . viral tropism is thus largely dictated by ace and tmprss coexpression. analysis of human, nonhuman primate, and mouse single-cell rna-sequencing (scrna-seq) data sets generated from healthy or diseased individuals revealed that expression of ace is primarily restricted to type ii pneumocytes in the lung, absorptive enterocytes within the gut, and goblet secretory cells of the nasal mucosa [ ] . interestingly, this meta-analysis identified an association between ace expression and canonical isgs or components of the ifn-signaling pathway in different tissues. independent analyzes of publicly available data sets concluded that ace expression pattern is similar to isgs [ ] . in vitro validations were performed by treating primary human upper airway cells with numerous inflammatory cytokines. ifnα , and to some extent ifny, led to greater and more significant ace up-regulation compared with all other tested cytokines [ ] . substantial up-regulation of ace was also observed in primary skin and primary bronchial cells treated with either ifn-i or ifn-ii. moreover, ace expression was also up-regulated upon ex vivo influenza a infection in human lung explants isolated following surgical resection [ ] . because the majority of cells robustly up-regulating ace were epithelial, this observation potentially explains why previous analyses to define canonical isgs within immune populations did not identify ace as an induced gene [ ] . finally, stat , stat , irf , and irf binding sites were identified within − , to + bp of the transcription start site of ace [ ] . despite need for direct evidence that ifns up-regulate ace in target cells in vivo, altogether these studies suggest that ace could be an isg that enhances sars-cov- internalization in human epithelial cells [ , ] . elucidating tissue and cell type specificity of isgs, as well as their mechanisms of action, is essential for understanding the potential dual role of ifns during human sars-cov- infection. it may also guide the use of ifns in clinical trials. although ifn-i treatment gave some encouraging results against sars-cov and mers-cov in vitro and in animal models, including mice, marmosets, and macaques [ , , , , ] , additional knowledge to optimize its therapeutic efficiency in humans is required [ ] [ ] [ ] [ ] [ ] . previous information yielded from these animal studies provided guidance for treating the current pandemic virus. first, it became clear from these former studies that ifnβ is a more potent inhibitor than ifnα as shown both in vitro and in patients [ , ] . second, the timing of ifn-i treatment seems determinant for infection outcomes. indeed, as shown in mice and in macaques, ifn-i is protective when administered prior to sars-cov or mers-cov infection or early in the course of infection, whereas late administration could be either ineffective or detrimental [ , ] . in humans as well, ifn-i-based therapies were not beneficial to critically ill patients with multiple comorbidities and who were diagnosed late with mers-cov, thus pointing out that ifn-i has to be administered early after infection [ , ] . the first clinical trials using ifn-i alone or in combination with other antivirals are currently carried out in covid- patients in several countries. for instance, the multicenter, adaptive, randomized, open clinical trial discovery evaluates, among other treatment, the efficacy of ifnβ as a treatment for covid- in hospitalized adults in europe. a recent openlabel, randomized, phase trial performed in adults with covid- in hong kong showed that the triple combination of ifnβ- b, lopinavir-ritonavir, and ribavirin was safe and superior to lopinavir-ritonavir alone in alleviating symptoms and shortening the duration of viral shedding and hospital stay in patients with mild to moderate covid- [ ] . it has to be noted that the patients were treated in the early stages of the disease because the median number of days from symptom onset to start of study treatment was days, further reinforcing the fact that the timing of ifn-i treatment is key [ ] . other therapeutic approaches are under investigation to avoid the adverse effects of ifn-i therapy and/or its potential inefficacity when administrated too late postinfection. one strategy is to use aerosol formulations of recombinant ifn to deliver the cytokine directly inside the lung [ , ] . this approach has several benefits because it is a noninvasive route of administration, and the local concentration reached in the tissue can be higher than through systemic injection and is thus expected to minimize the adverse effects of ifn. nebulized ifnα- b was used on covid- patients in wuhan, alone or in combination with arbidol [ ] . the study, performed on adults, showed a significant reduction of the duration of detectable virus in the upper respiratory tract in ifnα- b-treated patients, with or without arbidol [ ] . another study currently ongoing in beijing aims at evaluating the efficacy and safety of recombinant human ifnα spray in preventing sars-cov- infection in highly exposed medical staffs (chictr ). type iii ifns (ifnλs or ifn-iii) are gaining an increased interest in antiviral therapies [ ] [ ] [ ] . like ifn-i, they activate the jak-stat signaling pathway. they do so via a receptor that is largely restricted to cells of epithelial origin, including respiratory epithelial cells (reviewed in [ ] ). ifn-iiis are induced upon viral infections, and they are growing evidence that they provide important first-line defense against viral infections of the respiratory and gastrointestinal tracts [ ] [ ] [ ] . in mice, ifn-iii was shown to protect epithelial cells of the respiratory and tract from infections with several respiratory viruses, including mers-cov [ ] . a study investigating sars-cov- infection of intestinal epithelial cells, using both colon-derived cell lines and primary colon organoids, showed that ifn-iii response was more efficient than ifn-i at controlling viral replication [ ] . however, ifn-iiis produced by dendritic cells in the lung were recently shown to cause barrier damage and to compromise host tissue tolerance and predispose to lethal bacterial superinfections [ ] . therefore, although the antiviral properties are promising, the benefit of ifn-iii to treat covid- patients awaits careful evaluation. the first clinical trials using ifn-iii are ongoing, including one launched at the massachusetts general hospital to evaluate the safety and efficacy of pegylated ifnλ on a small number of covid- patients (nct ). besides the use of recombinant ifn as a therapeutic treatment, one interesting alternative strategy would be to boost the natural innate immune defenses of covd- patients at early stages of the disease. because pdcs are seemingly crucial to control coronavirus infections [ , ] , a possibility would be to either amplify or prolong their activation to make them produce more ifn-i and ifn-iii. a number of negative feedback loops prevent an exacerbated activation of pdcs, which can be deleterious for the organism in the long term. thus, transitorily inhibiting these negative retrocontrols may increase the antiviral activity of pdcs. for instance, the bone marrow stromal cell antigen (bst ) is an isg that activates the immunoglobulin-like transcript (ilt ) inhibitory receptor expressed by pdcs to interrupt the ifn-i response [ ] . the blockade of this interaction using either antibodies or inhibitory molecules should thus increase the duration of pdc activation. one could also envisage to take advantage of viral proteins that counteract the antiviral activity of bst , such as hiv- viral protein u (vpu) [ ] . other pdc inhibitory molecules include natural monamines such as histamine, dopamine, or serotonin, which bind to the c-x-c motif chemokine receptor (cxcr ) at the surface of pdcs [ ] . because the cxcr antagonist amd (also known as plerixafor) blocks the binding of monoamines to pdcs, it can prevent the amine-dependent inhibition of pdc activation [ ] . amd is already used in clinics as an immunostimulatory molecule able to mobilize hematopoietic stem cells in cancer patients [ ] . finally, we recently reported that the peptidyl-prolyl isomerase peptidyl-prolyl cis-trans isomerase nimainteracting (pin ) switches off the ifn-i expression by pdcs by inducing irf degradation [ ] . a number of pin inhibitors have been developed and could be tested for their potential activity on human pdcs [ ] and could represent another possible therapeutic strategy to boost pdc-mediated ifn-i production. sars-cov- emerged in the human population around months ago, yet it seems well adapted to avoid and inhibit the ifn-i response in its new host. such efficient strategies allow the virus to replicate and disseminate in infected individuals without encountering the initial host defense. this modest ifn response could explain why viremia peaks at early stages of the disease, at the time of symptoms appearance, and not around to days following symptoms, like during sars-cov and mers-cov infections. ifnβ treatment would be expected to improve the antiviral response of patients at the early stage of covid- and, if possible, at plos pathogens the site of infection. indeed, ifnβ appeared to be pivotal to improve patient states in a combined therapy regiment of ifnβ, lopinavir-ritonavir, and ribavirin [ ] . nonetheless, ifnresistant viral mutants may arise and be able to control ifn even more efficiently than parental viruses. the exacerbated production of proinflammatory cytokines observed at later stage of covid- might challenge the efficiency of an ifnβ treatment administrated after appearance of symptoms. there is indeed an increasing appreciation of the detrimental effects of inappropriate, excessive, or mistimed ifn-i responses in viral infections [ ] . the underlying mechanisms by which ifn-i promote disease severity likely include immunopathology due to excessive inflammation and direct tissue damage. at the late stages of covid- , immunomodulatory drugs that diminish inflammation may benefit patients. the therapeutic benefits of such treatments have been demonstrated in the context of influenza infection in clinical trials [ ] and in mice models [ ] . discovery of host markers associated with disease progression will be instrumental to defining the appropriate treatment and time of administration. luckily, most covid- patients develop no or mild symptoms. in these patients, the virus ends up being cleared by the immune system, possibly through a partial protection conferred by cross-reactive cd + t cells that have been found in between % and % of unexposed individuals [ ] . it is therefore probable that a viral replication that is under control thanks to an efficient adaptive immunity prevents a systemic viral spread and the subsequent cytokine storm. prior and coinfections along with age, gender, immunological state, and comorbidities also likely play a key role in the ability of the patients to efficiently respond to sars-cov- infection. the current studies that aim to better understand the mechanisms that render some patients particularly sensitive to sars-cov- infections raise hope for the possibility of treating patients with drugs that either enhance the ifn response at the early stage of the disease or dampen it at later stages. a novel coronavirus outbreak of global health concern a novel coronavirus from patients with pneumonia in china a new coronavirus associated with human respiratory disease in china genomic characterization of the novel human-pathogenic coronavirus isolated from a patient with atypical pneumonia after visiting wuhan a sars-cov- protein interaction map reveals targets for drug repurposing stimulation of innate immunity by host and viral rnas interferon-stimulated genes: what do they all do? nuclear rnf inhibits interferon function by promoting k -linked stat disassociation from dna murine coronavirus cell type dependent interaction with the type i interferon response negative regulation of the rig-i signaling by the ubiquitin ligase rnf pkr: a kinase to remember protein synthesis inhibition and gadd control ifn-beta heterogeneous expression in response to dsrna integration of pkr-dependent translation inhibition with innate immunity is required for a coordinated anti-viral response middle east respiratory coronavirus accessory protein a inhibits pkr-mediated antiviral stress responses inhibition of stress granule formation by middle east respiratory syndrome coronavirus a accessory protein facilitates viral translation, leading to efficient virus replication single-cell analysis reveals that stochasticity and paracrine signaling control interferon-alpha production by plasmacytoid dendritic cells differential induction of interferon stimulated genes between type i and type iii interferons is independent of interferon receptor abundance single-cell analysis shows that paracrine signaling by first responder cells shapes the interferon-beta response to viral infection antiviral interferon response at single-cell resolution multi-layered stochasticity and paracrine signal propagation shape the type-i interferon response pathogenic influenza viruses and coronaviruses utilize similar and contrasting approaches to control interferon-stimulated gene responses a systems immunology approach to plasmacytoid dendritic cell function in cytopathic virus infections sars and mers: recent insights into emerging coronaviruses clinical progression and viral load in a community outbreak of coronavirus-associated sars pneumonia: a prospective study haematological manifestations in patients with severe acute respiratory syndrome: retrospective analysis dysregulated type i interferon and inflammatory monocyte-macrophage responses cause lethal pneumonia in sars-cov-infected mice pathogenesis of severe acute respiratory syndrome lung pathology of fatal severe acute respiratory syndrome how the sars coronavirus causes disease: host or organism? ifn-i response timing relative to virus replication determines mers coronavirus infection outcomes plasmacytoid dendritic cells: development, regulation, and function cell-cell sensing of viral infection by plasmacytoid dendritic cells plasmacytoid dendritic cells control t-cell response to chronic viral infection plasmacytoid dendritic cell ablation impacts early interferon responses and antiviral nk and cd (+) t cell accrual sensing of immature particles produced by dengue virus infected cells induces an antiviral response by plasmacytoid dendritic cells pacsin regulates the tlr / -mediated type i interferon response in plasmacytoid dendritic cells ap- , and hermansky-pudlak syndrome proteins are required for toll-like receptor signaling in plasmacytoid dendritic cells bifurcation of toll-like receptor signaling by adaptor protein . science plasmacytoid dendritic cells control dengue and chikungunya virus infections via irf -regulated interferon responses plasmacytoid dendritic cells and infected cells form an interferogenic synapse required for antiviral responses control of coronavirus infection through plasmacytoid dendritic-cell-derived type i interferon cellular immune responses to severe acute respiratory syndrome coronavirus (sars-cov) infection in senescent balb/c mice: cd + t cells are important in control of sars-cov infection treatment of sars with human interferons interferon-beta a and sars coronavirus replication severe acute respiratory syndromerelated coronavirus is inhibited by interferon-alpha inhibition of novel beta coronavirus replication by a combination of interferon-alpha b and ribavirin mers-coronavirus replication induces severe in vitro cytopathology and is strongly inhibited by cyclosporin a or interferon-alpha treatment sars-cov- is sensitive to type i interferon pretreatment antiviral activities of type i interferons to sars-cov- infection an infectious cdna clone of sars-cov- treatment with lopinavir/ritonavir or interferon-beta b improves outcome of mers-cov infection in a nonhuman primate model of common marmoset comparative therapeutic efficacy of remdesivir and combination lopinavir, ritonavir, and interferon beta against mers-cov. nat commun transcriptomic characteristics of bronchoalveolar lavage fluid and peripheral blood mononuclear cells in covid- patients heightened innate immune responses in the respiratory tract of covid- patients sars-cov- productively infects human gut enterocytes deconvolution of pro-and antiviral genomic responses in zika virus-infected and bystander macrophages the defective component of viral populations ifitm-family proteins: the cell's first line of antiviral defense ifitm proteins inhibit entry driven by the mers-coronavirus spike protein: evidence for cholesterol-independent mechanisms distinct patterns of ifitm-mediated restriction of filoviruses, sars coronavirus, and influenza a virus association of sars susceptibility with single nucleic acid polymorphisms of oas and mxa genes: a case-control study antagonism of dsrna-induced innate immune pathways by ns a and ns b accessory proteins during mers coronavirus infection the zinc finger antiviral protein restricts sars-cov- . biorxiv novel host restriction factors implicated in hiv- replication a diverse range of gene products are effectors of the type i interferon antiviral response inhibition of hiv- particle assembly by ', '-cyclic-nucleotide '-phosphodiesterase pan-viral specificity of ifn-induced genes reveals new roles for cgas in innate immunity ly e mediates an evolutionarily conserved enhancement of virus infection by targeting a late entry step ly e impairs coronavirus fusion and confers immune control of viral disease enveloped viruses disable innate immune responses in dendritic cells by direct activation of tam receptors zika virus induces massive cytoplasmic vacuolization and paraptosis-like death in infected cells the tim and tam families of phosphatidylserine receptors mediate dengue virus entry interferon induction of ifitm proteins promotes infection by human coronavirus oc sars-cov- cell entry depends on ace and tmprss and is blocked by a clinically proven protease inhibitor sars-cov- receptor ace is an interferon-stimulated gene in human airway epithelial cells and is detected in specific cell subsets across tissues increasing host cellular receptor-angiotensin-converting enzyme (ace ) expression by coronavirus may facilitate -ncov infection ribavirin and interferon-beta synergistically inhibit sars-associated coronavirus replication in animal and human cell lines increased sensitivity of sarscoronavirus to a combination of human type i and type ii interferons ribavirin and interferon therapy for critically ill patients with middle east respiratory syndrome: a multicenter observational study ribavirin and interferon alfa- a for severe middle east respiratory syndrome coronavirus infection: a retrospective cohort study sars: systematic review of treatment effects interferon alfacon- plus corticosteroids in severe acute respiratory syndrome: a preliminary study ifn-alpha a or ifn-beta a in combination with ribavirin to treat middle east respiratory syndrome coronavirus pneumonia: a retrospective study pegylated interferon-alpha protects type pneumocytes against sars coronavirus infection in macaques ribavirin and interferon therapy in patients infected with the middle east respiratory syndrome coronavirus: an observational study triple combination of interferon beta- b, lopinavir-ritonavir, and ribavirin in the treatment of patients admitted to hospital with covid- : an open-label, randomised, phase trial fundamentals of aerosol therapy in critical care the expanding role of aerosols in systemic drug delivery, gene therapy and vaccination: an update interferon-α b treatment for covid- covid- : lambda interferon against viral load and hyperinflammation weak induction of interferon expression by sars-cov- supports clinical trials of interferon lambda to treat early covid- covid- and emerging viral infections: the case for interferon lambda type iii interferons: balancing tissue tolerance and resistance to pathogen invasion interferon-lambda mediates non-redundant front-line antiviral protection against influenza virus infection without compromising host fitness interferon-lambdas: front-line guardians of immunity and homeostasis in the respiratory tract lambda interferon renders epithelial cells of the respiratory and gastrointestinal tracts resistant to viral infections critical role of type iii interferon in controlling sars-cov- infection, replication and spread in primary human intestinal epithelial cells type iii interferons disrupt the lung epithelial barrier upon viral recognition. science. : eabc regulation of tlr / responses in plasmacytoid dendritic cells by bst and ilt receptor interaction epstein-barr virusencoded ebna modulates the ap- transcription factor pathway in nasopharyngeal carcinoma cells and enhances angiogenesis in vitro natural amines inhibit activation of human plasmacytoid dendritic cells through cxcr engagement mozobil(r) (plerixafor, amd ), years after its approval by the us food and drug administration trim is required for virusinduced ifn response in human plasmacytoid dendritic cells development of pin inhibitors and their potential as therapeutic agents disease-promoting effects of type i interferons in viral, bacterial, and coinfections a critical role for the sphingosine analog aal-r in dampening the cytokine response during influenza virus infection endothelial cells are central orchestrators of cytokine amplification during influenza virus infection targets of t cell responses to sars-cov- coronavirus in humans with covid- disease and unexposed individuals we thank nathalie j. arhel for helpful discussion. key: cord- -kfkp zpn authors: duru, adil doganay; sun, renhua; allerbring, eva b.; chadderton, jesseka; kadri, nadir; han, xiao; peqini, kaliroi; uchtenhagen, hannes; madhurantakam, chaithanya; pellegrino, sara; sandalova, tatyana; nygren, per-Åke; turner, stephen j.; achour, adnane title: tuning antiviral cd t-cell response via proline-altered peptide ligand vaccination date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: kfkp zpn viral escape from cd (+) cytotoxic t lymphocyte responses correlates with disease progression and represents a significant challenge for vaccination. here, we demonstrate that cd (+) t cell recognition of the naturally occurring mhc-i-restricted lcmv-associated immune escape variant y f is restored following vaccination with a proline-altered peptide ligand (apl). the apl increases mhc/peptide (pmhc) complex stability, rigidifies the peptide and facilitates t cell receptor (tcr) recognition through reduced entropy costs. structural analyses of pmhc complexes before and after tcr binding, combined with biophysical analyses, revealed that although the tcr binds similarly to all complexes, the p p modification alters the conformations of a very limited amount of specific mhc and peptide residues, facilitating efficient tcr recognition. this approach can be easily introduced in peptides restricted to other mhc alleles, and can be combined with currently available and future vaccination protocols in order to prevent viral immune escape. viral escape mutagenesis correlates often with disease progression and represents a major hurdle for vaccination-based therapies. here, we have designed and developed a novel generation of altered epitopes that re-establish and enhance significantly cd + t cell recognition of a naturally occurring viral immune escape variant. biophysical and structural analyses provide a clear understanding of the molecular mechanisms underlying this reestablished recognition. we believe that this approach can be implemented to currently available or novel vaccination approaches to efficiently restore t cell recognition of virus escape variants to control disease progression. recognition of major histocompatibility complex class i (mhc-i)-restricted viral peptides is a prerequisite for cd + t-cell activation, control and/or clearance of viral infections. usually, cytotoxic t-lymphocyte (ctl) responses are directed towards a limited number of immunodominant viral peptides [ ] and selection pressure imposed by adaptive immune responses leads often to the emergence of viral populations with a limited number of recurring escape mutations [ ] [ ] [ ] . epitope mutations can impair ctl responses [ ] by e.g. altering antigen processing [ , ] , reducing the overall stability of peptide/mhc complexes (pmhc) [ , ] and/or disrupting t-cell receptor (tcr) recognition [ , ] . ctl escape variants correlate with disease progression [ , ] and represent a major hurdle for disease control as well as for the design of t-cell based vaccines [ ] . to our knowledge, previous use of wild-type and escape epitopes in vaccination experiments has not provided efficient ctl responses against mhc-restricted viral escape variants [ , ] . therefore, the design of altered peptide ligands (apls) that could promote such responses would represent a crucial step towards the development of efficient vaccines [ ] . although our understanding of the interactions between tcrs and pmhc has increased dramatically, the impact of individual peptide modifications on tcr recognition remains difficult to predict. even subtle peptide alterations can significantly impact on pmhc stability, and impair or abolish t cell recognition. a conventional and sometimes successful approach to design apls with enhanced pmhc stability and immunogenicity has been to optimize interactions between peptide anchor residues and mhc binding pockets [ ] [ ] [ ] . however, escape variants that target tcr recognition often exhibit optimal mhc anchor residues, reducing possibilities for such modifications. optimally, the introduced modifications should also not alter the conformation of apls compared to the original epitopes, in order to elicit efficient cross-reactive ctl responses towards the wild-type epitope [ , , ] . therefore, the design of a novel generation of apls that could promote such responses would represent a crucial step towards the development of efficient anti-viral t-cell based vaccines [ ] . we have previously demonstrated that the immunogenicity of the cancer-associated h- d b -restricted antigen gp [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] or the t cell epitope associated with impaired peptide processing (teipp) neo-epitope thr [ ] [ ] [ ] was dramatically improved following substitution of peptide position to a proline (p p). comparative structural analyses revealed that the conformation of the apls was similar to wild-type epitopes, and that the stabilizing effect of p p is accounted for by van der waals and ch-π interactions with the h- d b residue y , conserved among most known mouse and human mhc-i alleles, resulting in rigidification of the pmhc complex [ ] . importantly, vaccination with p p-modified apls elicited high frequencies of ctls from the endogenous repertoire that efficiently targeted h- d b /gp [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] and h- d b /trh complexes on melanoma cells [ , ] . in the present study, we addressed if we could restore endogenous t cell recognition of a naturally occurring viral escape variant following vaccination with a p p-modified apl. it is well established that infection of c /bl mice with lymphocytic choriomeningitis virus (lcmv) induces robust ctl responses towards the immunodominant h- d b -restricted epitope gp (kavynfatm) [ ] . upon ctl pressure, a limited number of mutations in gp emerge, with consistent patterns, allowing for viral cd + t-cell escape [ , , , ] . the main naturally occurring mutation that allows lcmv to efficiently escape immune recognition, is the y f substitution (kavfnfatm) which abrogates endogenous cd + t cell recognition as well as recognition by the h- d b /gp -specific tcr p . here, we demonstrate that peptide vaccination with v p_y f (kapfnfatm) restores p recognition of y f in lcmvinfected mice. furthermore, vaccination with influenza constructs that encode for v p_y f provokes significant endogenous cd + t cell cross-recognition of y f. comparison of crystal structures of an ensemble of pmhc complexes before and after binding to the tcr p revealed that i) p binds nearly identically to all complexes, ii) the conformations of peptide residues p k and p f as well as h- d b residues r , e and h are affected by the p p modification, predisposing pmhc complexes for enhanced tcr recognition. the p p modification also decreases the entropic penalty for tcr recognition. in conclusion, our results demonstrate the possibility to vaccinate with modified peptides and/or proteins for enhanced t cell recognition, and may form an alternative basis for novel strategies to target viral escape mutants. the p p modification enhances pmhc stability without altering structural conformation, restoring p tcr recognition circular dichroism (cd) measurements revealed a consistent increase in pmhc complex thermal stability for the p p-substituted peptides v p (kapynfatm) and v p_y f (kapfn-fatm) compared to the wildtype gp (kavynfatm) and escape variant y f (kavfnf atm) epitopes, respectively ( fig a, table ). importantly, h- d b /gp and h- d b /y f display equivalent thermal stability ( fig a) . furthermore, surface plasmon resonance (spr) analyses revealed significantly higher binding affinity of soluble p tcr to h- d b /v p compared to h- d b /gp and significantly higher binding affinity of soluble p tcr to h- d b / v p_y f compared to h- d b /y f ( fig b, table ). in contrast to an undetectable affinity to h- d b /y f, p bound to h- d b /v p_y f. interactions between soluble p and h- d b / gp and h- d b /v p were also characterized using isothermal titration calorimetry (itc), revealing that binding of p to h- d b /v p was mainly enthalpy-driven with almost null contribution of entropy, whereas binding to h- d b /gp was entropically unfavorable (s fig, table ). in conclusion, the p p modification increases pmhc stability, resulting in recognition of v p_y f by p and enhances tcr affinity by decreasing the entropic cost for binding. next, p tcr down-regulation was assessed upon exposure to gp , v p, y f or v p_y f-loaded h- d b+ rma cells (fig c) . while h- d b /gp induced significant tcr down-regulation, minimal tcr downregulation was detected with y f, even at high peptide concentrations. exposure of p t cells to v p equaled or increased tcr internalization compared to gp . importantly, exposure to v p_y f increased p tcr down-regulation compared to y f (fig c) . the crystal structures of h- d b /v p and h- d b /v p_y f were determined to . and . Å resolution (s table) , and compared with h- d b /gp [ ] and h- d b /y f [ ] (fig d, s fig) . the overall structures of all pmhcs are nearly identical, and the amount of hydrogen bond and van der waals interactions formed between h- d b and each p p-apl was equivalent to each wild-type epitope counterpart. the backbone of the p p-apl corresponds exactly to the wild-type peptides, indicating that the p p modification does not alter the conformation of apls compared to wild-type peptides ( fig d) . the root mean square deviation values for main chain atoms are . - . Å and . - . Å for the backbone of the h- d b heavy chain and the peptides. the only significant conformational differences between wildtype and p p-apls were side chain movements of peptide residues p k and p f towards the nterminal and middle section of the peptide-binding cleft of h- d b (fig d, s fig) . first, we assessed the functional effects of all peptides on p t-cell activation by comparing intracellular tnf and ifnγ production, t cell degranulation (cd a) as well as target cell lysis. cd + t cells, isolated from spleens of naïve or gp -immunized p transgenic mice (p -tg), were co-cultured with rma cells pulsed with each peptide. peptides gp , v p and v p_y f induced significant production of tnf and ifnγ, as well as cd a expression, while y f failed to induce any p t cell response ( melting temperatures (t m ) corresponding to % protein denaturation are indicated. b. the soluble tcr p binds to the apl v p_y f. in contrast to y f, v p_y f is bound by p . binding affinity of the soluble tcr p to each pmhc was measured using spr. k d values are indicated. c. the p p modification increases tcr internalization. tcr downregulation was measured following exposure of p t cells to h- d b in complex with each peptide at indicated concentrations on rma cells. cd + cd + cd and vα + cells were gated to quantify tcr internalization and p values calculated by using two-way anova with turkey's multiple comparison test. ���� represents p< . ; ��� . and �� . . the h- d b -restricted influenza-derived peptide asnenmetm (asn) was used as negative control. d. the p p modification does not alter the conformation of the backbone of apls compared to native counterparts. superposition of the crystal structures of h- d b /v p and h- d b /v p_y f with h- d b /gp and h- d b /y f demonstrates that the p p modification does not alter backbone conformations. significant conformational changes are only observed for the side chains of peptide residues p k and p f following the p p substitution. tuning antiviral cd t-cell response via proline-altered peptide ligand vaccination we thereafter assessed the in vivo impact of the p p modification on lcmv-activated p t cells. p t-cells were adoptively transferred into c bl/ mice, thereafter infected with the lcmv clone (fig ) . six days post-infection, p t-cells isolated from spleens (fig a and b ) were either stained with pmhc tetramers or re-stimulated with − m gp , y f or v p_y f. tetramer staining demonstrated that a significant amount of the activated p t cells recognized the h- d b /v p_y f complex (fig c- e) . furthermore, while v p_y fand gp -stimulated p t-cells produced tnf and ifnγ, y f was not recognized (fig d and e ). altogether, these results demonstrate that, in contrast to y f, v p_y f is efficiently recognized by p t cells in vivo-activated by lcmv infection. next, we assessed if vaccination with v p_y f could elicit endogenous t cells that cross-react and recognize y f. we engineered y f and v p_y f into the stalk region of the influenza a neuraminidase (hkx ). this well-established model results in efficient processing and presentation of epitopes on infected cells [ ] . c /bl mice were infected with the modified viruses flu(y f) or flu(v p_y f) (fig a) . days following infection, cd + cd + spleno- (apc) tetramers in order to detect cross-reactive t cell populations (fig b and c ). vaccination with flu(v p_y f) elicits endogenous t cell populations that bind to both h- d b /y f and h- d b /v p_y f tetramers equally well ( fig b) . interestingly, flu(v p_y f) vaccination also elicits endogenous t cell populations with dual specificity to h- d b /gp and h- d b /y f tetramers. in contrast, h- d b /gp -, h- d b /y f-and h- d b /v p_y f-tetramer staining after vaccination with flu(y f) failed to identify any significant t cell population ( fig b) . intracellular expression of ifnγ and tnf in cd + cd + endogenous t cells was assessed following stimulation with peptides gp , y f or v p_y f (fig c) . in contrast to flu(y f), vaccination with flu(v p_y f) results in significantly enhanced ifnγ and tnf levels towards both y f and v p_y f. however, vaccination with neither flu(y f) nor flu(v p_y f) induced any elicitation of ifnγ and tnf towards gp . this is well in line with previous studies in which the y f-specific t cell clone yf.f killed efficiently targets presenting gp but did not produce ifnγ [ ] . similar results were obtained using bronchoalveolar lavage (bal)derived t cells (s fig). in conclusion, vaccination with flu(v p_y f) induces endogenous t tuning antiviral cd t-cell response via proline-altered peptide ligand vaccination cell populations that respond strongly to h- d b /v p_y f and efficiently cross-react with h- d b /y f. in order to assess the molecular bases underlying the effects of the p p modification on t cell recognition, we determined the crystal structures of the ternary complexes p /h- d b /gp , p /h- d b /v p and p /h- d b /v p_y f to . , . and . Å resolution (s table, the immune escape mutation y f abrogates the hydrogen bond network formed with p the p cdr β residues d , g and r form a network of hydrogen bonds with the side chains of the gp residues p y and p t, as well as with the backbone of p n and p t (s fig) . the side chain of r β runs parallel with the peptide, stretching out to reach to the tip of p y, forming van der waals interactions with the side chain of p f, forcing its rotation in the case of gp . the tcr residue y α, which side chain is positioned between p k and p y, forms a hydrogen bond with the h- d b residue e , which also forms a hydrogen bond with p y ( s fig). thus, the hydroxyl group of p y plays a key role in a net of hydrogen bond and van der waals interactions formed with tcr residues n α and y α as well as the h- d b residue e . the introduction of the y f mutation will abrogate all these interactions, abolishing p recognition (s fig). furthermore, the y f mutation should introduce higher hydrophobicity within this key tcr/pmhc interface, composed mainly by polar residues. altogether, this explains why p does not bind nor recognize the immune escape variant h- d b /y f. the three ternary tcr/mhc/peptide structures were compared with each corresponding tcr-unbound pmhc (fig , s fig) . the side chain of p y, essential for recognition by p [ , , ] , rotates down following p binding to both h- d b /gp and h- d b /v p (fig a and b) . a similar rotation was also observed for residue p f in h- d b /v p_y f upon binding to p (fig c) . the side chain of p f in gp is also affected upon binding to p ( fig a) . interestingly, the p p modification resulted in a similar conformation for p f in both h- d b /v p and h- d b /v p_y f prior to binding to p (fig d and fig ) . furthermore, the side chain of residue p k in h- d b /gp also moves towards the n-terminal of the peptide the p p modification results in conformational changes of peptide residues p k and p f, predisposing pmhcs for optimal binding to p . a. comparison of gp before binding (in green) and after binding (in white) to p reveals major conformational changes in gp following binding to p . these include a movement of the p -p backbone of gp that is pushed down in the cleft combined with a degrees rotation of the isopropyl moiety in residue p v. furthermore, the side chain of peptide residues p k, p y and p f all take new conformations following binding to p . all movements are indicated by blue arrows. b. the introduction of p p in v p results in optimal positioning of the side chains of residues p k and p f prior to binding to p (in orange). the only observed conformational difference was taken by residue p y following v p binding to p (in cyan). c. similarly to v p, the only conformational difference observed for v p_y f before (in orange) and after (in violet) binding to p is at peptide residue p y. d. peptides gp (in white), v p (in cyan) and v p_y f (in violet) take nearly identical conformations when bound to p . https://doi.org/ . /journal.ppat. .g tuning antiviral cd t-cell response via proline-altered peptide ligand vaccination binding cleft following p binding (fig a) , taking an identical conformation as in both p psubstituted peptides (fig d) . one of the most significant differences in h- d b /gp , before and after binding to p , is a shift of the p -p backbone of gp when bound to p , towards the binding cleft of h- d b . following p docking, p v in gp extends . Å deeper into the d-pocket of h- d b , combined with a ˚rotation (fig a) . in contrast to gp , the p -p section is more constrained in both v p and v p_y f, following tcr binding (fig b and c ). however, it should be noted that the final conformations of all three peptides in the ternary complexes is nearly identical (fig d) . in conclusion, residues and in p p-apls take the same conformations prior to tcr binding as found in the ternary complexes, potentially enabling a more favorable surface for p tcr binding. the crystal structures of tcr-unbound and tcr-bound pmhcs also revealed that conformational differences in h- d b residues were observed only for residues r , e and h (figs , s , s and s ). the large movement of p f in gp following binding to p induces the counter wise reorientation of the side chain of residue h towards the tcr (fig a) . the redisposition of h and p f in h- d b /gp promotes the adequate positioning of the key tcr residue r β, which runs longitudinally along the length of the n-terminal part of the peptide (s fig). in contrast, residues p f and h are already optimally positioned in both the tcr unbound and bound forms of the h- d b /v p and h- d b /v p_y f complexes (fig b and c ), most probably predisposing for optimized interactions with p . furthermore, p k in gp also takes a different conformation upon binding to p , bending backwards towards the h- d b residues r and e , which conformations are affected (fig a, fig a) . here again, the side chain of p k takes exactly this conformation in both v p and v p_y f already before tcr binding (fig , fig ) . altogether, p k, p f and heavy chain residues r , h and e have already adopted in the unbound v p and v p_y f complexes similar conformations to those observed in all three ternary structures (fig and fig ) . thus, the p p substitution potentially facilitates tcr recognition by positioning specific key peptide and mhc residues prior to the formation of the ternary complexes. this is well in line with our spr and itc results, which indicate that the energy required for p recognition of v p is reduced compared to gp (table , comparison of h- d b /gp before (in green) and after p binding (in white) reveals that the conformation of a very limited amount of pmhc residues is affected (shown as sticks). following binding to p , the side chain of peptide residue p k moves towards the n-terminal part of the peptide binding cleft while the side chain of p f rotates. as a consequence, conformational changes are observed only for heavy chain residues r , h and e . b. in contrast to gp , the introduced p p modification already positions most peptide and heavy chain residues in optimal conformations, limiting significantly the required movements following binding to p . pmhc residues before and after binding to p are colored orange and cyan. c. similarly to v p, the p p modification in v p_y f results in optimal positioning of all key peptide and heavy chain residues prior to binding to p . pmhc residues before and after binding to p are colored orange and violet. https://doi.org/ . /journal.ppat. .g tuning antiviral cd t-cell response via proline-altered peptide ligand vaccination [ , , ] . heteroclitic subdominant viral t cell determinants were also used to enhance both pmhc stability and t cell avidity towards the mouse hepatitis virus-specific subdominant s determinant [ , ] . most, if not all studies performed in other laboratories have focused their efforts on introducing peptide mutations that would significantly increase the stability of pmhcs with as little alteration as possible of peptide conformation. here, instead of mutating a key anchor position, we targeted interactions between peptide position and the mhc residue y , conserved among most known mouse and human alleles. indeed, besides h- d b , we have previously demonstrated that the p p modification enhances significantly the stability of h- k b in complex with different taas [ ] . thus, the p p modification could potentially enhance stabilization of other mhc-i alleles that comprise the heavy chain residue y , leading to enhanced tcr recognition. here, we addressed if we could increase cd + t cell avidity and restore recognition of the viral escape variant y f that binds to h- d b with the same high affinity as gp [ ] . the tcr p is specific for h- d b /gp and it has been previously demonstrated that p recognition is abolished by the y f mutation [ , ] . comparison of the crystal structures of h- d b /gp and h- d b /y f demonstrated that the only structural difference between these two pmhcs was the removal of the hydroxyl tip from the peptide residue p [ ] . we demonstrate that the p p modification in v p_y f overcomes the restrictions imposed by the y f mutation, reestablishing p recognition of this structural mimic of y f. furthermore, we show that it is fully possible to restore endogenous cd + t cell recognition of y f following vaccination with v p_y f. possibly, the higher avidity of subsets of the endogenous t cell population for h- d b /v p_y f pushes them over a certain threshold of activation, and the molecular similarities between h- d b / v p_y f and h- d b /y f allow for cross-reactivity, resulting in significant cytokine secretion towards y f. however, in vitro re-stimulation with gp of endogenous cd + t cells isolated from flu(v p_y f)-vaccinated mice did not induce any significant secretion of cytokines, although these endogenous cd + t cells recognized both v p_y f and gp -loaded mhc tetramers. martin et al have previously provided evidence for selective activation of different effector functions in cd + t cells by apls. more specifically, the results of their study show that the h- d b /y f-specific t cell clone yf.f killed efficiently targets presenting gp but did not produce high amounts ifnγ against gp [ ] . this is well in line with the results presented in this study. altogether, this suggests to us that vaccination with a cocktail of epitopes could provide wider protection against both immunodominant and immune escape targets. so how does it possibly work at the molecular level? the rigidification of p p-modified peptides could enhance tcr recognition by decreasing entropic costs. indeed, we have previously demonstrated in taa models that peptide rigidification enhanced considerably tcr recognition [ , ] . overall the effects of proline replacement on protein stability and function are well established for a large ensemble of proteins [ ] , revealing that protein-protein interactions often occur through regions enriched with proline residues [ ] . proline substitutions increase overall protein stability as well as the stability of specific protein regions [ ] . indeed, proline replacement of specific residues in tcr cdr loops can increase significantly recognition of antigens [ ] . the importance of the interaction of peptide residue p p with residue y , conserved amongst most known mhc-i alleles, has been previously described [ ] , revealing that p p reduces significantly the flexibility of the pmhc complex, thus decreasing unfavorable entropic change upon complex formation. such reduced entropic penalties for tcr recognition following p p mutation were confirmed here by itc measurements, which indicated reduced unfavorable entropic contribution for recognition of h- d b /v p by p tuning antiviral cd t-cell response via proline-altered peptide ligand vaccination compared to h- d b /gp . the importance of the reduction of peptide conformation heterogeneity for enhanced tcr has been described, using a combination of crystal structure and molecular dynamic studies [ ] . a peptide that must move to optimize the interactions with the bound tcr will increase the entropic cost for binding, resulting in slower binding, lower affinity and less efficient recognition [ ] . consequently, although many tcrs bind with unfavorable entropy changes [ , ] , reduction of conformational heterogeneity coupled with rigidification of the peptides may lead to enhanced t cell recognition. in this study, the p p mutation reduces motion and therefore enhances t cell recognition by increasing t cell association rate and decreasing entropic costs for binding. although x-ray structural studies of proteins provide accurate snapshots of protein complexes, crystal structures provide relatively little information about the dynamic bases underlying protein-protein interactions. the dynamic motions of both pmhc and tcr impact on recognition by t cells, clearly influencing function and recognition [ ] . here, we compared the crystal structures of each studied pmhc complex before and after p tcr binding (besides the p /h- d b /y f complex that could not be obtained since p does not bind to this pmhc). peptides tune the motions of mhc heavy chains and reduced motions may lead to enhanced recognition. besides the peptide rigidification imposed by the p p modification, comparison of a structural snapshot for each ternary structure with each tcr-unbound pmhc variant indicated an additional structural reason for the increased tcr recognition of p p-modified epitopes. in all cases, conformational differences were observed in peptide residues p k and p f in v p_y f and v p, compared to y f and gp , before tcr binding. in all p p cases, the side chains of peptide residues p k and p f took the same conformation, as observed in the ternary structures, prior to tcr binding. in line with this, others [ , ] and we [ ] have previously demonstrated the importance of residue p k for recognition by the tcr p . the crystal structure of the semi-agonist y a (kavanfatm) also revealed a similar conformation for both p k and p f prior to binding to p tcr [ ] . furthermore, the conformation of the mhc "tcr footprint" heavy chain residues r , h and e [ , ] was also affected following p p substitution, possibly due to the movements of p k and p f. altogether, prior to tcr landing, the p p modification alters the conformation of residues both in the peptide and the mhc heavy chain similar to conformations taken upon binding to the tcr, thus predisposing the pmhc for facilitated tcr recognition. the results presented within this study indicate in our opinion that i) docking of p to p p-modified peptides is facilitated since the conformations of key residues in both peptide and heavy chain are already optimal prior to tcr binding (ready-to-go conformation); ii) consequently, the energetic costs for tcr recognition should be reduced since there is no need for any major movement in the rigidified epitope besides the conformational change for residue p y. as vaccination with v p_y f restored endogenous t cell recognition of y f, the p p modification could thus represent a novel way to increase the immunogenicity of a large array of h- d b -restricted epitopes as well as possibly viral epitopes restricted by other mhc alleles. we thus describe here a successful approach to restore recognition of viral escape peptide that can be easily coupled to already existing vaccination protocols, including vaccination with full-length proteins as well as e.g. modified mrna vaccines, by introducing the p p modification in a selection of viral epitopes. h- d b+ /h- k b+ rma cells, kindly provided by prof. klas kärre, were used as target cells in the functional assays described below. pathogen-free wild-type (wt) c bl/ (b ) and tuning antiviral cd t-cell response via proline-altered peptide ligand vaccination rag / -deficient (rag / -/-) p -transgenic mice were bred and maintained within the facilities of the mtc department, karolinska institute. vα + t cells from p mice were used as effector cells for in vitro experiments. p mice were used for in vivo t-cell stimulation assays. peptides gp , y f, v p and v p_y f as well as the control peptide np (asnenmetm, abbreviated as asn) were purchased from genscript (piscataway, nj, usa). antibodies - . (anti-cd α), - . (anti-cd β), xmg . (anti-ifn-γ), mp -xt (anti-tnf), - c (anti-cd ε), d b (anti-cd a), bp- (anti-ly . /cd ), im (anti-cd ) and b . (anti-tcr vα ) were purchased from bd biosciences (san diego, ca, usa). antibodies gk . (anti-cd ) and h - (anti-tcr cβ) were purchased from abcam (cambridge, uk) and ebioscience (san diego, ca, usa). refolding of all pmhcs was conducted as previously described [ ] . p was produced and refolded by dilution and thereafter-purified using ion exchange and size exclusion chromatography. crystals for h- d b /v p and h- d b /v p_y f were obtained by hanging drop vapor diffusion in . data collection was performed at beam lines id - and id - at esrf (grenoble, france). diffraction data were processed and scaled using mosflm . . and scala [ ] . crystal structures were determined by molecular replacement using phaser [ ] . the crystal structure of h- d b /gp (pdb id: s u) [ ] , with omitted peptide, was used as search model for h- d b /v p and h- d b /v p_y f. p /h- d b /gp , p /h- d b /v p and p /h- d b / v p_y f were determined using pqy [ ] . in all cases, poorer electron density was displayed for the tcr cα domain, probably due to high flexibility, as previously observed [ ] . random % reflections were used for monitoring refinement by r free cross-validation [ ] . the model was rebuilt in coot where necessary. the stereochemistry of the final models was verified using procheck [ ] or coot [ ] . measurements were performed in mm k hpo /kh po (ph . ) using . - . mg/ml protein concentrations. melting temperatures (tm) were derived from changes in ellipticity at nm as previously described [ ] . curves and tm values are an average of at least three measurements from at least two independent refolding assays per pmhc. spectra were analyzed using graphpad prism (la jolla, usa). all measurements were performed on biacore (ge healthcare, usa) at ˚c. soluble p ( μg/ml) was non-covalently coupled to the anti-c β antibody h - . rus of h - were coupled to a cm -chip, resulting in rus immobilized p . a control surface without antibody was used as reference. concentration series of pmhcs were injected over the chip. the surface was regenerated with μl . m glycine-hcl, mm nacl, ph . . unspecific binding was corrected for by subtracting responses from reference flow cells. tuning antiviral cd t-cell response via proline-altered peptide ligand vaccination data were analyzed with biaevaluation (biacore ab, uppsala, sweden). k d -values were obtained from steady-state fitting of equilibrium binding curves from at least two independent measurements. measurements were performed on a microcal itc (ge healthcare, usa) at ˚c. μl h- d b /v p ( μm) or h- d b /gp ( μm) in mm hepes, mm nacl, ph . were titrated into μl of p ( . - μm) in injections under rpm stirring rate. data analysis was performed using origin, fitted to a non-linear curve in an iterative process. the reported constants are an average of two independent experiments. p -splenocytes were mixed with peptide-pulsed rma cells at : effector:target (e:t) ratio. cells were co-incubated at ˚c for h and stained with anti-cd β and -tcr vα antibodies. flow cytometry was performed using facscalibur (bd biosciences) and changes in mean fluorescence intensity (mfi) of the vα staining were used to estimate tcr down-regulation. data was analyzed using flowjo (tree star, inc., ashland, or, usa). p tcr-transgenic mice were injected subcutaneously (sc) with μg gp in pbs combined with . ng phosphorothioate-modified cpg-odn (invivogene, sweden). mg aldara cream was applied at site of injection ( % imiquimod, meda ab, sweden). animals were sacrificed days later and spleens were recovered. target rma cells, labeled with cr , were pulsed with indicated peptide concentrations for h at ˚c and subsequently mixed with in vivo-stimulated negatively selected (macs cd + t cell isolation kit, miltenyl biotec, germany) p cd + t cells at : e:t ratio followed by a standard h cr -release assay. radioactivity was measured on a γ-counter (wallac, uppsala, sweden). percentage of specific lysis was calculated as [cr release in test well-spontaneous cr release] / [maximum cr release-spontaneous cr release] x . cd + t cells isolated from spleens of naïve (for tnf production assays) or in vivo-stimulated p transgenic mice were co-cultured for h with − m or − m peptide-pulsed rma cells in the presence of anti-cd a antibody for degranulation assays. golgistop (bd biosciences) was added after h co-incubation. h later, cells were stained with anti-cd α and -cd ε antibodies. for intracellular cytokine staining assays, cells were fixed and permeabilized using the cytofix/cytoperm kit (bd biosciences) according to instructions. cells were thereafter stained for ifnγ and tnf expression. facs sampling was performed on cyan (dako, glostrup, denmark) and analyzed with flowjo. h- d b molecules with a biotinylation tag were refolded with peptides and mβ m in the presence of protease inhibitors and purified as previously described [ ] . each obtained monomeric h- d b /peptide complex ( . mg/ml) was tetramerized at a : ratio with streptavidin-pe or streptavidin-apc (bd biosciences) in order to create each of the following tetramers h- tuning antiviral cd t-cell response via proline-altered peptide ligand vaccination p t cells (cd low , ly . + ), isolated from spleens of p transgenic mice, were adoptively transferred intravenously (in pbs) into c bl/ mice three days prior to intraperitoneal infection with x pfu of lcmv (clone ). spleens were harvested on day post infection. cd + t cells were enriched by b cell panning and red blood cell lysis and stimulated with il- ( units/ml), brefeldin a ( μg/ml) (bd biosciences) and − m peptide (gp , y f or v p_y f or no peptide) in complete rpmi for h at ˚c, % co . washed cells were surface stained with anti-cd , -cd and -ly . , fixed and permeabilized using bd cytofix/cytoperm kit ( min at ˚c). intracellular staining of ifnu, tnf and il- (at : ) was performed for min at ˚c. endogenous t cells were distinguished by congenic marker ly. . from ly. . + p t cells. cells were resuspended in facs buffer after enrichment and stained at : for hr at rt with gp , y f or v p_y f tetramers. washed cells were surface stained for cd , ly . , cd a and cd for min at ˚c. data was collected using lsr fortessa (bd biosciences) and analyzed with flowjo. the peptides y f and v p_y f were introduced into the influenza a virus by inserting/replacing a region in the stalk of neuraminidase (na) using the cloning system as described. reverse genetics, generation of modified influenza: briefly, ug of each plasmid (np, ns , pb , m, pa, pb , ha and na) was mixed with ug of lipofectomine and optimem and added to a mix of co-cultured mdck/ t cells, in the presence of tpck-trypsin. the transfection was allowed to proceed for - h in % co at ˚c. the virus was thereafter propagated in chicken eggs for days at ˚ [ ] . viral rna was isolated from virus particles with rneasy-kit (qiagen, valencia, ca). access rt-pcr kit (promega) was used for characterization of recombinant influenza viruses. naive c bl mice were adoptively transferred with p t cells one day prior to infection. with x pfu or i.p. with . x pfu of influenza a virus following anesthesia with isofluorane, then used for analysis of primary immunity at day post infection. kinetics, magnitude and phenotype of primary virus-specific cd + t cell responses were measured by flow cytometry. gp -and apl-specific cd + t cell populations were characterized using h d b / gp , y f and v p_y f tetramers. splenocytes were incubated with tetramers for min at room temperature. washed cells were stained for cd + and cd for min at ˚c. intracellular ifnγ and tnf staining ( : ) was performed for min at ˚c. data was collected using lsr fortessa (bd biosciences) and analyzed with flowjo. tuning antiviral cd t-cell response via proline-altered peptide ligand vaccination data were routinely shown as mean ± sd. unless stated otherwise, statistical significance was determined by the student's t test or analysis of variance (anova) using graphpad prism . . � p < . ; �� p < . ; ��� p < . ; ���� p < . . all experimental animal procedures were performed under swedish national guidelines (n / ) and following approval from the university of melbourne animal ethics experimentation committee (ethics number . ). tuning antiviral cd t-cell response via proline-altered peptide ligand vaccination s immunodominance in major histocompatibility complex class i-restricted t lymphocyte responses determination of structural principles underlying three different modes of lymphocytic choriomeningitis virus escape from ctl recognition a structural basis for lcmv immune evasion: subversion of h- d(b) and h- k(b) presentation of gp revealed by comparative crystal structure viral escape by selection of cytotoxic t cell-resistant virus variants in vivo hepatitis c virus-t-cell responses and viral escape mutations effect of epitope flanking residues on the presentation of n-terminal cytotoxic t lymphocyte epitopes hepatitis c virus mutation affects proteasomal epitope processing viral escape by selection of cytotoxic t cell-resistant variants in influenza a virus pneumonia the outcome of hepatitis c virus infection is predicted by escape mutations in epitopes targeted by cytotoxic t lymphocytes structural and biological basis of ctl escape in coronavirus-infected mice natural variants of cytotoxic epitopes are t-cell receptor antagonists for antiviral cytotoxic t cells mutational escape from cd + t cell immunity: hcv evolution, from chimpanzees to man. the journal of experimental medicine hiv and siv ctl escape: implications for vaccine design eventual aids vaccine failure in a rhesus monkey by viral escape from cytotoxic t lymphocytes protective efficacy of cross-reactive cd + t cells recognising mutant viral epitopes depends on peptide-mhc-i structural interactions and t cell activation threshold more than one reason to rethink the use of peptides in vaccine design determinant selection of major histocompatibility complex class i-restricted antigenic peptides is explained by class i-peptide affinity and is strongly influenced by nondominant anchor residues. the journal of experimental medicine increased immunogenicity of an anchor-modified tumor-associated antigen is due to the enhanced stability of the peptide/mhc complex: implications for vaccine design amino-terminal alteration of the hla-a* -restricted human immunodeficiency virus pol peptide increases complex stability and in vitro immunogenicity functional and structural characteristics of ny-eso- -related hla a -restricted epitopes and the design of a novel immunogenic analogue single amino acid replacements in an antigenic peptide are sufficient to alter the tcr v beta repertoire of the responding cd + cytotoxic lymphocyte population prevention of cytotoxic t cell escape using a heteroclitic subdominant viral t cell determinant design of agonistic altered peptides for the robust induction of ctl directed towards h- db in complex with the melanomaassociated epitope gp . cancer research tap-independent self-peptides enhance t cell recognition of immune-escaped tumors. the journal of clinical investigation the mhc class i cancer-associated neoepitope trh linked with impaired peptide processing induces a unique noncanonical tcr conformer the immunogenicity of a proline-substituted altered peptide ligand toward the cancer-associated teipp neoepitope trh is unrelated to complex stability proline substitution independently enhances h- d(b) complex stabilization and tcr recognition of melanoma-associated peptides immunobiology of cytotoxic t-cell escape mutants of lymphocytic choriomeningitis virus in vitro selection of lymphocytic choriomeningitis virus escape mutants by cytotoxic t lymphocytes in vivo selection of a lymphocytic choriomeningitis virus variant that affects recognition of the gp - epitope by h- db but not h- kb addition of a prominent epitope affects influenza a virus-specific cd + t cell immunodominance hierarchies when antigen is limiting selective activation of cd t cell effector functions by epitope variants of lymphocytic choriomeningitis virus glycoprotein cd + t cell activation is governed by tcr-peptide/mhc affinity, not dissociation rate viral escape at the molecular level explained by quantitative t-cell receptor/peptide/mhc interactions and the crystal structure of a peptide/mhc complex heteroclitic immunization induces tumor immunity. the journal of experimental medicine structural and functional correlates of enhanced antiviral immunity generated by heteroclitic cd t cell epitopes unexpected t-cell recognition of an altered peptide ligand is driven by reversed thermodynamics stereospecific interactions of proline residues in protein structures and complexes another role of proline: stabilization interactions in proteins and protein complexes concerning proline and tryptophane the role of proline substitutions within flexible regions on thermostability of luciferase backbone flexibility of cdr and immune recognition of antigens peptide and peptide-dependent motions in mhc proteins: immunological implications and biophysical underpinnings structure of the complex between human t-cell receptor, viral peptide and hla-a thermodynamics of t-cell receptor-peptide/mhc interactions: progress and opportunities peptidic termini play a significant role in tcr recognition identification of a crucial energetic footprint on the alpha helix of human histocompatibility leukocyte antigen (hla)-a that provides functional interactions for recognition by tax peptide/hla-a -specific t cell receptors. the journal of experimental medicine strategic mutations in the class i major histocompatibility complex hla-a independently affect both peptide binding and t cell receptor recognition murine class i major histocompatibility complex h- dd: expression, refolding and crystallization scaling and assessment of data quality phaser crystallographic software structural basis for enabling t-cell receptor diversity within biased virus-specific cd + t-cell responses directed evolution of human t cell receptor cdr residues by phage display dramatically enhances affinity for cognate peptide-mhc without increasing apparent cross-reactivity free r value: a novel statistical quantity for assessing the accuracy of crystal structures main-chain bond lengths and bond angles in protein structures coot: model-building tools for molecular graphics visualization of inhibitory ly receptor specificity with soluble major histocompatibility complex class i tetramers a dna transfection system for generation of influenza a virus from eight plasmids plos pathogens tuning antiviral cd t-cell response via proline-altered peptide ligand vaccination plos pathogens tuning antiviral cd t-cell response via proline-altered peptide ligand vaccination tuning antiviral cd t-cell response via proline-altered peptide ligand vaccination key: cord- - fip l authors: labadie, thomas; roy, polly title: a non-enveloped arbovirus released in lysosome-derived extracellular vesicles induces super-infection exclusion date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: fip l recent developments on extracellular vesicles (evs) containing multiple virus particles challenge the rigid definition of non-enveloped viruses. however, how non-enveloped viruses hijack cell machinery to promote non-lytic release in evs, and their functional roles, remain to be clarified. here we used bluetongue virus (btv) as a model of a non-enveloped arthropod-borne virus and discovered that the majority of viruses are released in evs. based on the cellular proteins detected in these evs, and use of inhibitors targeting the cellular degradation process, we demonstrated that these extracellular vesicles are derived from secretory lysosomes, in which the acidic ph is neutralized upon the infection. moreover, we report that secreted evs are more efficient than free-viruses for initiating infections, but that they trigger super-infection exclusion that only free-viruses can overcome. a a a a a lipid envelopes surrounding viruses are likely the results of a convergent evolution, potentially acquired during the adaptation of non-enveloped virus to animals [ ] . the envelope plays essential roles, such as the fusion with cellular membranes to enter in cells, or the assembly of new virus particles during budding at the plasma membrane. in contrast, non-enveloped viruses developed alternative mechanisms, such as cell entry by membrane penetration [ ] and exit by cell lysis. in both cases, free virus particles remain the canonical unit of virus infection, with a single virus particle entering a cell to replicate and amplify its genome. recent studies in the field of non-enveloped viruses have drawn attention on previously unknown cell to cell spread mechanisms, in which released virus particles are not naked. a seminal work on hepatitis a virus revealed that virus particles are released both in enveloped and non-enveloped states, with the envelope protecting virus particles from neutralising antibodies [ ] . more recently, it was reported that other non-enveloped lytic viruses, such as coxsackievirus b [ ] , hepatitis e virus [ ] , poliovirus [ , ] , polyomavirus [ , ] , rotavirus and norovirus [ ] , can also exit the cells as cloaked in extracellular vesicles (ev). further, it was observed that evs could contain multiple virus particles, allowing infection of a unique cell with many virus particles at a time [ , ] . this new transmission mechanism, reported as en block transmission, allows viral genome complementation during the infection and could be a regulator of fitness evolution for a virus population [ , ] . however, the mechanisms by which viruses hijack the host cellular secretion machinery, or the benefit of vesicular transmission are still unclear [ ] . understandings these might alter the rigid definition of non-enveloped viruses. to this end, here we investigated ev associated virus release using a model non-enveloped multi-layered capsid virus, bluetongue virus (btv), of the orbivirus genus, with a segmented double stranded rna genome. btv is an insect-borne virus that periodically causes epidemics among ruminants worldwide, with a particularly high mortality rate in sheep. although classified as a non-enveloped virus, btv is able to exit cells by both lytic and non-lytic mechanisms [ ] [ ] [ ] . here, we report that evs containing multiple virus particles are the main infectious unit of btv. our results revealed a mechanism of en block transmission in which both mvbs and the late stages of autophagy are essential to cloak virus particles in secreted lysosomes. our results explain how both forms of the virus (free and vesicular) contribute to its life cycle and demonstrate their importance. further, we observed that ev associated virus induces a more efficient infection and synthesis of viral protein than does infection with free virus particles, while free virus particles are essential to overcome super-infection exclusion. btv egresses infected cells not only by cell lysis but also by a non-lytic mechanism [ , ] . since btv particles were shown to be associated with mvbs components during trafficking [ ] and the purified virus particles are associated with ns [ ] , a membrane protein, it is possible that multiple btv particles are released within evs. to determine this, we infected sheep cells (pt cell line) and culicoides insect cells (kc cell line) at a multiplicity of infection (moi) of , and cell culture supernatants were centrifuged to isolate free virus particles from large evs (~ μm) which sediment as pellets under the conditions used. viral supernatants were harvested at hours post-infection (hpi) as the released virus content is high at this time point yet there is almost no detectable cell death (s fig). virus titrations ( fig a) and viral genome quantification by quantitative pcr (fig b) both revealed that the majority of infectious virus particles and viral genomes were sedimented after a , xg centrifugation. further, western blot analysis of the structural protein content, both in the pellet and the supernatant revealed that vp , vp and vp were at higher levels in the pellet (fig c) , consistent with the virus titres. moreover, specific infectivity, defined as the ratio of the number of genomes per pfu [ ] , was significantly lower in the , xg pellets when compared to the supernatant (fig d) , suggesting that the form of virus present in the pelleted fraction was more infectious than btv present in the supernatant. to localise the btv proteins within purified evs, we used a super resolution microscopy approach that could visualise both structural protein vp and non-structural protein ns with the phosphocholine bodipy, indicating that btv is associated with lipid membranes in evs ( fig e) . moreover, direct observations of both purified evs secreted from infected cells (fig f) , and ultrathin sections of evs embedded in agar ( fig g) by negative stain electron microscopy revealed the presence of large evs (ranging from . to . μm) containing multiple virus particles, corroborating our fluorescence imaging observations. altogether, these data established that the majority of btv virus particles are released in large evs secreted from mammalian and insect infected cells in vitro. to further analyse the btv particles found in the , xg supernatant, this supernatant was centrifuged again at , xg, and the infectivity was measured in both the , xg pellet and its supernatant ( fig h) . most of the infectivity was then associated with the , xg pellet, that was analysed by electron microscopy for the form of virus present ( fig i) . we observed that the majority of virus was in the form of aggregates, made of particles embedded in a lipid membrane, or in the form of naked virus particles. the lipid membrane in the case of the , xg pellet may be a transient lipid membrane acquired from single particles budding at the plasma membrane, as already described [ ] , so we refer to the , xg supernatant as the free virus fraction in this report. to determine the origin of evs containing btv virus particles, we tested the presence of specific cellular markers. among the different markers of evs assessed, we determined the presence of annexin a , the tumour susceptibility gene (tsg ), lc -i and lc -ii, and the lysosomal associated protein lamp by western blot (fig b and s a fig). note that tsg is a mvbs marker and lc b an autophagy related protein. to identify the cellular mechanisms involved in virus packaging in evs, we first investigated the potential role of autophagy, using the -methyladenin ( -ma) that inhibits class iii pi k activity. we observed that -ma failed to significantly decrease the secretion of infectious evs from sheep cells infected at a moi of ( fig b) , suggesting that the induction of autophagy is not essential for the secretion of and viral genome quantification (b) in the pellet or in the supernatant after isolation of evs secreted by infected mammalian sheep cells and insect cells. data are presented as mean ± sd. each point indicates the value of independent replicates (n = ; unpaired t-test, � p< . , �� p< . , ��� p< . , ���� p< . ). (c) outer capsid proteins vp and vp , and inner capsid protein vp , detected in the pellet and supernatant after isolation of evs secreted by infected mammalian cells. (d) specific infectivity, which is the number of genome copies per plaque forming unit, measured for virus particles in the pellet and the supernatant after a , xg centrifugation. data are presented as mean ± sd. each point indicates the value of independent replicates (n = ; unpaired t-test, � p< . ). (e) immunofluorescence microscopy images of purified evs secreted from infected (two top rows) or mock (bottom row) mammalian cells labelled with anti vp (orange) and ns (red) antibodies, and a fluorescent lipid membrane marker (green). scale bar: μm. (f and g) electron microscopy images of whole evs (f) and sectioned evs (g) secreted from infected mammalian cells. yellow arrows indicate the virus particles positions in the sections. scale bar: nm. (h) virus particles titration in the pellets and the final supernatant after centrifugation of , xg and , xg. data are presented as mean ± sd. each point indicates the value of independent replicates (n = ; unpaired t-test, ���� p< . ). (i) representative electron microscopy images of btv particles observed in the , xg pellet. scale bar: nm. https://doi.org/ . /journal.ppat. .g virus released in secretory lysosomes infectious evs. however, inhibition of autophagosome-lysosome fusion with chloroquine (cq), led to a significant reduction of infectious evs released as compared to the control ( fig b) , indicating that the late steps of autophagy are necessary for infectious evs. in addition, inhibition of mvbs regulator protein hsp using geldanamycin in btv-infected cells (moi = ) also led to a significant reduction of infectivity measured in the evs fraction, as compared to the control, indicating a possible role for mvbs in the release of infectious evs. in contrast, gw , a drug that inhibits the release of exosomes (small vesicles~ nm) derived from mvbs, did not affect the secretion levels of evs containing btv ( fig b) . as expected, except for gw , these drugs also had an inhibitory effect on the virus replication (s b fig) , affecting virus released in both ev and free viruses, albeit to various levels. these cumulative inhibitory strategies of autophagy and mvbs provide evidences that mvbs and the late stages of autophagy are both involved in the release of evs containing virus particles. however, these evs cannot be exosomes, based on the results obtained with the gw inhibitor. following recent description that autophagy and mvbs share common molecular machinery and organelles such as amphisomes [ ] , we asked if evs could be derived from secreted lysosomes after the fusion with amphisomes. therefore, we purified the evs using an isopycnic ultracentrifugation and found that most of the infectivity was associated with the low-density fractions (~ . to . g/ml), along with the cathepsin enzymes, a lysosomal marker ( fig c) . interestingly, the ratio of cell surface lamp to cytosolic lamp was also significantly higher in infected cells (fig d) , indicating an increase of lamp located at the plasma membrane in infected cells. the presence of viral proteins was then investigated in the lysosomes of btv infected cells. using super resolution microscopy, we observed that in infected cells only, outer capsid protein vp as well as lamp co-localised with the mvbs marker hsp (fig e) , indicating a fusion between mvbs and the autophagy compartments. supporting these results, we also observed the co-localisation of lamp and vp with tsg , another mvbs marker (s d fig) , and the co-localisation of vp with the lysosomal markers lamp and cathepsins, indicating the presence of viral proteins within lysosomes ( fig f) . altogether, increased lamp localisation at the plasma membrane, a hallmark of increased lysosomal exocytosis [ , ] , and the presence of btv proteins within lysosomes indicate that btv infection could reprogram lysosomal degradation pathways to facilitate virus secretion. ns is the only viral membrane protein and it is likely to be involved in the mechanism of ev secretion. therefore, we tested the presence of virus in evs ( fig a) following infection with available ns mutants [ , , ] . two of these mutants impair the synthesis of the two ns isoforms, respectively ns (mutant ns m ) and ns a (mutant ns m ), while another with a modified late domain motif (ns gaap ) cannot bind tsg and one, unable to bind the outer capsid protein vp (ns stop ). of the four mutant viruses, only btv ns m significantly reduced virus particle release in evs (fig a) . to understand this phenotype, we virus released in secretory lysosomes analysed the localisation of the mutant virus particles in infected cells and observed that the outer capsid protein vp , and ns , were still co-localised with the lysosomal markers lamp and cathepsin ( fig b, s fig) , suggesting that the absence of btv ns m in evs was not due to a defect in virus trafficking. we then examined lysosomes in cells infected with wild type btv (btv wt ) and non-infected cells expressing a lamp -gfp fusion protein in the presence of a fluorescent lysotracker specific to acidic organelles ( fig c) . confocal microscopy revealed a significant decrease of lysotracker fluorescence intensity in the lysosomes of btv wt infected cells, when compared to non-infected cells, suggesting that btv wt neutralises the acidic ph of lysosomes ( fig d) . the number of lysosomes detected in infected cells was also significantly higher than in non-infected cells (fig e and g ). in contrast, cells infected with btv ns m showed comparable levels of lysotracker fluorescence to non-infected cells, suggesting that btv ns m was unable to counter lysosomes acidification. to provide further confirmation, cells infected with btv ns m and non-infected cells were examined in the presence of the lysosomal v-atpase inhibitory drug bafilomycin a (fig e and g ). interestingly, bafilomycin a significantly increased the levels of btv ns m in evs (fig h) , while not affecting the intracellular replication of btv ns m , whereas the drug had a strong inhibitory effect on the replication of btv wt (s b fig) . consistent with this model, we observed that the intracellular calcium levels of cells infected with btv wt were significantly higher than the calcium levels in btv ns m and non-infected cells (fig i and j ). together, our data strongly support that newly synthesised btv particles are released via evs derived from secretory lysosomes after ph neutralisation. along with their cellular origin, the role of the secreted form of non-enveloped viruses remains unclear. to better understand the role of evs during btv infection, we compared the infectivity of evs and free virus particles, by analysing the kinetic of total virus production in cells infected either by evs or free-virus particles. infection of mammalian cells with infectious evs or free virus particles led to a similar amount of virus released from cells at hpi. however, at hpi, we found that cells infected with infectious evs released more virus (fig a) , and showed a higher level of viral genome in their cytoplasm ( fig b) than cells infected with free virus released in secretory lysosomes virus particles. this suggests that infectious evs are a more efficient form of infecting agents than are free virus particles. to test this possibility, we investigated the kinetic of viral protein synthesis by following the formation of viral inclusion bodies (vibs, virus assembly factories) in the cytosol of cells infected with either evs or free virus particles (moi = ). we examined the size and number of vibs in infected cells between to hpi, by fluorescence microscopy (fig c) . a frequency analysis of the vibs surface area distribution (fig d and e ) revealed that at hpi, vibs median surface areas were . ± . μm in free-virus infected cells against . ± . μm in evs infected cells. at hpi, mean vib size surface was clearly smaller in free-virus infected cells with . ± . μm as compared to evs infected cells ( . ± . μm ). this result suggests that virus-directed protein synthesis is considerably more efficient in evs infected cells. an analysis of the frequency distribution of vibs formed per cell at hpi (fig virus released in secretory lysosomes f) indicated that evs infected cells had a geometric mean of . vibs/cell whereas in free virus infected cells, the geometric mean was . vibs/cell, supporting the idea that infectious evs allow the formation of multiple viral factories soon after the infection. together, these data indicate that ev infected cells produce more vibs, that expand faster, than vibs produced by free-virus infected cells. thus, in addition to containing the majority of infectious virus particles released from infected cells, evs are also more efficient than free-virus particles to initiate an infection. we next considered if differences between evs and free virus particles are due to cell defence mechanisms, such as super-infection exclusion. although not always understood, this cell defence mechanism in which infected cells become resistant to a second infection has been observed for several viruses. we first evaluated the presence of a super-infection exclusion mechanism in sheep cells infected with btv, using two different viruses, a non-modified btv (btv wt ) and a fluorescent btv (btv unag ) encoding a fluorescent fusion protein vp -unag. after controlling that btv unag virus particles are also mainly released in evs (fig a) , we first infected sheep cells with btv wt, followed by a second infection with btv unag immediately ( hpi) or at , , , or hpi (fig b) . hours after the initial infection with btv wt , cells were then labelled with an anti vp antibody. unag fluorescence correspond to the detection of the btv unag virus only, whereas indirect fluorescence with vp antibody detects both viruses (fig c) , although less efficiently in the case of vp -unag (fig d) . using flow cytometry, we quantified unag fluorescence in cell population gated for vp positivity (from the antibody-based detection). these cells were either infected with btv wt (unag negative), btv unag (unag positive) or both viruses (unag positive). we used a constant moi across all experiments, so that the number of btv unag single infected cells should remain constant. therefore, variations of the unag fluorescence level reflect variations of co-infection with btv wt and btv unag and a superinfection exclusion of btv unag . a strong decrease of the unag fluorescence was observed in cells with longer incubation times between the two infections ( fig e) . this result provides evidence that btv infection induces super-infection exclusion in sheep cells. we repeated this experiment by infecting sheep cells with btv wt (total virus particles), and then with free-virus particles or evs of btv unag . sheep cells were first infected with btv wt , and then btv unag free virus particles or infectious evs. a super infection exclusion mechanism was observed in cells co-infected with btv wt and evs containing btv unag (fig f) , with a significant decrease of unag fluorescence intensity dependent on the incubation times between the two infection. conversely, no significant differences of unag fluorescence were detected in cells with secondary infection of btv unag free virus particles over time ( fig g) . altogether, these results indicate that while btv infection led to a super-infection exclusion mechanism in sheep cells, btv free virus particles were able to overcome this mechanism. while recent discoveries regarding evs containing viruses have changed our perception of non-enveloped viruses, such observations were lacking for a non-enveloped arthropod-borne virus. moreover, it has been hypothesised that lipid envelopes are the hallmark of virus adaptation to animals [ ] . among all the arthropod-borne viruses infecting vertebrates, arthropodborne reoviruses are the only non-enveloped viruses. based on these results and the recent literature on picornavirus, polyomavirus, calicivirus and rotavirus [ , , , ] , it is tempting to suggest that the vesicular transmission could be another example of convergent evolution for a non-enveloped virus adapting to animals. in this study, we demonstrated that the majority of btv virus particles are clustered in evs, secreted by both mammalian and insect cells. despite virus released in secretory lysosomes the absence of envelope, our results highlight the presence of a lipid bilayer protecting virus particles and providing a more efficient infection initiation compared with free virus particles infection. it has been reported that hepatitis a virus and hepatitis e virus exit cells inside exosomes derived from the mvbs [ , , ] , that are characterised by the presence of endosomal compartments and vesicular transport markers, such as flotillin , cd or members of the escrt protein family. these exosomes are around - nm in size [ ] . in contrast, it has been shown that coxsackievirus b and poliovirus are released in vesicles of approximately nm in diameter, through an autophagy-mediated mechanism [ , , ] . moreover, a recent study reported that rotavirus egress cells in evs derived from the plasma membrane, independently of mvbs and autophagy, and that norovirus are release in exosomes [ ] . a clear picture of different mechanisms used by non-enveloped virus for secretion in evs is lacking. our study contributes to understand better the diversity of release mechanisms. we showed that the lysosomal markers lamp and cathepsin are present in infectious evs, in addition to the mvb markers tsg and hsp . based on our results, it is clear that mvbs are involved in the release of btv in evs, because of the presence of the tsg in these evs, the co-localisation of tsg with viral proteins in infected cells, and the deleterious effect of geldanamycin on the secretion of evs containing virus. however, based on the size of these evs, the failure of inhibition by gw , the exosome inhibitor, and the high level of infectious evs released by the btv ns gaap mutant (unable to interact with tsg [ , ] ), we conclude that the evs release by our ovine cells cannot be exosomes. however, the presence of lysosomal markers in the evs suggests that they could originate from the fusion between mvbs and autophagosomes that lead to the recently discovered secretory amphisome [ ] , virus particles could be subsequently exported in lysosomes after the amphisomes-lysosomes fusion. supporting this mechanism, we observed the presence of the lysosomal cathepsin proteins in the same low-density fractions as the viral particles after isopycnic density centrifugation, as well as co-localisation of the lysosomal markers lamp and cathepsin with the viral proteins. interestingly, the btv ns m virus, which has a mutated first start codon and is only able to synthesis an ns a isoform of ns (lacking the n-terminal amino-acids) [ ] , released significantly less virus particles in evs than btv wt . conversely, the btv ns m mutant (with a mutation in the second start codon preventing the synthesis of the ns a isoform) showed comparable levels of infectious evs to the btv wt . we did not observe a change in the co-localisation of vp or ns with the lysosomal markers in cells infected with btv ns m compared with btv wt . in contrast, we found that lysosome acidity was higher in non-infected cells and cells infected with btv ns m , compared with btv wt . this suggests that ns m is unable to counter lysosomal acidification as shown for wt ns . we were able to revert this phenotype using bafilomycin a , an inhibitor of the vacuolar type h+-atpase, and showed that lysosomal ph disruption in btv ns m infected cells restored the secretion of btv particles in evs. this phenomenon suggests that btv infection induces lysosomal disfunctions similar to what is observed in lysosomal storage diseases, commonly associated with an increase of lysosome biogenesis, an increased lamp expression at the cell surface, as well as lysosome enlargement [ ] [ ] [ ] . similarly, the levels of free calcium in infected cells were lower in non-infected cells and in cells infected with btv ns m , compared with btv wt. interestingly, it has been proposed that the endoplasmic reticulum, in close spatial association with lysosomes, regulates the calcium storage in these organelle [ , ] . we previously showed that ns transits though the endoplasmic reticulum (er), and that disrupting ns trafficking through the er causes release of immature particles. it would be thus interesting to explore the interplay between ns , the er and lysosomes in order to determine how ns could regulate the secretion of btv particles in evs. altogether, our data support an original model of a virus particles release mechanism involving mvbs and secretory lysosomes. interestingly, this phenomenon is not restricted to viruses, as it has also been described for intracellular uropathogenic e. coli [ ] . related with our results, a recent preprint describes how beta-coronaviruses, which are enveloped viruses, could exploit the lysosomes for virus egress by disrupting lysosomal acidification [ ] . these results, and ours, reveal a potentially unanticipated wide diversity of egress mechanism using lysosomes by different viral families. additionally, we also showed that evs containing virus particles offer an efficient platform for entry and infection of naive cells. however, we observed that btv infection triggers a superinfection exclusion phenomenon for evs containing virus particles, whereas free virus particles can overcome it, underlying the importance of both viral forms during btv cycle. previously, we showed btv vp , the outer capsid spike binds sialic acid, a likely receptor for the free virus particles [ ] . however, reports from others showed that infection with virus particles cloaked in evs did not necessarily rely on their known virus receptor [ , , , ] . here, we discovered evidences of a superinfection exclusion mechanism during btv infectious, supporting the existence of a specific ev receptor. we observed that only free virus particles could enter in cells already infected, suggesting that evs and free virus particles use different entry routes. in such a scenario, the receptor is not required to interact with viral outer capsid proteins, as ev secretion is already an efficient cell-to-cell communication system [ ] . however, the difference in virus tropism would certainly have consequences on the virus pathogenicity. it is thus necessary to study the impact of infectious evs on btv induced pathogenicity, as infected ruminants variably respond to btv infection, with cattle being mainly asymptomatic and sheep exhibiting more severe clinical signs, such as haemorrhage and ulcer in the gastro-intestinal tract or muscle necrosis [ ] . in natural hosts, btv transmission requires blood feeding midges (culicoides species) to take a blood meal from an infected animal. our results demonstrated that culicoides cells, for which btv is not cytopathic, also secrets evs containing virus particles. it would be thus interesting to investigate whether the uptake of evs secreted by infected insects and evs secreted by infected mammals can infect in mammals or insects respectively. in our electron microscopy study of the viruses present in the k pellet we observed the free virus fraction to be heterogeneous by electron microscopy. the observed particles were either naked or aggregated and enveloped in a lipid membrane. it is likely that this membrane is transient and acquired by the budding of individual virions at the plasma membrane, as described previously [ , ] . however, further investigation will be necessary to understand the importance of this heterogeneity in the virus life cycle. overall, the findings described here highlight an original aspect of virus release leading to the secretion of a non-enveloped virus in host-derived secretory lysosomes, of which the acidic ph is neutralized upon the infection. such mechanism is distinct to the proposed mechanism for the release of rotavirus in evs [ ] , suggesting a divergent evolution of insect-borne double-stranded rna viruses. investigation of host-pathogen interactions for insect-borne viruses can significantly enhance our knowledge on the diversity of virus-containing extracellular vesicles origins. this may highlight different cellular tropisms and immunity evasion strategies between the free and vesicular forms of non-enveloped viruses and can have important implications for the development of innovative approaches for virus control. virus released in secretory lysosomes supplemented with % fcs and antibiotics ( units/ml penicillin, mg/ml streptomycin, gibco, life technologies). mammalian cells were incubated at ˚c in a humidified % co incubator and insect cells at ˚c. btv- and btvunag (with a segment encoding a vp -unag fusion protein) stocks were obtained by infecting sheep cells at a multiplicity of plaque forming units per cell (pfu/cell) for min under gentle agitation at room temperature (rt). the inoculum was then removed, and cells were maintained for h in dmem with % fcs for h at ˚c. the harvested supernatants were clarified ( min at xg) twice, and stored at ˚c. all the ns mutants were described in previous studies [ , , ] . stocks for these mutants were amplified as described above for btv wt , and a sanger sequencing of ns confirmed the presence of all studied mutations. viral infectivity was estimated using tissue culture infectious dose of per ml (tcid /ml) titration, for which viral suspensions were diluted from − to − in d-mem medium and inoculated to bsr cells in -well plates. titres were calculated according to the spearman-karber method. for analysis of replication, sheep cells were inoculated with purified evs or free virus particles in -well plate (moi = ) for h at rt under gentle rotation. the inoculum was then removed and replaced by d-mem ( % fcs). cell culture media was harvested at , , and hpi, clarified by centrifugation ( xg- min), and infectivity was quantified by a tcid method. pt infected cells were lysed by three cycle of freeze thawing. lysed cells were resuspended in pbs and cell debris was spun down at g for min. viral rna extraction was then performed on the harvested supernatant as indicated below. extraction of the viral rna was performed with a qiamp viral rna mini kit (qiagen), followed by a reverse transcription using a universal degenerated oligo specific to all non-coding sequences of btv- segments (btv/uni ; gttaaawhdb ) and the goscript reverse transcription (rt) system (promega, madison, wi, usa). the cdnas obtained for the segment were then quantified with a real time quantitative pcr (qpcr), using the x sybr green qpcr master mix (bimake, houston, tx, usa) and segment specific primers (btv s / f, gacgccagagataccttttac ; btv s / r, cttgaatca tatccggaccac ) or segment specific primers (btv s / f, ' cattcgcatcg-tacgcagaa '; btv s / r, ' gcttaaacgccacgctcata '). for absolute quantification of the cdna copy numbers, a standard curve was produced using a -fold serial dilution of a pt -s plasmid of known concentration as a template for amplification. evs were isolated from infected cells cultured in dmem % fcs (mammalian cells) or schneider s insect medium ( % fcs), except for the analysis of protein markers in evs by western blot and immunofluorescence, for which cells were cultured in % fcs medium in order to avoid detection of evs present in the fcs. to identify the optimal time point for cell culture supernatant harvesting, corresponding to the highest possible viral titre with the lowest cell mortality, we used a cell viability imaging kit based on the detection of total cells number (using dapi staining) versus cells with a compromised plasma membranes (r , thermofisher scientific). following harvesting of the, evs were purified from free virus particles using a differential centrifugation protocol adapted from one used on encephalomyocarditis virus [ ] . cell debris was first removed by two steps of low speed centrifugation at xg for min. large evs virus released in secretory lysosomes were pelleted by centrifuging initial volumes of . ml at , xg for min. free virus particles remained in the supernatant, and pelleted evs were resuspended in either μl or μl volumes of d-mem without fcs, depending on the concentration needed for the subsequent analysis. for all quantitative comparisons with free viruses, a correction factors were applied if the ev resuspension volumes were not equivalent to the volume prior centrifugation. when necessary, the , xg centrifugation was performed for h at ˚c using a sw ti swinging bucket rotor (beckman coulter). the pellet was then resuspended in μl or μl of d-mem for subsequent analysis. evs purification by isopycnic gradient centrifugation was performed at , xg for hours at ˚c, using a sw ti swinging bucket rotor (beckman coulter). evs resuspended in μl d-mem and diluted with μl iodixanol ( %) to a final % concentration. evs were layered on top of a - % iodixanol discontinue gradient and harvested in fractions, from the bottom of the tube using a syringe. fraction density was calculated by measuring the absorbance at nm after a : , dilution and using a standard curve obtained by serial dilution of iodixanol. specific infectivity of btv in evs and free virus fractions correspond to the number of particles required to infect a cell. we approximated the particles/ml as the genome copy number/ ml measured by qpcr using s specific primers after extraction and reverse transcription of the viral genome. the number of pfu/ml was obtained by virus titration using a plaque assay. proteins were detected from either resuspended evs or cell lysates. cell monolayers were washed twice with a pbs solution and lysed for min at ˚c with a ripa lysis buffer, x protease inhibitor cocktail, and mm edta ( , thermofisher scientific). cell lysates, or evs, were diluted in x nupage lds sample buffer (thermofisher scientific) to a final x concentration and incubated at ˚c for min. proteins were then separated on a % sds gel and blotted on a pvdf membrane. when indicated, the membranes were cut horizontally for incubation with different antibodies. the anti vp , vp and vp antibodies (made in the pr. roy's laboratory [ ] ), anti lc b (nb - , novus bio), anti annexin a ( , bd bioscience), anti tsg (t , sigma aldrich), anti lamp (ab , abcam) and anti pan-cathepsin (sc- , santa cruz biotechnology), were incubated overnight at ˚c, followed by a hours incubation with respective horseradish peroxidase-coupled secondary antibody (anti-mouse igg ab , anti-guinea pig igg ab and anti-rabbit igg ab , abcam). luminescence was then detected using supersignal west pico plus chemiluminescent substrate (thermo fisher scientific). the imagej software was used to perform linear contrast enhancement. to image evs without sectioning, purified evs were resuspended in d-mem, adsorbed onto a carbon-coated copper grid, and stained with a % solution of uranyl acetate. for imaging of evs sections, purified evs were resuspended in μl d-mem ( % fcs) and mixed with a low volume of glutaraldehyde % (final concentration = . %) ( , electron microscopy sciences). after min of fixation at rt, evs were briefly warmed to ˚c, and μl of evs virus released in secretory lysosomes were dropped on μl of pre-melted low-melting agar ( % in water) and kept at ˚c before the addition of evs. samples were quickly mixed by pipetting and stored at ˚c before sectioning. after post-fixation in % osmium tetroxide- . % sodium cacodylate, evs were dehydrated in increasing concentrations of ethanol and embedded in epoxy resin (taab laboratories equipment ltd., aldermaston, united kingdom). ultrathin sections were stained with reynolds lead citrate and mounted onto nickel grids. images were visualised with a transmission electron microscope (jem- , jeol akishima, tokyo, japan). sheep cells in well-plates were infected (moi = ) with btv- , hours post seeding. viral or mock inoculum were incubated min at rt under gentle rotation. the inoculum was then removed and replaced by d-mem ( % fcs), and cells were incubated at ˚c in a humidified incubator ( % co ). at hpi, cell culture medium was replaced with d-mem containing mm -methyladenine (m , sigma aldrich), nm geldanamycin (invivo-gen), μm chloroquine (c , sigma aldrich), μm gw (d , sigma aldrich), μm bafilomycin a (sml , sigma aldrich), so that drugs did not interfere with viral entry. these concentrations were selected because they were already validated by previous studies on btv from us and others [ , , [ ] [ ] [ ] . at hpi, cell supernatants were harvested for isolation of evs and analysis of infectivity. for intracellular virus particle quantification in presence or absence of the different drugs, the media culture of pt infected cells was removed at hpi, and cell monolayers were rinsed twice with pbs. cells were then lysed by successive cycles of freeze/thaw at - ˚c/ ˚c. cell lysates were then resuspended in μl dmem and virus particles were quantified using a tcid method. cell surface protein expression of lamp was measured by immunofluorescence using flow cytometry. pt cells were seeded in -well plates for h before infection with btv- (moi = ). after hpi, cell monolayers were washed twice with pbs, re-suspended using trypsin-edta (thermo fisher scientific). cells used for analysing the lamp cytosolic content were fixed with paraformaldehyde % (w/v) for min and permeabilised with a pbs-triton x ( . % v/v) solution for min. the cells were then blocked using bovine serum albumin (pbs-bsa %) for min and incubated with rabbit anti lamp (ab , abcam) targeting the luminal domain, or an isotope control antibody (ab , abcam) for hours at room temperature. cells used for analysing the cell surface lamp content were labelled with the anti lamp antibody for hours at ˚c prior fixation. then all labelled cells were incubated for h at rt in the presence of a species-specific a conjugated secondary antibodies (a , thermo fisher scientific). after centrifugation and washing, cells were re-suspended in pbs and analysed with a bd lsr ii flow cytometer (bd biosciences, san jose, ca, usa). for each replicate, , cells were analysed using the same parameters. flow cytometry data were analysed with flowjo (flowjo llc, ashland, or, usa) software. for the analysis, a gating strategy discriminating doublet cells by plotting fsc-a vs. fsc-h was used, and the threshold of background fluorescence was determined using cells labelled with the isotope control antibody (s c fig). for imaging, evs were resuspended in d-mem ( % fcs) + fluorescent bodipy phosphocholine analogue at μg/ml (d , thermofisher scientific) and incubated overnight at ˚c. evs were then dropped on poly-l-lysine (p , sigma aldrich) pre-coated chambered # . borosilicate coverslips (nunc lab-tek , thermofisher scientific), and adsorbed for hour at ˚c. the residual medium was removed, and attached evs were fixed with pbs-pfa ( %) for min at rt, rinsed with pbs and permeabilised with pbs-triton x ( . %) for min at rt. evs were then rinsed twice with pbs, incubated for hour at rt with a solution of pbs-bsa ( %), and incubated overnight at ˚c with primary antibodies anti vp and ns (made in pr. roy's laboratory), followed by fluorophore a and a -conjugated antibodies (a and a , thermofisher scientific) for hours at rt and mounting with aqueous mounting medium (f , sigma aldrich). for sheep cells imaging, cells were cultured in chambered tissue culture treated polymer coverslips (μ-slide angiogenesis or μ-slide well , ibidi), infected with btv- (moi = ) h post-seeding, and at hpi, inoculated with a baculovirus encoding lamp -gfp at concentration of particles / cell (celllight lysosomes-rfp, bacmam . , thermofisher scientific). at hpi (unless specified), cell monolayers were rinsed twice in pbs, fixed in pbs-pfa ( %) for min, permeabilised in pbs-triton x ( . %) for min, incubated in pbs-bsa ( %) for min. cells were labelled with primary antibodies anti hsp ( - -ap, proteintech), anti pan-cathepsin (sc- , santa cruz biotechnology), anti vp and anti ns overnight at ˚c, and labelled with alexa fluor a , a and a -conjugated antibodies (a , a and a , thermofisher scientific) for hours at rt. nuclei were labelled with a pbs-hoescht solution at μg/ml for min at rt and samples were finally mounted in mounting medium (f , sigma aldrich). labelled cells and evs were analysed with an inverted lsm confocal microscope with airyscan (carl zeiss ltd.), or with an inverted widefield nikon ti eclipse microscope (nikon) for the vibs surface analysis. for the co-localisation analysis, three independent replicates (with more than cells imaged for each replicate) were analysed with a statistical object distance analysis (soda, icy software) on the whole cells [ ] . after infection with btv- (moi = ), cells were incubated at rt under gentle rotation for min, before the inoculum was replaced with d-mem containing baculovirus expressing lamp -gfp at concentration of particles / cell (celllight lysosomes-rfp, bacmam . , thermofisher scientific). at hpi, cells were incubated with hoescht at μg/ml for min at rt, followed by an acidic compartment marker nm (lysotracker dnd , thermofisher scientific) for min at rt. cells were then washed twice with a pbs solution and analysed with a lsm confocal microscope. images were analysed with icy (https://icy.bioimageanalysis. org). cells were counted by detection of stained nuclei and lysosomes by detection of the fusion lamp -gfp fluorescent protein. each lysosome detected was considered as a region of interest, and lysotracker fluorescence intensity detected in these regions were then quantified. after infection with btv (moi = ), cells were incubated at rt under gentle rotation for min, before the inoculum was replaced with d-mem. at hpi, cells were incubated with hoescht at μg/ml for min at rt, followed by incubation with the fluorescent labelling reagent fluo- am (at μm in d-mem) for the detection of intracellular calcium (gr , abcam) for hour at ˚c. fluo- am/d-mem solution was then replaced by d-mem only to remove any non-specific binding, and left a further min at ˚c, followed by two rinsing with a pbs solution and imaged with a lsm confocal microscope (fluo- am excitation at nm). for image analysis, all cells were detected using the hk mean plug- virus released in secretory lysosomes in in icy (https://icy.bioimageanalysis.org) applied on the fluo- channel, and detected cells were considered as regions of interest in which the fluo- am intensity was then measured. cells were infected and fixed at different times post infection as above, and labelled with an anti ns (made in pr. roy laboratory) antibody, a secondary alexa fluor a -conjugated antibody (a , thermofisher scientific), and a -conjugated wheat germ agglutinin as a membrane label (w , thermofisher scientific) for min at rt prior cells permeabilisation. microscopy images were analysed with icy, and vibs were detected using the spot detector plugin, which provided ns spot surfaces. cell counting was performed based on the number of nuclei detected in images. for data analysis, a cut-off was applied to keep all detected ns spots with a surface above μm . the frequency distribution of vibs surface values were performed with a bin width of μm and the lognormal regression of the frequency distribution of the number of vibs per cells were performed after confirming that data were following a lognormal distribution. the total number of vibs measured at each time points is indicated in the s table. for superinfection exclusion analysis, sheep cells in well-plates were infected with btv- (btv wt , moi = ) as described above. simultaneously ( hpi), or at , , , or hpi, cells were inoculated with purified evs or free virus particles of btv unag (moi = ) and placed at ˚c in a humidified incubator ( % co ) for hour. btv unag inoculum was then removed and replaced with d-mem ( % fcs). at hpi ( hpi of btv unag infection), cell monolayers were washed twice with pbs, re-suspended using trypsin-edta (thermo fisher scientific), fixed with paraformaldehyde % (w/v) for min, permeabilised with a solution of pbs-triton x ( . % v/v) for min, and blocked with a pbs-bsa ( %) solution for min. the cells were then incubated with an anti vp antibody (made in pr. roy's laboratory) overnight at ˚c, followed by an incubation of h at rt in the presence of a species-specific alexa fluor a conjugated secondary antibodies (a , thermo fisher scientific). after centrifugation and washings, cells were resuspended in pbs and analysed with a bd lsr ii flow cytometer (bd biosciences, san jose, ca, usa). for each replicate, , cells were analysed using the same parameters. the detection of alexa fluor fluorescence corresponded to the total vp detected in infected cells, and the unag detected fluorescence to vp expressed by btv unag infection only. numerical data were analysed with python (v . . ), the scipy library (v . . ) and prism software version . . (graph pad software, san diego, ca, usa). for all sets of data, two-tailed t-tests or anova tests were performed for statistical comparison. when necessary, outlier values removal was performed using the ellipticenvelope from the scikit-learn library ( . . ). figures were prepared using jupyter notebooks (project jupyter, https://jupyter.org) and inkscape (https://gitlab.com/inkscape/inkscape) cell walls and the convergent evolution of the viral envelope how non-enveloped viruses hijack host machineries to cause infection a pathogenic picornavirus acquires an envelope by hijacking cellular membranes coxsackievirus b exits the host cell in shed microvesicles displaying autophagosomal markers hepatitis e virus egress depends on the exosomal pathway, with secretory exosomes derived from multivesicular bodies nonlytic viral spread enhanced by autophagy components phosphatidylserine vesicles enable efficient en bloc transmission of enteroviruses bk polyomavirus hijacks extracellular vesicles for en bloc transmission vesicle-cloaked virus clusters are optimal units for inter-organismal viral transmission extracellular vesicles: vehicles of en bloc viral transmission cooperation between different variants: a unique potential for virus evolution localization of the non-structural protein ns in bluetongue virus-infected cells a viral nonstructural protein regulates bluetongue virus trafficking and release bluetongue virus nonstructural protein orchestrates virus maturation and drives non-lytic egress via two polybasic motifs influence of cellular trafficking pathway on bluetongue virus infection in ovine cells viral genome segmentation can result from a trade-off between genetic content and particle stability the interplay between exosomes and autophagy-partners in crime regulated lysosomal exocytosis mediates cancer progression a trp channel senses lysosome neutralization by pathogens to trigger their expulsion interaction of calpactin light chain (s a /p ) and a viral ns protein is essential for intracellular trafficking of nonenveloped bluetongue virus nonstructural protein of bluetongue virus assists virus release by recruiting escrt-i protein tsg jc virus infected choroid plexus epithelial cells produce extracellular vesicles that infect glial cells independently of the virus attachment receptor hepatitis a virus structural protein px interacts with alix and promotes the secretion of virions and foreign proteins through exosome-like vesicles extracellular vesicle heterogeneity: subpopulations, isolation techniques, and diverse functions in cancer progression lysosome enlargement during inhibition of the lipid kinase pikfyve proceeds through lysosome coalescence a gene network regulating lysosomal biogenesis and function transcriptional activation of lysosomal exocytosis promotes cellular clearance the endoplasmic reticulum, not the ph gradient, drives calcium refilling of lysosomes lysosomes shape ins( , , )p -evoked ca + signals by selectively sequestering ca + released from the endoplasmic reticulum β-coronaviruses use lysosomal organelles for cellular egress bluetongue virus coat protein vp contains sialic acid-binding domains, and vp resembles enveloped virus fusion proteins extracellular vesicles mediate receptor-independent transmission of novel tick-borne bunyavirus extracellular vesicles: a new communication paradigm? the pathology and pathogenesis of bluetongue persistent infection of bhk /wi- cells with rubella virus and characterization of rubella variants the viral envelope is not sufficient to transfer the unique broad cell tropism of bungowannah virus to a related pestivirus picornavirus infection induces temporal release of multiple extracellular vesicle subsets that differ in molecular composition and infectious potential in vitro reconstitution of bluetongue virus infectious cores hsp chaperones bluetongue virus proteins and prevents proteasomal degradation autophagy activated by bluetongue virus infection plays a positive role in its replication neutral sphingomyelinases control extracellular vesicles budding from the plasma membrane mapping molecular assemblies with fluorescence microscopy and object-based spatial statistics the authors thank eiko matsuo (kobe university, kobe city, japan) for providing the btv u-nag fluorescent virus. we acknowledge microscopy support from mark turmain (the biosciences em core facility, ucl, london uk). the authors have declared that no competing interests exist. key: cord- -bdw ke i authors: guo, hongbo; rabouw, huib; slomp, anne; dai, meiling; van der vegt, floor; van lent, jan w. m.; mcbride, ryan; paulson, james c.; de groot, raoul j.; van kuppeveld, frank j. m.; de vries, erik; de haan, cornelis a. m. title: kinetic analysis of the influenza a virus ha/na balance reveals contribution of na to virus-receptor binding and na-dependent rolling on receptor-containing surfaces date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: bdw ke i interactions of influenza a virus (iav) with sialic acid (sia) receptors determine viral fitness and host tropism. binding to mucus decoy receptors and receptors on epithelial host cells is determined by a receptor-binding hemagglutinin (ha), a receptor-destroying neuraminidase (na) and a complex in vivo receptor-repertoire. the crucial but poorly understood dynamics of these multivalent virus-receptor interactions cannot be properly analyzed using equilibrium binding models and endpoint binding assays. in this study, the use of biolayer interferometric analysis revealed the virtually irreversible nature of iav binding to surfaces coated with synthetic sialosides or engineered sialoglycoproteins in the absence of na activity. in addition to ha, na was shown to be able to contribute to the initial binding rate while catalytically active. virus-receptor binding in turn contributed to receptor cleavage by na. multiple low-affinity ha-sia interactions resulted in overall extremely high avidity but also permitted a dynamic binding mode, in which na activity was driving rolling of virus particles over the receptor-surface. virus dissociation only took place after receptor density of the complete receptor-surface was sufficiently decreased due to na activity of rolling iav particles. the results indicate that in vivo iav particles, after landing on the mucus layer, reside continuously in a receptor-bound state while rolling through the mucus layer and over epithelial cell surfaces driven by the ha-na-receptor balance. quantitative bli analysis enabled functional examination of this balance which governs this dynamic and motile interaction that is expected to be crucial for penetration of the mucus layer and subsequent infection of cells by iav but likely also by other enveloped viruses carrying a receptor-destroying enzyme in addition to a receptor-binding protein. introduction specificity, avidity and dynamics of influenza a virus (iav)-receptor interactions are determining factors in host tropism and pathogenesis. virus attachment to sialic acid (sia) receptors on host cell surfaces and decoy mucins is mediated by hemagglutinin (ha) [ ] [ ] [ ] , while neuraminidase (na) removes receptors by cleaving sias [ ] [ ] [ ] . a precisely tuned functional ha/na balance [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] is required for efficient infection of, and replication in, a specific host. ha and na properties affect host-and cell-specificity but have been studied in much more detail for ha because of the relative lack of accurate na assays [ ] . iavs that infect humans bind preferentially to α , sialosides, having an α , -linkage between sia moieties and the penultimate residue, whereas avian iavs prefer binding to α , -linked sias [ ] [ ] [ ] . cell surface glycan composition, as well as branching, modification and linkage-type of the internal carbohydrate chain residues, is variable between host and cell type and also strongly affects binding affinity (reviewed in [ ] ). nas of avian iavs are highly active and prefer cleavage of α , -linked sialoglycans, while human virus nas cleave α , -and α , -linked sias with lower activity [ ] [ ] [ ] . the ha/na balance is important for initiating iav infection. abundantly sialylated mucins in the mucus layer covering the airway epithelial cells bind ha and may trap iavs before they reach the epithelial cells [ , ] . this needs to be counteracted by na activity sufficiently matching ha binding specificity. this is also required to prevent the virus-trapping at sites on the cell surface that do not support efficient endocytosis [ , ] . na activity-dependent de-sialylation of the surface of the infected cell and the virus envelope glycoproteins facilitates budding and release of new virus particles and prevents virus aggregation [ ] . as a consequence, viral fitness depends on continuous fine-tuning of the ha/na balance during virus evolution. replication of viruses, harboring mismatched pairs of ha and na or propagated in the presence of inhibitory decoy receptors or na inhibitors, could be rescued by adaptive mutations in the ha, na or both proteins [ , , , ] . cross-species transmission events, leading to human pandemics in the past, required adaptions in both ha and na [ , ] . when infecting a novel host species, a non-optimal ha/na balance is for instance adapted by evolving increased na activity as well as decreased binding by ha to the decoy glycan-receptor repertoire on mucins present in the mucus, and which differs between different hosts. [ , [ ] [ ] [ ] . there is no straightforward method to determine the ha/na balance of iavs. hemagglutination of erythrocytes, which harbor poorly defined receptor repertoires, does not well reflect binding avidity and dynamics. current setups of solid phase binding assays (e.g. elisas, glycan arrays) as endpoint binding assays do not handle well the polyvalent aspects of virus binding by masking the dynamics of virus binding, even more so as precise variation of receptor density is difficult. moreover, besides a wide range of synthetic glycans, only very few glycoproteins (mostly fetuin) are being employed in current binding studies, thus limiting studies on the effects of diversity and clustering of glycans as naturally presented on glycoproteins. na activity assays often use soluble substrates poorly reflecting natural sialosides [ , ] . the soluble substrate -( -methylumbelliferyl)-α-d-n-acetylneuraminic acid (munana) can be used to determine the na activity on a soluble substrate but ignores the effects of the virion context on na activity on a receptor-coated surface (e.g. binding of the virion to the polyvalent substrate via its ha). thus, methods that determine the dynamic effects of ha and na activity urgently need improvement to allow integration into a model that accurately provides a quantitative description of the dynamic interaction between iav particles and (decoy) receptors. biolayer interferometry (bli) is increasingly being used for analyzing virus-receptor interactions [ ] [ ] [ ] [ ] , but standard methods for quantifying kinetic parameters describing iavreceptor interactions are lacking. here we have used this label-free real-time binding analysis method to study the dynamics of iav-receptor interactions. our results indicate that the initial virus binding rate is the prime, and physiologically most important kinetic parameter of ha-dependent iav particle binding to synthetic as well as natural glycoprotein receptors. na was shown to contribute to virus binding and to be absolutely required for virus dissociation. in turn, na activity is critically dependent on the ability of virions to bind to a receptor-coated surface. the ha/na balance of different virus-receptor combinations was determined by measuring empirical virus binding and elution parameters. prior to virus elution, na-dependent morphological changes in receptor-associated virus particles were observed as well as rolling of virus particles over a receptor-coated surface in a na activity-dependent manner. bli is a potentially versatile tool to obtain mechanistic and quantitative insight into the dynamics of virus-receptor interaction by real-time recording of virion binding to, and release from, receptor-coated surfaces. these interactions are highly polyvalent by nature and expected to be poorly described by equilibrium binding models. in addition, receptor density and identity are key determinants of the interaction. we therefore established a flexible experimental set-up, using synthetic sialoside receptors as well as n-linked or o-linked sialoglycoproteins which carry sialoglycan receptors attached in the natural linkage-conformation that is encountered in vivo. we first evaluated the applicability of the set-up to a quantitative description of iav-receptor binding kinetics. to assist interpretation of the results, the approximate geometric properties of a streptavidin (sa) bli sensor surface, to which biotinylated glycans or glycoproteins can be tightly attached (k d = − ), and of iav virions are depicted in s fig (fig ) . virus association was performed in the presence of the na inhibitor oseltamivir carboxylate (oc) and the lack of virus dissociation after transfer of sensors to buffer containing oc ( fig a and fig b) showed that virus binding is virtually irreversible (off-rate constant k off % ) due to highly polyvalent interactions between virus particles and the receptor-coated bli sensor surface. pr mtsin is a faster binder than pr cam , (fig a and fig b) , but reached a maximum binding signal of~ nm at the highest concentration only after hours (s a fig and s b fig) . a fully occupied sensor surface theoretically accommodates . e+ spherical virus particles ( nm diameter; s fig and [ ] [ ] [ ] [ ] ). this fitted well with the experimentally determined number of virus particles bound to a maximally loaded sensor as determined by quantification of na activity (s c fig). importantly, sensor-regeneration followed by re-binding of virus from the same well could be consecutively repeated at least times yielding identical curves (s fig) . thus, substantial virus decay during the assay or binding of a limited subset of virus particles with particular properties did not occur. analysis of iav-receptor interactions has predominantly been focused on quantification of an apparent dissociation constant (k d = k off /k on ) by measuring polyvalent binding of virus particles or artificial ha polymers (e.g. by antibody complexation) after a fixed binding time to geometrically poorly characterized receptor-coated surfaces with often unknown receptor density and orientation. however, as virus binding is virtually irreversible, equilibrium binding models do not apply and the binding rate equation ( ) and virus concentration was observed for the fast (pr mtsin ; r = . , p = . ) and the slow binding (pr cam , ; r = . , p = . ) virus ( fig c) . the effect of receptor density on iav association and dissociation rates can be precisely determined as bli provides quantitative recording of receptor loading levels. lowering the receptor density, resulting in less potential ha-receptor contacts between virus and sensor surface, gradually reduced maximum binding levels ( fig d and s d and s e fig) as well as the v obs ( fig e) . remarkably, irreversible binding was observed at any receptor density that supported binding (fig d) . a~ -fold higher fractional receptor loading was required for the weaker binder pr cam , ( . ± . ) than for pr mtsin ( . ± . ) to reach % of the maximum fractional initial binding rate (p< . ) ( fig e) . pr mtsin reached a maximum binding rate at~ % receptor loading whereas the weaker binder pr cam - reached its maximum binding rate close to maximum receptor density. a~ -fold reduction in receptor loading was already sufficient to reduce the v obs from maximal to zero for both viruses. this suggests a narrow margin between the number of interacting ha-receptor pairs, below which binding is undetectable due to high reversibility (high k d ) and above which binding is irreversible (see fig d, no dissociation at any receptor density). we conclude that v obs , which is directly related to virus concentration and receptor density, is the most suitable parameter for quantification of virus binding strength over a range of receptor densities and is likely to reflect in vivo virus binding, which probably occurs to levels very far from saturation. as the initial binding rate is linear with virus concentration, the relative specificity for two receptors (v rel = v obs / v obs ) is independent of virus concentration and the relative-binding specificity of viruses can therefore be quantitatively compared without the need for elaborate virus quantitation procedures. as an example, we quantified the effect on receptor-specificity of amino acid substitution e d in ha of pr which is predicted to cause a shift in binding from α , -to α , -linked sia receptors in h [ ] . indeed glycan array analysis of the ha proteins of lab strains pr cam , and pr cam , , which only differ by substitution d e, showed an absolute specificity shift (fig a) . in contrast, virus binding quantified by bli showed a relative specificity shift (v rel = v 'slnln /v 'slnln ) of . ± . for pr cam , to . ± . for pr cam , ( fig b and fig c) . remarkably, strain wsn ha, which carries e plus some additional mutations in comparison to pr cam , , displayed efficient binding to α , -as well as α , -linked synthetic glycans on the microarray, but wsn wt virus did not bind to synthetic glycans in the bli assay. possibly, soluble ha proteins can have access to short glycans on glycan arrays whereas virus envelope-embedded has cannot, depending on virus strain, bind to the same glycans on bli sensors. to test this, we examined binding to glycoproteins carrying α , -or α , -linked sias on n-linked sialoglycans ( 'n fetuin or 'n transferrin bt; fig d and fig e) . pr cam , virus displayed absolute specificity for 'n fetuin. however, an absolute α , -to α , -linked sia specificity shift by mutation e d, as observed above by glycan array analysis, did not occur , pr cam , or wsn wt was determined by glycan array analysis. binding to mono-, bi-and tri-antennary glycans carrying one, two or three lacnac repeats terminated with a α , -or α , -linked sia is indicated. means of independent replicates are graphed, standard errors of the means are indicated. biotinylated 'slnln or 'slnln for virus binding to glycoproteins (v rel = v 'n fetuin /v 'n transferrin = . ± . for pr cam , ), similar to what was observed for the synthetic glycans ( fig b and fig c) . glycoproteins, in contrast to synthetic glycans, supported wsn wt virus binding to bli sensors which displayed dual binding specificity (v rel = . ± . ) in agreement with the glycan array results for wsn ha proteins. in conclusion, determination of v obs allows quantification of relative receptor specificity differences for virus particles, which is independent of virus concentration (s g fig, s h fig and s i fig) . the application of (tailor-made) glycoproteins enables to elucidate quantitative differences in iav receptor-binding fine specificity, which is a step forward to mimicking virus-receptor interactions that occur in vivo. the versatility of the latter is further illustrated by showing the relative specificity for n-linked versus o-linked glycans, using engineered fetuin constructs (s fig). o-linked glycans are abundantly present on soluble mucins present in respiratory mucus. as an initial experiment to explore potential differences in binding dynamics to o-or n-linked glycans we expressed fetuin variants containing three n-linked and/or three olinked glycans and compared the binding of pr cam , and pr mtsin to these receptors (s a- na activity contributes to the dynamic interaction of virions with receptor-coated surfaces. we first determined whether non-bound virions are able to cleave receptors loaded on bli sensors. we used viruses carrying the same na but a different ha (pr mtsin (fig b) . in the presence of na activity, the binding rate of tx namtsin became increasingly lower in time, presumably reflecting virion release due to ongoing receptor depletion. regeneration of sensors, which removes all virus particles but leaves the biotinylated glycans on the sensors, was followed by a second round of tx namtsin binding in order to determine the extent of de-sialylation that occurred in the first round of binding ( fig c) . only tx namtsin binding in the first round in absence of oc led to receptor desialylation as detected by inefficient binding of tx namtsin in the second round. in contrast, α , specific pr mtsin did not bind to the sensor and thereby could not remove sias, as shown by efficient re-binding of tx namtsin upon regeneration. we conclude that non-bound virions do not contribute to sialidase activity and do not have to be taken into account when studying the effect of virion-associated na activity on iav receptor binding dynamics by bli. we determined the quantitative effect of na activity on the dynamics of iav-receptor interactions of two viruses carrying the same ha, but different na proteins (wsn hamtsin and pr mtsin ; s e fig) . whereas the viruses displayed similar binding to both 'n fetuin and 'sln when na was inhibited (fig a and d ), only pr mtsin binding was strongly reduced by its associated na activity (fig b and e ). this observation corresponded well to thẽ -fold lower na activity of wsn hamtsin per virus particle (s i fig and s j fig) . we conclude that, in addition to ha binding properties, na activity drastically affects virus binding dynamics in the absence of na inhibitors. the binding curves reflect the ha/na balance which is quantifiable by empirical parameters like initial binding rate, the area under the curve (unit is min nm) and x,y coordinates of the peak (time and binding level). na activity-dependent self-elution of viruses bound in presence of oc provides quantification of other descriptive parameters that characterize the balance between ha, na and receptor. of note, the concentration of released particles is too low for their re-association to take place. as expected, pr mtsin eluted much faster from 'n fetuin or 'sln than wsn hamt-sin (which does not elute from 'sln). for both viruses the self-elution rate from 'n fetuin was higher than from 'sln ( fig c and fig f) . this likely results from differences in specific na-dependent influenza a virus rolling on receptor surfaces activity of the nas for these receptors but, as ha and na are in competition for binding/cleavage of the same receptor, might also be affected by the k d of monovalent ha-receptor interactions and thus be a reflection of ha/na balance on a specific receptor. this possibility is further corroborated by an experiment where we compared the elution rates from 'n fetuin and 'n transferrin bt for two viruses (pr cam , and wsn wt ) carrying the same na (from wsn wt ) but a different ha (s fig). both viruses bind at similar, but relatively low, rate to 'n transferrin bt ( fig e) whereas wsn wt displays a~ -fold faster binding rate to 'n fetuin than pr cam , ( fig d) . ha clearly affects the self-elution rate as, in combination with the same na, self-elution from 'n fetuin is much more efficient in companion of the weaker binding ha of pr cam , (s b fig thus, whereas α , sia versus α , sia specificity of na is seemingly opposite for pr cam , and wsn wt , this is not caused by the na itself (which is identical for both viruses) but by differences in their has. also a change of receptor (in this case to fucosylated 'sln, giving sialyl-lewis x ; sle x ) was shown to have differential effect on the observed binding rate ( fig g) and na-driven self-elution ( fig h and i ). while v obs is similar for sle x and 'sln in the absence of na activity ( fig g) , an active na resulted in a lower v obs and maximum binding level and a smaller area under the curve for 'sln ( fig h) and in a faster virion self-elution from 'sln ( fig i) . thus, receptor binding dynamics on 'sln and sle x differ due to a lower specific na activity towards sle x which shifts the relative ha/na balance on these receptors. in conclusion, bli can be used to quantify changes in the dynamics of receptor binding due to an altered ha/ na/receptor balance. in a small increase in reflection resulting in negative virus dissociation values, which cannot be explained by additional virus particle binding, was consistently observed during the initial phase of self-elution (e.g. fig c, f and i). the effect was most prominent when na activity was low. the possible role of na activity herein was further examined by performing self-elution of pr mtsin from 'sln and wsn hamtsin from 'n fetuin in the presence of an oseltamivir concentration range (fig a and b ). duration and magnitude (maximally~ . nm) of the increase in the apparent binding level, which was not observed when na was fully inhibited, depended on the degree of na activity. the effect must reflect a neuraminidase activitydependent change in the receptor-bound virus particles as free virus particles that can bind are absent. cleavage of sias by na is expected to gradually decrease the number of ha-sia contacts between receptor-coated surface and virus until the particle dissociates. relaxation of virus particle binding (by less contacts) could result in an altered binding conformation of a particle that, at the maximal number of contacts, might be tightly squeezed against the receptor surface. such a morphological change likely explains the observed, na-activity dependent, changes in reflection. remarkably, the initial binding rate of wsn hamtsin to 'n fetuin was higher in absence than in presence of a high concentration of oc (compare fig a and b ). several avian na genotypes have been shown to possess a nd sia-binding site, alternatively referred to as hemadsorption site [ , ] , but such a site is not conserved in wsn na and oc binding to this site has never been demonstrated. however, active site mutations that abolish catalytic na activity have resulted in na-dependent hemagglutination, which could be inhibited by oc [ , ] . we therefore examined the effect of oc on binding of wsn hamtsin (low na activity) and pr mtsin (high na activity) virions to 'sln and 'n fetuin. complete na inhibition gave binding curves displaying a continuous increase of virus binding (fig c- f , blue lines). however, the v obs of wsn hamtsin increased at lower oc concentrations in a concentrationdependent way (fig d and fig f) . in time, the curves bent down by depletion of sia receptors due to na activity. in contrast, the v obs of pr mtsin , which has a highly active na in comparison to wsn hamtsin , directly decreased strongly at lower concentrations of oc ( fig c and fig e) . the results imply that nas with a relatively low cleavage rate (low k cat ) contribute to the v obs by binding with their active site to the sia receptor. to strengthen this conclusion, we quantified the enhancement of the initial binding rate by wsn wt viruses carrying the same na but four different has ( fig g and s fig) . whereas all four viruses displayed an effect of na on v obs , the virus with the lowest ha-dependent initial binding rate (pr cam , ), was relatively most enhanced in initial binding rate by na-dependent binding (s e fig and s f fig) . this demonstrates that the degree of the contribution of na to v obs is influenced by its ha partner. in conclusion, the na protein contributes to the initial binding rate balance by binding with its active site to sia receptors and the magnitude of the effect is probably influenced by the ha/na balance. all four viruses showed faster dissociation when more virus was loaded, indicating that there is positive cooperativity between viruses in respect to self-elution rate. the only plausible mechanism by which viruses can assist each other in self-elution is by exerting na activity on a larger surface area than their own contact area, implicating that virus particles move over the receptor-coated surface. to test this hypothesis a concentration series ( . pm to pm) of pr mtsin, harboring a highly active na, was bound to 'n fetuin in presence of oc resulting in virus saturation levels on the sensor surface ranging from~ % to~ % (fig e) , based on a maximal binding level of nm after prolonged binding (s a fig). subsequent na-dependent self-elution ( fig f) confirmed that higher virus loading levels result in faster self-elution as shown by plotting fractional virus dissociation against time (fig g) . we next determined whether virus particles are ) for minutes in presence of μm oc after which the sensors were washed in pbs and dissociation was examined at a range of oc concentrations as indicated in the panels. (c-f) pr mtsin (c, e) and wsn hamtsin (d, f), carrying the same ha mtsin but either na mtsin (high na activity) or na wsn (low na activity), respectively, were bound at pm concentration to biotinylated 'sln (c, d) or fc-tagged 'n fetuin (e, f) loaded to maximum level. binding was performed in absence (red lines) or in the presence of a range of oc concentrations as indicated in the panels. significant differences between initial binding curves shown in panels c-f were analyzed by ibm spss statistic . in panel c, there was no significant difference between curves with μm oc and nm oc (p> . ), whereas the curves of μm oc and nm oc were significantly different from nm, . nm and nm (p< . ). in panel d, the curves of μm oc and nm were significantly different from those of nm, . nm and nm. there also was a significant difference between nm and . nm. in panel e, the nm curves significantly differed from the other three oc concentrations. in panel f, there were significant differences between the nm (and nm) and . nm and nm curves. (g) the ratio (initial virus binding rate v obs in absence of oc)/(initial virus binding rate v obs in presence of μm oc) of four viruses carrying the wsn wt na in the background four different has was plotted (the individual binding curves are shown in s fig) . standard deviations and significant differences between the mean initial binding rate ratios are indicated ( à indicates p< . ). indeed able to clear sias from a larger surface area than their own contact area during self-elution. after self-elution, sensors were regenerated and re-bound with a high concentration ( pm) of virus ( fig h) . clearly, even at a level where only~ % of the surface was bound with virus, self-elution created a surface to which only very limited re-binding of virus particles could take place indicating extensive removal of sias from the complete surface. as the concentration of virus released from the surface is too low for re-association and non-bound virus does not contribute to receptor cleavage, we conclude that virus particles exert na activity while moving over the receptor-coated surface. migration of attached iav particles over a receptor surface necessarily depends on the very high k d of monovalent ha-sia interactions resulting in their rapid formation and dissociation. we hypothesize that na, in combination with the highly dynamic formation and release of individual ha-sia interactions, drives virus rolling by the generation of a receptor gradient due to its receptor destroying activity. rolling of virus particles is predicted to be faster for virus particles with higher na activity. this was tested by comparing the binding and dissociation dynamics of pr mtsin and wsn hamtsin , which have the same ha but have high and low na activity, respectively. the experimental set-up shown in fig a links sensors to bli graphs by color coding. first (fig b) , biotinylated 'n+o fetuin -coated sensors were loaded with wsn hamtsin to a~ % saturation level in the presence of oc (blue and red) or incubated in buffer (green). in the next step (fig c) , these sensors were incubated with pr mtsin in the presence or in the absence of oc. in presence of oc (blue) efficient binding of pr mtsin to the large virus-free area takes place. in the absence of oc, high na activity prevents long na-dependent influenza a virus rolling on receptor surfaces lasting binding of pr mtsin to an empty sensor (green). the slightly negative slope of the wsn hamtsin loaded sensor (red) represents minor dissociation of wsn hamtsin in absence of oc, as expected on basis of its low na activity. this also implicates that wsn hamtsin elution is not appreciably assisted by pr mtsin . in the last step of the experiment (fig d) , the sensors were regenerated to remove all bound viruses followed by probing the residual sia content by its association capacity with wsn hamtsin in the presence of oc. as expected, the blue sensor, to which both viruses were bound in presence of oc, was as efficiently bound by wsn hamtsin as in the first round. also as expected, the green sensor to which no virus was bound in the first step and pr mtsin in absence of oc in the second step was efficiently cleared from sias, as demonstrated by its poor capacity to re-bind to wsn hamtsin . in contrast, binding of wsn hamtsin to~ % of sensor surface in the first step (red) prevented complete removal of sias by pr mtsin in the second step as demonstrated by considerable re-binding of wsn hamt-sin in the third step (red). this implies that wsn hamtsin , by marginal rolling over the surface because of its low na activity, protects its contact surface with the sensor against the na activity of pr mtsin , which cleaves sias of all the non-protected surface area. we conclude that na activity is the driver of virus rolling on a receptor-coated surface. the ménage a trois between iav ha, na and (decoy) receptors largely determines viral fitness and host cell tropism. here, by studying their highly dynamic but poorly understood interplay using bli, we obtained novel mechanistic and quantitative insights into iav-host interactions. we combined the key findings into a schematic model (fig ) . the initial monovalent ha-sia interaction is virus concentration-dependent and governed by a binding rate constant k on (m − s − ) and a dissociation constant k off (s − ). its high k d (k on /k off % . to~ mm [ ] [ ] [ ] cannot be easily determined and makes monovalent virus-receptor interactions undetectable by bli too. in combination with picomolar virus concentrations, this high k d inevitably results in the observed low virus binding rate. subsequent ha binding steps are intramolecular (determined by a different k on and k off with the unit s − ), resulting in multivalent binding. remarkably, and counterintuitive to its receptor destroying activity, na can contribute to the initial binding rate. receptor residence time in the na substrate binding site is determined by the binding constant (k d = k on /k off , range is μm~ μm [ ] and catalytic rate constant (k cat ). obviously, a low k cat promotes the chance on secondary binding events (mostly of ha), which will also be affected by the na/ha virus incorporation ratio (s k fig) . bli-detectable virus binding is, due to multivalency, virtually irreversible but highly dynamic as individual ha-and na-sia interactions are rapidly formed and broken in a virus concentration independent mode. the number of simultaneous interactions required for virtually irreversible (k off % ) binding is low and logically depends on receptor density and the k off and k on values of a virus (fig e) . the rapid interconversion of binding states via the association/dissociation events shown in fig enables a virus to roll over the surface. na activity (curled arrows) is required to drive rolling, most likely by creating a receptor gradient that forces a virus to roll away from the empty receptor positions. receptor cleavage eventually results in virus dissociation when receptor density becomes too low to support tight multivalent binding. this model clearly sketches important biological as well experimental consequences. quantification of iav-receptor binding usually focuses on determination of the dissociation constant k d . however, equilibrium binding models did not apply, even at low receptor densities, and binding curves were dominated by concentration-independent avidity effects resulting in virtually irreversible binding. the binding curves are in agreement with a random sequential adsorption model [ , ] . in such models irreversible particle binding proceeds to a plateau at~ % occupation of the binding surface (the jamming limit). however, iav particle binding could slowly proceed to complete saturation of the surface (fig and s a fig). we attribute this to virus rolling over the surface, a mechanism by which at an eventually very low rate sufficient space for new binding events is created. current, inherently complex, models for polyvalent binding lack general applicability [ , ] and cannot be used to determine the kinetic constants of the different binding events shown in part of the viral envelope, containing ha (red symbol) and na (blue symbol), and the sensor surface, coated with sia (purple diamond)-containing receptors (r), is shown. kinetically different steps lead to multivalent interaction between iav and a receptor-coated surface. the initial ha-dependent binding event (step in red) is a virus concentration-dependent intermolecular process governed by a binding rate constant k on (with the unit m − s − ) and a dissociation constant k off (with the unit s − ). subsequent ha binding steps (e.g. steps and ) are intramolecular with a k on (not necessarily the same as in the first step) and k off (both with the unit s − ). for iav, having a k d (k off /k on ) of~ . to~ mm for a monovalent ha-receptor interaction, binding at pm concentrations inevitably results in a low binding rate that is mostly determined by the first binding event. na can also contribute to the initial binding rate. this contributory effect can be inhibited by oc and is therefore attributed to binding of receptor to a na catalytic site. the contribution of na to receptor binding is determined by a dissociation constant (k d = k off /k off ) for the substrate (step i in blue) and a catalytic rate constant (k cat ; bold blue arrow) determining the receptor cleavage rate. a lower k cat will result in prolonged receptor binding before cleavage or dissociation takes place. this will enhance the chance for additional binding events (mostly by ha, which is present at higher density than na), thereby promoting the cascade of multivalent interactions responsible for tight virus binding. given the lower k d ( μm~ μm) [ ] of na, in comparison to ha, for interaction with sialosides, a considerable contribution to the initial binding rate by na is expected even whereas the na/ha ratio of a virus particle is generally quite low. longer lasting, bli-detectable, binding requires the formation of additional ha-and/or na-sia bonds, which is indicated by the grey shaded area. initial binding events will be hardly detected due to the low levels of equilibrium binding in step and i. during the bli-detectable phase of binding, ha-and na-sia interactions are formed and broken in a virus concentration independent mode with the result that all binding states can rapidly interconvert via binding/dissociation events to and ii to iv. the number of simultaneous interactions that can occur is logically dependent on receptor density and k off /k on ratios but how many simultaneous interactions suffice to keep a virus particle bound to the surface remains unknown. the experiments shown in fig suggest the number of interactions required is very low. theoretically, the dynamics of ha-sia interactions allow a virus to roll over the surface but experiments shown in fig show that na activity strongly stimulates rolling (and eventually leads to virus dissociation). this is schematically indicated by the curled arrows where na cleavage activity creates receptor-free positions on the surface. the receptor gradient caused in this way is probably the driving force for virus rolling but the direction in which the virus rolls (away from the empty position or "reaching over" the empty position) still needs further research. an endpoint binding assay (shown for our data in s f fig) falsely assumes an equilibrium binding model and further errors into apparent k d determinations are introduced by low binding rates (as observed for weak binders or at low virus concentration, s a fig and s b fig) that prevent reaching a binding plateau within min. iav binding to cells is poorly reversible [ ] and adherence of only a few particles is already sufficient for productive cell infection. thus, where equilibrium binding levels or binding to saturation are not an issue, the initial binding rate v obs is a physiologically relevant parameter for iav binding. it can be determined by bli, but not easily by endpoint assays like conventional glycan arrays, more recently developed shotgun glycan arrays [ ] or receptor-coated -well plate based assays. direct proportionality between virus concentration and v obs during the initial binding phase allows quantitative comparison of viruses of unknown concentration by determination of the relative binding rate constants for different receptor pairs. coating of sensors with recombinant glycoproteins, in a homogeneous orientation by virtue of their nterminal tag (e.g. fc-tag, biotinylated bap-tag), provides analysis of receptor surfaces on which the sialoglycans receptors are attached in protein-linked conformations as encountered in vivo. genetic engineering enables binding studies to glycoproteins carrying specific glycantypes (n-linked, o-linked) or glycan-density whereas further tuning of glycan structure can be accomplished by glycoprotein expression in cell lines with specific differences in their glycosyltransferase expression patterns. this will enable a much more refined analysis than the often used biotinylated polyacrylamide molecules carrying randomly distributed glycans and supposed to adopt a spherical configuration with a~ nm diameter [ ] . the recent emergence of h n strains displaying na-dependent hemagglutination hint at a role for na in determining the changes in receptor binding that accompany and/or drive virus evolution. this phenomenon is thought to reflect the gained capacity of na to bind receptors that are refractory to cleavage by na [ , , ] . by using highly sensitive bli assays we showed that the na of strain wsn wt contributes to the initial binding rate even though it is capable of receptor cleavage leading to virus elution. this effect is exerted by binding of the receptor to the na catalytic site, as demonstrated by the inhibitory effect of oc, and is negatively correlated with the specific activity of the na. changes in ha-receptor binding specificity and avidity are thought to be prime factors in causing, or responding to, antigenic change [ , ] or infection of an altered host cell range. secondary changes in na have been proposed to restore a critical ha/na functional balance. now, considering the evidence for a role of na in receptor binding, more complex scenarios should be considered. for instance, changes in na might facilitate (transient) functional changes in ha by (temporarily) contributing to the initial virus binding rate. bli allows quantification of such effects by determination of the relative increase by na of ha-dependent virion binding (fig and s fig). a functional ha/na balance has primarily been described by weighing separately determined ha binding and na activity properties, using isolated proteins or virions. bli enables quantification of the separate contributions of ha and na to virus binding rates. the strength of bli lays in studying the simultaneous effect of na and ha, and thereby their balance, on the dynamics of virus-receptor interactions. virtually irreversible binding is the result of multiple ha-sia interactions that rapidly associate and dissociate, thereby providing access for na to temporarily unbound sias. sia cleavage by na creates a receptor density gradient that drives virus rolling, most likely away from the receptor-free spot in the direction of higher receptor density, or alternatively, by taking "a large step" to a site beyond the de-sialylated receptor. ultimately, when receptor density becomes too low, virus dissociation will occur. the resulting binding/dissociation profiles can be recorded by bli and are a quantitative reflection of the ha/na balance. yet lacking a mathematical model, these profiles can be described by empirical parameters (initial binding rate, area under the curve and x,y coordinates of the peak). kinetic binding rate constants for ha and na (k on and k off ) and the na catalytic rate constant (k cat ) will determine virion rolling characteristics (fig ) . in addition, the ha/na ratio and distribution pattern in the virus envelope will determine the frequency at which na will be present in the contact area between virus and receptor surface where receptor cleavage can take place. as such, ha/na ratio and distribution pattern are additional variables that can be involved in functional evolution of the ha/na balance and its effect on virus rolling. whereas mostly a random distribution of ha over the viral envelope has been observed, the less abundant na has been shown to occur as singular tetramers as well as small clusters thereof. the latter has mostly been observed for iavs with a filamentous morphology [ ] [ ] [ ] . whereas iavs harvested from cell culture usually display a spherical (~ to nm diameter) or slightly pleomorphic shape, filamentous morphology is frequently, but not always, observed in clinical samples (reviewed in [ , ] ) although the loss of a filamentous phenotype is not absolutely required for adaptation to growth in cell culture. filamentous iavs usually have a diameter of to nm and a length up to μm. patches of na clusters at the tip and the base of long filaments have been described [ , ] but other reports suggest the presence of na along the length of the filament [ ] . several functions for a filamentous morphology have been proposed (reviewed in [ , ] ) including a role in clearance of sias from mucus by long budding filaments that have not pinched of from the cell surface [ ] . results obtained in this study are confined to the behavior of spherical iavs and additional studies are required to see if filamentous iavs can move over a surface by lateral rolling, crawling or a caterpillar-like motion. interestingly, unidirectional motility of a filamentous influenza c virus over a receptor-coated glass-slide was recently demonstrated by microscopy [ ] where as a more random movement of spherical iav particles was observed on fetuin-coated glass slides by total internal reflection microscopy. directional movement of virus particles at two different velocities, both dependent on na activity, was reported [ ] . earlier, the combination of a receptor-binding and a receptor-destroying enzyme has been proposed to enable iav movement over a cell surface by a mechanism of repeated cycles of receptor binding, receptor release and receptor cleavage [ ] . spherical and filamentous iav particles probably both occur in vivo and may very well have different functions. whereas na-dependent motility of filamentous particles through a mucus layer might be difficult, they might be better suited for spreading the infection to neighboring cells or clearing the mucus layer from their sia content. spherical iav particles on the other hand, are likely obtained during in vivo infection of humans by inhalation of aerosols containing a relatively low number of virions that somewhere get stranded on the mucus layer covering epithelial cells of the respiratory tract. thus, whereas quantitative measurements of spherical iav binding kinetics by bli should yield valuable data for modeling and testing the movement of such particles through a mucus layer and over epithelial cell surfaces, additional experiments are required to determine whether this can be extended to filamentous iav particles. iav was shown to require na activity for penetrating through a mucus layer in vitro [ ] . the irreversible but highly dynamic binding mode that leads to virus rolling results in virus self-elution from a receptor-coated surface only upon efficient clearance of receptors from the complete surface. considering the low amount of virions confronted by a large amount of mucus and epithelial cells, we propose that virions, once attached to the mucus, remain receptor bound for their entire extracellular life. soluble, densely sialylated mucins in the mucus layer ( - μm thickness) form, by polymerization, a mesh-like structure with an average pore-size of~ nm [ ] through which the virions need to move to reach the pericilliary layer. in this layer, the cilia of columnar epithelial cells ( . to . μm in diameter; to μm in length) are covered by membrane-spanning mucins and tethered muco-polysaccharides that protrude into the narrow (~ nm) space between cilia from which soluble mucins or nm beads were shown to be excluded [ ] . we consider it likely that rolling is necessary for gaining the directionality and even traction required for efficiently penetrating this heavily sialylated maze to reach sites on the epithelial cell membrane that permit virus entry. whether, and to which extent, rolling also takes place over the epithelial cell surface in order to arrive at a site that permits entry by endocytosis remains an open question. the overall density of sias on the cell surface is estimated to be high [ , ] , but also very heterogeneous. iav entry by clathrin-mediated endocytosis was shown to take place by de novo formation of clathrincoated pits at sites where a virus particle was bound [ ] . thus, it seems likely that in case of rolling the virus becomes arrested at a specific place in time. tight binding to specific receptors might be required for this but these have so far not been identified. note that the virus may also surf together with a tightly-bound glycoprotein over the cell surface. rolling might also be important for cell-to-cell virus spread. virions budding from de-sialylated cells, resulting from na activity, may even utilize sialylated mucins covering the epithelial cell layer as a rolling track to neighboring cells. in this perspective, an optimal ha/na balance should be considered as the combination of ha and na kinetic parameters, including factors like ha/na ratio and distribution, that optimally supports virion rolling over distinct surfaces coated with a diversity of receptors. also protective antibodies targeting iav receptor binding sites will function within this context with an important role for the ha/na/receptor balance as they will compete with receptors for interaction with iav [ , ] . the bli-based methodology that we have established here is well-suited to quantify such processes using a highly versatile, modifiable and controllable receptor repertoire. [ ] as well as all other viruses were grown in mdck-ii cells as described previously [ ] and stored at − ˚c. virus titers were determined by measuring the tcid on mdck-ii cells. sequences of the ha and na genes were confirmed by sequence analysis (macrogen). for quantification, virus samples were concentrated by tca precipitation [ ] and applied to standard % sds-page gels for separation of viral proteins followed by silver staining. silver-stained polymerase bands were quantified by densitometry on silver staining gels as outlined and shown in supplementary s a fig, s b fig and s c fig. ha and na amounts were quantified by western blotting of standard % sds/page gels. monoclonal antibodies used for detection and quantification by densitometry were fi (for quantification of ha) [ ] , n - d (for quantification of pr mtsin n ) [ ] and gt (for quantification of wsn n ). recombinant purified ha and na proteins (see below) were used for standard curves (s d- s i fig) . before the application of samples, -mesh copper grids with a pure carbon film were exposed to a glow discharge in air for s to make them hydrophilic. ten microliters of the virus preparations was applied to the grids and incubated for min. excess sample was blotted with a filter paper. for negative staining, μl of % phosphotungstic acid at ph . was applied. after min, excess stain was blotted and grids were left to dry. the specimens were examined in a jeol jem transmission electron microscope at kv and images were taken at a magnification of . x with a matataki k x k camera. human codon-optimized h -encoding and n -encoding cdnas of pr mtsin and wsn wt (accession no. p . for wsn ha, acf . for wsn na, adx . for pr ha, and p . for pr na) were cloned in pcd (ha) or pfrt (na) expression vectors flanked by cd signal peptide-, gcn -isoleucine-zipper trimerization (for ha) or tetramerization (for na) domains, and strep-tag ii-encoding sequences similarly as described previously [ , ] . codon-optimized cdna fragments encoding the variable domains of the heavy and light chains of antibody fi [ ] were synthesized by genscript usa inc and cloned inframe into pcaggs vectors containing human igg heavy and light constant domains, respectively, similarly as described previously [ ] . codon-optimized bos taurus fetuin-encoding cdna (accession no. np_ . ) was cloned in the pcaggs expression vector fused in frame to sequences encoding a human fc-tag with or without a tandem repeat of the -amino-acid biotin acceptor peptide (bap)-tag [ ] . biotinylated fetuin is referred to as fetuin bt. mutations for knocking out o-linked glycosylation sites were introduced into the fetuin gene by using the q site-directed mutagenesis kit (neb) and confirmed by sequencing. human codon-optimized cdna encoding biotin protein ligase (bira) [ ] was cloned in pcd in frame with sequences encoding a cd signal peptide and a his tag. expression vectors were transfected into hek t (atcc crl- ),cho k (atcc ccl- ) and gnti-deficient cho b cells [ ] (from ineke braakman, utrecht university, the netherlands) using polyethyleneimine i (pei) similarly as described previously [ , ] . cho cells are deficient in α , sialyltransferases and therefore exclusively synthesize , sia linkages [ , ] . tissue culture supernatants were harvested - days post transfection. recombinant ha and na proteins were purified using strep-tactin sepharose beads according to the manufacturer's instructions (iba, germany). fc tag-containing proteins were purified using protein a sepharose beads (ge healthcare), similarly as described previously [ ] . for an overview of the different fetuin constructs see s c fig. the concentration of purified protein was determined by using a nanodrop spectrophotometer (isogen life sciences) according to the manufacturer's instructions and analyzed by % sds-page followed by visualization of protein bands using a colloidal blue staining kit (invitrogen). biotinylated transferrin (sigma) contains two glycan chains exclusively carrying α , sias [ , ] (referred to as 'n transferrin bt). the activity of iav virus particles as well as recombinant soluble nas was determined by using a fluorimetric assay similarly as described previously [ ] . in short, viruses and na preparations were subjected to -fold serial dilutions in reaction buffer ( mm tris-hcl, mm cacl , ph . ) in a flat-bottom -well black plate (greiner bio-one). subsequently, a similar volume of reaction buffer containing μm -( -methylumbelliferyl)-α-d-n-acetylneuraminic acid (munana; sigma) was added to each well, mixed well, and incubated at ˚c for min. the reaction was terminated with the stop solution ( . m glycine, % ethanol, ph . ). the fluorescence of the -mu reaction product was immediately determined in relative fluorescence units (rfus) using a fluostar optima plate reader (bmg labtech, mornington, australia) with excitation and emission wavelengths at and nm, respectively. microarrays were printed as described previously [ ] . the glycan array analysis of the ha proteins was performed as previously described [ ] . briefly, μg/ml recombinant ha was precomplexed with a horseradish peroxidase-linked anti-streptavidin tag antibody and an alexa fluor anti-mouse antibody ( : : molar ratio) for min at ˚c, prior to incubation for min on the microarray slide under a microscope cover glass in a humidified chamber at room temperature. after repeated washes with phosphate-buffered saline (pbs) with . % tween, pbs, and deionized water, the slides were immediately subjected to imaging. bli analysis was performed on an octet qk machine using standard streptavidin (sa) or protein a bio-sensors. pbs with calcium and magnesium (pbs+/+) was used as standard assay buffer. receptor loading was performed by loading biotinylated receptors (synthetic glycans or proteins) to sa sensors or fc-tagged glycoprotein receptors to protein a sensors. unless otherwise specified, sensor were loaded with receptor to maximum levels (no further increase in reflection) using nm synthetic glycan or μg/ml glycoprotein as loading sample concentration. after loading sensors were washed in pbs+/+ until a stable baseline was obtained. virus binding was performed by moving receptor-loaded sensors to wells containing μl virus sample at the indicated concentrations (the use and concentration of oc is indicated where applicable). then virus-loaded sensors are usually moved to pbs+/+ in presence of μm oc to examine virus dissociation (consistently producing a flat line), or washed times for seconds in pbs+/+ to remove oc and next transferred to pbs+/+ without oc to measure na activity-driven self-elution. alternatively, in order to determine simultaneous action of ha and na virus binding was analyzed from the start in pbs+/+ in the absence of oc. regeneration of sensors, preserving the binding of biotinylated receptors but removing all bound virus, was performed by dipping sensors briefly in mm tris/glycine buffer ph . pr mtsinspecific antibody ( / from nibsc) was used for detection of virus binding in presence of oc with or without prior sensor generation. fetuin-coated sensors were also analyzed for their lectin-binding properties. to this end, fetuin-coated sensors were incubated with different lectins ( ng/ul; sna, mal-i, mal-ii, eca, all from vector labs) and lectin-binding curves were obtained. each bli experiment was repeated at least twice. representative experiments were graphed. initial binding rates, corresponding to the sloops of the binding curves during the first few minutes of the virus-binding experiments, were determined by second order polynominal equation (graphpad prism . ). the correlation between virus particle numbers and the initial binding rate was determined by linear regression and pearson r analysis using graphpad prism . software. significant differences between curves were analyzed by univariate analysis of variance model using ibm spss statistic . fractional receptor densities correlating with half maximum initial binding rates were determined by non-linear regression analysis using graphpad prism . software. significance analysis was based on two tailed unpaired t test or one way anova followed by tukey's multiple comparisons test (graphpad prism . ). streptavidin-coated (sa) biosensors contain biotin binding sites (pall-fortebio). sa tetramers carry four biotin binding sites, ordered two by two at opposing planes of the cubic structure [ ] . only two binding sites (spaced at . nm distance as determined by x-ray crystallography [ ] ) on one side of a surface-coated sa tetramer ( nm center to center distance assuming regular hexagonal packaging) are assumed to have exposed biotin binding sites [ ] . ha trimers are closely packaged on the virus surface (s fig, in agreement with [ ] [ ] [ ] [ ] ) and the center to center distance has been determined at nm [ ] [ ] [ ] [ ] . a fully loaded streptavidin can, in principle, form a bivalent interaction with an ha-trimer in which the sia-binding sites are spaced at nm distance [ , ] . lowering the receptor-density results in a non-homogenous sensor surface with streptavidins carrying , or receptor molecules. as a result, increasing amounts of surfacearea will have a receptor density too low to bind virus at decreasing receptor concentrations thus contributing to the observed decrease in maximum binding levels and initial binding rate when lowering receptor density (fig d and fig e) . (b) labstrains pr and wsn wt are spherical viruses with a diameter of~ nm (s fig) [ ] [ ] [ ] [ ] . when virus particles can be flattened for . times the radius % of the virus surface will be in contact with the sensor. (c) when % of the virus surface is in contact with the sensor,~ ha trimers can interact with receptor-loaded sa molecules at the virus-sensor contact interface (inner red circle). in principle two receptor molecules on a sa molecule can interact with an ha trimer but whether this occurs simultaneously will depend on the exact geometry of the specific glycan that was loaded. (d) at saturating levels of virus binding (hexagonal packaging) the majority of sa molecules are not present at the contact interface and therefore cannot be cleaved by na without virus movement. [ , ] . biotinylated fetuin was made by expressing a construct encoding a bap-tag fused to fetuin that, by co-transfection with a plasmid carrying a biotinylation enzyme, yields c-terminally biotinylated 'n+o fetuin ( 'n+o fetuin bt) upon expression in cho k cells. (d) confirmation of sia linkage-type specificity of glycoproteins using lectin binding. the glycoproteins were analyzed for linkage type specificity of their sialic acids using lectins mal i (specific for siaα , galα , glcnac linkages abundantly present on n-linked glycans), mal ii (specific for siaα , galα , galnac linkages abundantly present on o-linked glycans), sna (specific for siaα , galα , glcnac linkages abundantly present on n-linked glycans), and eca (specific for terminal galα , glcnac epitopes present on non-sialylated n-linked glycan antennae). (e) viruses used for binding to receptor-loaded sensors are wild type pr mtsin , wild type wsn wt and recombinant viruses carrying the ha encoding segments of pr mtsin (wsn hamtsin ) or pr cam (pr cam , ) in the background of seven wsn segments. pr cam , is identical to pr cam , except for a substitution (d e) that was introduced in ha to obtain a shift from α , to α , linkage-type binding specificity. tx namtsin carries the ha encoding segment of a/bilthoven/ / (h n ) in the background of seven pr mtsin segments [ ] . whereas both pr mtsin and pr cam , bound to fetuin containing n-glycans (a and b), only pr cam , was able to bind fetuin containing o-glycans, albeit at a -fold lower initial binding rate than to n-glycosylated fetuin (c). when n-and o-linked glycans are both present (a), the initial binding rate seems to be determined by the stronger binding to n-linked glycans as binding of pr cam , is not accelerated despite its ability to bind α , sialylated o-linked glycans. (e-g) confirmation of presence of n-and/or o-glycan on recombinant fetuin. the glycoproteins were treated with pngase f (neb) for hours at ˚c under non-denaturing conditions, which effectively removes almost all n-linked oligosaccharides from glycoproteins. the glycoproteins were analyzed for linkage type specificity using lectins mal i (specific for siaα , galα , glcnac linkages abundantly present on n-linked glycans), mal ii (specific for siaα , galα , galnac linkages abundantly present on o-linked glycans), and eca (specific for terminal galα , glcnac epitopes present on non-sialylated n-linked glycan antennae). (e) after pngase f treatment, the binding level of mal i to ' n+o (blue) and ' n fetuins (red) significantly reduced, whereas the binding level to ' o fetuin (green) remains the same, indicating the presence of sialylated n-linked oligosaccharides specifically on ' n+o and ' n fetuin. (f) after pngase f treatment, the binding level of eca to ' n+o (blue) and ' n fetuins (red) dramatically decreased, whereas the binding level to ' o fetuin (green) remains the same, indicating the presence of non-sialylated n-linked oligosaccharides specifically on ' n+o and ' n fetuin. (g) similar binding of mal ii to glycoproteins was observed after treatment with pngase f, indicating that o-linked oligosaccharides were not removed by pngase f treatment. reliable determination of v obs depends on precise determination of virus concentration. hemagglutination titers or infectious titers do not reveal absolute or relative particle of different viruses. quantitative pcr or western blotting (usually targeting np) are sensitive to variation caused by rna or protein contamination of virus preparations and, when comparing different viruses, to differences in specificity of probes or antibodies. therefore, densitometric quantification of the polymerase content of a virus preparation was used, assuming that every particle carries eight c) and (e). the obtained numbers/particle fit well to numbers obtained by different methods by others [ , ] . (g, h, i) quantification of na by similar procedures as applied for ha in panel d-f. antibodies gt -gtx (wsn) and n - d (pr ) were used and quantification was calibrated using a western blot of a standard concentration series of recombinant soluble na of pr mtsin and wsn expressed in hek t cells run in parallel. the na proteins were expressed similarly are described previously [ ] . (j) na activity of pr mtsin and wsn wt virus particles and expressed recombinant na soluble tetramers was determined using a two-fold dilution series in a soluble substrate na activity assay (munana assay). the activity of recombinant wsn na was . -fold lower than of pr mtsin na, whereas the na activity of the cognate virus particles showed a . -fold lower activity for wsn wt particles. this difference is explained by the . -fold higher incorporation level of na in pr mtsin particles as determined by western blotting (g-i). the ha content of both viruses was similar (d-f). (k) ha/na ratio was determined from panels f and i. both viruses bind at similar, but relatively low, rate to 'n transferrin bt (fig e) whereas wsn wt displays a~ -fold faster binding rate to 'n fetuin than pr cam , ( fig d) . ha clearly affects the self-elution rate as, in combination with the same na, self-elution from 'n fetuin is much more efficient in companion of the weaker binding ha of pr cam , (b) than in companion of the stronger binding ha of wsn wt (a). self-elution rates from 'n transferrin bt are more similar for both viruses. thus, whereas α , versus α , sia specificity of na is seemingly opposite for pr cam , and wsn wt , this is not caused by the na itself (which is identical for both viruses) but by differences in their has. in absence of oc, ongoing receptor cleavage reduces receptor density in time and thus the binding rate of additional virus particles, resulting in bending of the curves. as receptor cleavage by na is in competition with receptor binding by ha, the weakest binder (pr cam , ), which is assisted most in initial binding rate by na (fig g) , will also suffer most from receptor destruction by its na and as a consequence display a binding curve that bends down fastest in absence of oc (f). receptor binding and membrane fusion in virus entry: the influenza hemagglutinin structure of the haemagglutinin membrane glycoprotein of influenza virus at a resolution the structure and function of the hemagglutinin membrane glycoprotein of influenza virus influenza virus neuraminidase: structure and function influenza neuraminidase structure of the influenza virus glycoprotein antigen neuraminidase at . a resolution balanced hemagglutinin and neuraminidase activities are critical for efficient replication of influenza a virus influenza a virus reassortants with surface glycoprotein genes of the avian parent viruses: effects of ha and na gene combinations on virus aggregation phenotypic expression of ha-na combinations in human-avian influenza a virus reassortants functional balance of the hemagglutinin and neuraminidase activities accompanies the emergence of the h n influenza pandemic glycosylation of haemagglutinin and stalk-length of neuraminidase combine to regulate the growth of avian influenza viruses in tissue culture the n neuraminidase of human influenza virus has acquired a substrate specificity complementary to the hemagglutinin receptor specificity postreassortment changes in influenza a virus hemagglutinin restoring ha-na functional match generation and characterization of variants of nws/g c influenza virus after in vitro passage in -amino-neu a-c en and -guanidino-neu ac en generation and characterization of a mutant of influenza a virus selected with the neuraminidase inhibitor bcx- identification of residues that affect oligomerization and/or enzymatic activity of influenza virus h n neuraminidase proteins receptor binding properties of human and animal h influenza virus isolates receptor specificity in human, avian, and equine h and h influenza virus isolates receptor determinants of human and animal influenza virus isolates: differences in receptor specificity of the h hemagglutinin based on species of origin glycan receptor specificity as a useful tool for characterization and surveillance of influenza a virus amino acid residues contributing to the substrate specificity of the influenza a virus neuraminidase characterization of sialidase from an influenza a (h n ) virus strain: kinetic parameters and substrate specificity oligosaccharide specificity of influenza h n virus neuraminidases a beneficiary role for neuraminidase in influenza virus penetration through the respiratory mucus influenza a penetrates host mucus by cleaving sialic acids with neuraminidase roles of neuraminidase in the initial stage of influenza virus infection neuraminidase is important for the initiation of influenza virus infection in human airway epithelium interdependence of hemagglutinin glycosylation and neuraminidase as regulators of influenza virus growth: a study by reverse genetics pandemic swine h n influenza viruses with almost undetectable neuraminidase activity are not transmitted via aerosols in ferrets and are inhibited by human mucus but not swine mucus receptor binding by a ferret-transmissible h avian influenza virus an immunofluorescence study of influenza virus filament formation the m and m proteins of influenza a virus are important determinants in filamentous particle formation the genetic aspects of influenza virus filamentous particle formation densities and sizes of the influenza virus-a (pr strain) and virus-b (lee strain) and the swine influenza virus structure of the uncleaved human h hemagglutinin from the extinct influenza virus functional significance of the hemadsorption activity of influenza virus neuraminidase and its alteration in pandemic viruses neuraminidase hemadsorption activity, conserved in avian influenza a viruses, does not influence viral replication in ducks neuraminidase receptor binding variants of human influenza a(h n ) viruses resulting from substitution of aspartic acid in the catalytic site: a role in virus attachment? a mutant influenza virus that uses an n neuraminidase as the receptorbinding protein hemagglutinins from two influenza virus variants bind to sialic acid derivatives with millimolar dissociation constants: a -mhz proton nuclear magnetic resonance study a surface plasmon resonance assay for the binding of influenza virus hemagglutinin to its sialic acid receptor glycans on influenza hemagglutinin affect receptor binding and immune response influenza virus neuraminidases with reduced enzymatic activity that avidly bind sialic acid receptors random sequential adsorption of human adenovirus onto polyvinylidene fluoride surface influenced by extracellular polymeric substances time scale of random sequential adsorption a simple model of multivalent adhesion and its application to influenza infection a model for describing the thermodynamics of multivalent host-guest interactions at interfaces comparison of the optimal culture conditions for cell growth and tissue plasminogen activator production by human embryo lung cells on microcarriers chemistry of natural glycan microarrays polyacrylamide-based glycoconjugates as tools in glycobiology role of secondary sialic acid binding sites in influenza n neuraminidase genetic evolution of the neuraminidase of influenza a (h n ) viruses from to and its correspondence to haemagglutinin evolution single hemagglutinin mutations that alter both antigenicity and receptor binding avidity influence influenza virus antigenic clustering hemagglutinin receptor binding avidity drives influenza a virus antigenic drift structural organization of a filamentous influenza a virus distribution of surface glycoproteins on influenza a virus determined by electron cryotomography filamentous influenza viruses filamentous influenza viruses filament-producing mutants of influenza a/puerto rico/ / (h n ) virus have higher neuraminidase activities than the spherical wild-type cryotomography of budding influenza a virus reveals filaments with diverse morphologies that mostly do not bear a genome at their distal end influenza a virus hemagglutinin and neuraminidase act as novel motile machinery drug delivery across physiological barriers a periciliary brush promotes the lung health by separating the mucus layer from airway epithelia influenza virus infection of desialylated cells sialic acids on the plasma membrane of cultured human lymphoid cells. chemical aspects and biosynthesis assembly of endocytic machinery around individual influenza viruses during viral entry rescue of influenza a virus from recombinant dna substitutions near the receptor binding site determine major antigenic change during influenza virus evolution dissection of the influenza a virus endocytic routes reveals macropinocytosis as an alternative entry pathway trichloroacetic acid (tca) precipitation of proteins a neutralizing antibody selected from plasma cells that binds to group and group influenza a hemagglutinins antibodies directed toward neuraminidase n control disease in a mouse model of influenza the influenza a virus hemagglutinin glycosylation state affects receptor-binding specificity a protective and safe intranasal rsv vaccine based on a recombinant prefusion-like form of the f protein bound to bacterium-like particles identification and characterization of a proteolytically primed form of the murine coronavirus spike proteins after fusion with the target cell deficient uridine diphosphate-n-acetylglucosamine-glycoprotein n-acetylglucosaminyltransferase activity in a clone of chinese-hamster ovary cells with altered surface glycoproteins aberrant metabolic sialylation of recombinant proteins expressed in chinese hamster ovary cells in high productivity cultures comparative-study of the asparagine-linked sugar chains of human erythropoietins purified from urine and the culture-medium of recombinant chinese-hamster ovary cells screening for n-glycosylated proteins by liquid chromatography mass spectrometry site-specific carbohydrate profiling of human transferrin by nano-flow liquid chromatography/electrospray ionization mass spectrometry arraying the post-translational glycoproteome (ptg) only two residues are responsible for the dramatic difference in receptor binding between swine and new pandemic h hemagglutinin structural origins of high-affinity biotin binding to streptavidin streptavidin-biotin binding in the presence of a polymer spacer. a theoretical description kinetic evidence from image analysis of hemagglutinin-reconstituted vesicles zernike phase contrast electron microscopy of ice-embedded influenza a virus electron microscopy of the influenza virus submembranal structure structure of influenza haemagglutinin at neutral and at fusogenic ph by electron cryo-microscopy influenza virus pleiomorphy characterized by cryoelectron tomography polypeptides specified by the influenza virus genome i. evidence for eight distinct gene products specified by fowl plague virus mutation of the second sialic acid-binding site, resulting in reduced neuraminidase activity, preceded the emergence of h n influenza a virus oseltamivir carboxylate was kindly provided roche. recombinant pr virus tx namtsin was kindly provided by dr. r. fouchier (erasmus medical center, rotterdam, the netherlands). n - d was generously provided by dr. x. saelens (molecular virology unit, university of ghent). cho b cell was kindly provided by ineke braakman (utrecht university, the netherlands). key: cord- - zwq do authors: guo, kejun; shen, guannan; kibbie, jon; gonzalez, tania; dillon, stephanie m.; smith, harry a.; cooper, emily h.; lavender, kerry; hasenkrug, kim j.; sutter, kathrin; dittmer, ulf; kroehl, miranda; kechris, katerina; wilson, cara c.; santiago, mario l. title: qualitative differences between the ifnα subtypes and ifnβ influence chronic mucosal hiv- pathogenesis date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: zwq do the type i interferons (ifn-is) are innate antiviral cytokines that include different ifnα subtypes and ifnβ that signal through the ifn-i receptor (ifnar), inducing hundreds of ifn-stimulated genes (isgs) that comprise the ‘interferome’. quantitative differences in ifnar binding correlate with antiviral activity, but whether ifn-is exhibit qualitative differences remains controversial. moreover, the ifn-i response is protective during acute hiv- infection, but likely pathogenic during the chronic stages. to gain a deeper understanding of the ifn-i response, we compared the interferomes of ifnα subtypes dominantly-expressed in hiv- -exposed plasmacytoid dendritic cells ( , , , and ) and ifnβ in the earliest cellular targets of hiv- infection. primary gut cd t cells from donors were treated for hours ex vivo with individual ifn-is normalized for ifnar signaling strength. of , ifn-regulated genes, ‘core isgs’ were induced by all ifn-is tested. however, many ifn-regulated genes were not shared between the ifnα subtypes despite similar induction of canonical antiviral isgs such as isg , rsad and mx , formally demonstrating qualitative differences between the ifnα subtypes. notably, ifnβ induced a broader interferome than the individual ifnα subtypes. since ifnβ, and not ifnα, is upregulated during chronic hiv- infection in the gut, we compared core isgs and ifnβ-specific isgs from colon pinch biopsies of hiv- -uninfected (n = ) versus age- and gender-matched, antiretroviral-therapy naïve persons with hiv- (pwh; n = ). core isgs linked to inflammation, t cell activation and immune exhaustion were elevated in pwh, positively correlated with plasma lipopolysaccharide (lps) levels and gut ifnβ levels, and negatively correlated with gut cd t cell frequencies. in sharp contrast, ifnβ-specific isgs linked to protein translation and anti-inflammatory responses were significantly downregulated in pwh, negatively correlated with gut ifnβ and lps, and positively correlated with plasma il and gut cd t cell frequencies. our findings reveal qualitative differences in interferome induction by diverse ifn-is and suggest potential mechanisms for how ifnβ may drive hiv- pathogenesis in the gut. a a a a a the type i interferons (ifn-is) are innate antiviral cytokines that include ifnα ( different subtypes) and ifnβ [ ] . these cytokines significantly inhibited hiv- replication in vitro, but human clinical trials with ifnα showed only moderate or no inhibitory effects on hiv- infection [ , ] . all ifn-is bind to the same ifn-i receptor that is composed of two subunits, ifnar and ifnar , resulting in phosphorylation of jak and tyk . this in turn results in the phosphorylation of stat and stat that associate with irf to form the transcriptional activator, isgf [ ] . isgf translocates to the nucleus, where it binds to promoters of genes encoding ifn response elements (isres), resulting in the induction of hundreds of ifn stimulated genes (isgs), collectively referred to as the 'interferome' [ ] . recent studies highlighted ifnα as a potential adjunct to an hiv- curative strategy [ - ]. however, almost all hiv- clinical trials with ifnα were performed with only one subtype, ifnα , with mixed results (reviewed in [ , ] ). more recently, ifnα treatment of siv rhesus macaques under antiretroviral therapy did not reduce the latent hiv- reservoir [ ] . our group and others reported that ifnα only moderately inhibited hiv- in humanized mice and primary cd + t cells compared to the more potent subtypes ifnα , ifnα and ifnα [ ] [ ] [ ] [ ] . interestingly, the anti-hiv- potency of ifnα subtypes correlated with their binding affinity to ifnar , suggesting that the antiviral differences between the ifnα subtypes were due to quantitative differences in ifnar signaling strength [ , , ] . however, several human ifnα subtypes exhibit strong signals of purifying selection [ ] , suggesting that these ifnα subtypes may have evolved to have specific, nonredundant functions. some ifnα subtypes were better at inducing certain immune responses in vivo [ , ] and may trigger distinct intracellular signaling pathways [ ] . nevertheless, the notion of qualitative differences between ifnα subtypes remains controversial [ , ] . one reason is that most comparative studies normalized ifn-is using protein amounts or units/ml based on inhibition of vesicular stomatitis virus or encephalomyocarditis virus [ ] . to unravel qualitative differences between the ifn-is, it would be important to normalize ifn-is for quantitative differences in ifnar signaling strength. it is widely accepted that ifn-i signaling can prevent acute retrovirus infection. genetic ablation of ifnar resulted in higher friend retrovirus replication in mice co-infected with lactate-dehydrogenase elevating virus, a potent ifn-i inducer [ ] . moreover, ifnar blockade increased siv replication in rhesus macaques [ , ] . administration of ifnα decreased retrovirus replication in mice, monkeys and humans [ , , ] . however, in persistent virus infections, chronic ifn-i stimulation was linked to pathogenic outcomes [ ] [ ] [ ] [ ] [ ] . although clinical administration of ifnα increased the number of low-dose mucosal inoculations needed for breakthrough siv infection in rhesus macaques, once the infection was established, lower cd t cell counts were observed in ifnα -treated monkeys relative to untreated controls [ ] . these findings highlight that hiv- infection shares features with 'interferonopathies' such as aicardi-goutières syndrome [ ] , which are currently being targeted through ifn-i blockade strategies (clinicaltrial.gov nct ). in fact, ifnar blockade during chronic hiv- infection in humanized mice restored immune function, leading to better hiv- control [ , ] . neutralization of (most) ifnα subtypes in siv-infected rhesus macaques prior to infection increased viral loads as expected, but also decreased subsequent immune activation profiles [ ] . by contrast, blockade of ifn-i signaling in chronic sivtreated and untreated rhesus macaques decreased inflammation profiles associated with isgs but did not reverse t cell exhaustion or activation [ ] . the basis for the protective versus pathogenic effects of ifn-is remains unclear. one hypothesis is that the initial ifn-i response is protective due to the induction of antiviral factors, whereas chronic stimulation promotes inflammation through other isgs with sustained, elevated expression. distinct ifn-is present in the acute versus chronic stages of persistent viral infection may induce divergent cellular immune responses. tissue compartmentalization may also play a role. the gut is a critical site not only for early hiv- infection, but also in driving chronic immune activation [ ] . epithelial barrier dysfunction, partly due to the loss of th cells, leads to the translocation of enteric bacteria from the gut lumen to the lamina propria and systemic circulation, resulting in chronic immune activation [ ] [ ] [ ] . we recently reported increased ifnβ gene expression in the gut, but not the blood, in persons with hiv- (pwh) infection compared to age/gender-matched hiv- uninfected controls [ ] . by contrast, ifnα subtypes were downregulated in pwh, and ifnλ, a type iii ifn linked to mucosal immunity in mouse models [ ] , was undetectable in these samples [ ] . these findings suggest that among the diverse ifn-is, ifnβ may play a dominant role in the gut during chronic hiv- infection. here, we utilized transcriptomic approaches to evaluate whether the ifnα subtypes and ifnβ exhibit qualitatively different effects on gene expression. we then tracked how interferon-regulated genes were altered during chronic hiv- infection in the gut. our analyses highlight potential mechanisms driven by ifnβ that may influence mucosal hiv- pathogenesis. the relative anti-hiv- activity of ifnβ in primary lpmcs remains unclear, though studies using pbmcs suggest that ifnβ is relatively potent [ ] . we previously reported a spectrum of antiviral potencies of the ifnα subtypes against hiv- in primary lpmcs [ ] . these data were extended to show a -fold difference in % inhibitory concentrations (ic ) between ifnα and ifnα [ ] . using the same lpmc donors used to calculate the ic s of ifnα and ifnα , we titrated the anti-hiv- potencies of ifnα (weak), ifnα (potent) and ifnβ. the ifn-is were added into lpmc cultures at the time of infection with hiv- bal . at d post-infection, the percentage of hiv- gag p + cells were evaluated by flow cytometry. sigmoidal curves were fitted into the average inhibition data for donors, and used to calculate ic values (pg/ml protein concentration). we also calculated a metric known as 'vres', which corresponds to the level of residual virus replication at maximal doses of ifn-is [ ] . for comparison, previously reported inhibition curves for ifnα and ifnα are also shown [ ] . ifnα showed ic concentrations over -fold lower than that of ifnα (fig ) . notably, ifnβ had a similar potency as ifnα and ifnα . we also calculated ic s for individual donors (s a fig) and show significantly lower inhibition by ifnα compared to ifnα and ifnα (s b fig). ifnα and ifnα reduced hiv- infection levels to~ % at the maximal doses tested ( ng/ml), whereas ifnα , ifnα and ifnβ had higher vres between - %. however, these differences in vres were not significant (s c fig) . these findings validate prior results on the relative anti-hiv- activity of these ifnα subtypes and highlight ifnβ as a potent anti-hiv- ifn. quantitative differences were evident, as increasing the dose of weaker ifnα subtypes should enable these cytokines to inhibit hiv- just as well as the potent ifnα subtypes. the stronger anti-hiv- potencies of ifnα and ifnα compared to ifnα and ifnα were associated with higher isg induction [ ] , but it remained unknown how the other ifnα subtypes and ifnβ compare. to test the isre signaling activity of ifn-is, we used a commercially-available ilite assay (see methods). the ilite cells are human u monocytic cell lines transduced with a luciferase reporter downstream of an isre from isg , a canonical isg. serial -fold dilutions of the ifnα subtypes and ifnβ were added into the ilite cells and relative light units were measured after h. % effective concentrations (ec ) were then calculated from best-fit sigmoidal plots. fig a highlights the -fold ec difference in isre-activity between ifnα and ifnα . the ec s of the other ifnα subtypes and ifnβ fell in-between ifnα and ifnα data were normalized to mock (untreated) as % for each donor. dose-response curves were generated using a one-phase decay equation in graphpad prism . to determine ic values. vres, the percentage of cells that remain infected relative to the mock at the maximum dose tested, corresponded to plateau values from the decay equation. � note that data on ifnα and ifnα were previously published [ ] . https://doi.org/ . /journal.ppat. .g type i interferons in mucosal hiv- infection ( fig b) . notably, the isre ec of the ifnα subtypes significantly correlated with anti-hiv- potency data from our previous study (fig c) [ ] . a significant positive correlation was also observed between isre-activity and the ic values from the ifn-is tested in fig that includes ifnβ (r = . , p = . ). importantly, isre ec values correlated strongly with published ifnar binding affinity values when comparing the ifnα subtypes ( fig d) [ ] . however, the inclusion of ifnβ, which was reported to have a higher ifnar binding affinity than multiple ifnα subtypes [ , ] , abolished the correlation (fig e) . these data demonstrate that isre-activity as measured by the ilite assay can be used to evaluate quantitative differences between the ifn-is, particularly for the ifnα subtypes. our data in figs and , as well as data from other studies [ ] [ ] [ ] [ ] , provide strong evidence for quantitative differences among the ifn-is. to uncouple quantitative versus qualitative differences, we normalized our ifn-i treatments for isre-activity ( fig b) for unbiased transcriptomics. purified gut cd + t cells from different donors were treated with pg/ml ifnα , and the other ifn-is were added at higher concentrations that match the isre-activity of this ifnα dose (e.g., . ng/ml ifnα ). purified cd + t cells were evaluated instead ifnα subtypes that had potent activity against hiv- in a previous study are highlighted in black. ifnβ is highlighted in green. note that the ec s are negative log values; thus the higher the bar, the less concentration is needed to achieve an ec . (c) isre-activity versus hiv- inhibition. hiv- inhibition values were previously reported [ ] showing % inhibition of hiv- p + cells relative to mock in lpmc cultures. isre-activity versus ifnar binding affinity (d) without or (e) with ifnβ. ifnar binding affinity data were previously reported using surface plasmon resonance. for panels c to e, linear regression curves were plotted using prism . and evaluated using pearson statistics. https://doi.org/ . /journal.ppat. .g type i interferons in mucosal hiv- infection of total lpmcs to reduce potential confounders due to cell type heterogeneity in lpmcs when performing rnaseq. cd + t cells account for majority of cells in lpmcs ( %) [ ] and are the main cell types for the interaction between hiv- and antiviral isgs. given that only limited numbers of primary lpmcs can be obtained per donor, we selected ifnα , ifnα , ifnα , ifnα and ifnα , as these were highly expressed in hiv- -exposed primary pdcs in vitro and in pbmcs during chronic hiv- infection in vivo [ , ] . we also selected ifnβ because it is significantly upregulated in the gut during chronic hiv- infection on average, we obtained . million (range: . to million) sequence reads per sample (s table) . we first evaluated the transcripts per million (tpm) levels of isg , from which the isre was genetically linked to luciferase in the ilite assay. all ifnα subtypes tested and ifnβ induced isg to similar levels ( fig a, s b fig). in fact, the treatment dose used type i interferons in mucosal hiv- infection also induced the canonical isgs rsad (viperin) and mx (fig b) , as well as oasl, bst and apobec g (s table) to similar extents. these data confirmed that the treatments were indeed normalized for isre-activity and ifnar signaling strength. overall, the ifnα subtypes and ifnβ altered , genes. upregulated genes (isgs) were more prevalent ( - % of irgs) than downregulated genes across all ifn-is ( fig c) . we next compared our irg set with irgs catalogued in the interferome database [ ] . a majority ( . %) of the observed irgs were found in the interferome database ( fig d) . we then partitioned the data into individual ifn-is. strikingly, there were, on average, . -fold more ifnβ-regulated genes than those regulated by any of the individual ifnα subtypes tested (fig c) . on average, ifnβ upregulated . -fold and downregulated . -fold more genes than the individual ifnα subtypes (fig c) . there was a strong correlation between ifnar binding affinity and the number of irgs or isgs (r > . , p< . ), but these correlations were lost if ifnβ was removed (p< . ). in addition, our current analysis revealed novel irgs (s table) . many of these novel irgs may not encode protein products and/or have tentative gene designations, potentially explaining why these genes are not in the interferome database. however, some long non-coding rnas such as nrir (negative regulator of the interferon response) [ ] and bispr (bst interferon stimulated positive regulator) [ ] could have critical roles for modulating ifn-i responses. some repressed protein-coding genes such as ccr appeared specific to just one ifnα subtype (s table) . the ifnα subtypes are homologous genes that activate ifnar, raising questions on whether their differential effects were primarily quantitative. we postulated that if the differences between the ifnα subtypes are mainly quantitative, then we should observe a substantial overlap between the irgs of cells treated with different ifnα subtypes that were normalized for isre-activity. the number of irgs that overlapped between any two of the tested ifnα subtypes ranged from % (ifnα vs ifnα ) to % (ifnα vs ifnα ) ( fig a) . this included a core set of irgs altered by all ifnα subtypes tested (fig b; s table) . interestingly, many additional genes appeared to be specific to an ifnα subtype, particularly for ifnα and ifnα ( fig b and s table) . to test if the irgs unique to each ifnα subtype could have been regulated by other ifnα subtypes but excluded due to a stringent fdr cut-off of %, we investigated the median fdr of these unique genes against the other ifnα subtypes. this analysis is illustrated in s fig, where the fdr values of each of the ifnα -specific genes or ifnα -specific genes ( fig b) were plotted against the gene induction datasets for the other ifnα subtypes tested. as shown, majority of the ifnα or ifnα -specific genes had fdr values that were > % in the other ifnα subtypes. these analyses were expanded on s table, where ifnα-specific irgs had a median fdr of at least %, with most > %, when tested against genes sets from other ifnα subtypes. thus, irgs that were differently expressed by a specific ifnα subtype were unlikely to be significantly altered by the other ifnα subtypes. we next evaluated trends as to whether irgs altered by at least one ifnα subtype (a total of , genes) were similarly upregulated or downregulated. these comparisons were based on mean fold-induction values that can be visualized in the heatmap ( fig c) . surprisingly, a large number of genes (n = ) that trended to be upregulated by ifnα , ifnα , ifnα and ifnα appeared to be downregulated by ifnα (fig c, s table) . as the irgs were based on an arbitrary cut-off (� . × relative to mock, fdr � %), we next evaluated whether increasing our fc criteria changed the differential expression patterns. at ×, . × and × cut-offs, we still observed genes that were differentially regulated by ifnα to determine whether ifnα subtypes induced molecular programs distinct from each other, we subjected the irgs to ingenuity pathway analyses (ipa) [ ] . s table provides a ranked list of activated and inhibited pathways for each ifn-i. as expected, 'interferon signaling' and 'pattern recognition receptors' were the top induced pathways predicted for the . % of ifnα -regulated genes were found among ifnα -rgs, whereas . % of ifnα -rgs were found among ifnα -rgs. (b) euler diagram showing the interferome overlap between the ifnα subtypes tested. a core set of irgs altered by all ifnα subtypes were detected. ifnα-subtype specific genes were highlighted (e.g., for ifnα , for ifnα ). the total number of irgs (n = , ) include genes not shown that were shared between to ifnα subtypes. (c) heatmap of irgs from distinct ifnα subtypes. highlighted areas in red correspond to core isgs, whereas those in blue correspond to genes differentially regulated by ifnα relative to the four other ifnα subtypes tested. (d) ingenuity pathway analyses of ifnα subtypes, highlighting z-scores for shared pathways and those predicted to be specific to ifnα and ifnα . https://doi.org/ . /journal.ppat. .g ifnα subtypes tested ( fig d) . death receptor, nfκb and inflammasome signaling were also highly induced by all ifnα subtypes (s table) . interestingly, ipa also predicted distinct pathways induced by the ifnα subtypes. cd signaling, ctl and nk function, p mapk signaling and sumoylation were predicted for ifnα but not the other ifnα subtypes (s table and fig d) . only the ifnα interferome was associated with downregulated apoptosis, ppar and lxr/rxr activation, likely due to some downregulated genes such as casp (s table) . these differential predictions provide confirmatory evidence of qualitative biological differences in endpoint effector mechanisms induced by different ifnα subtypes. our data in fig c revealed~ more irgs for ifnβ than the individual ifnα subtypes. we pooled ifnα regulated genes independently of the subtype and compared them to ifnβ-regulated genes. almost half ( . %) of ifnβ interferome genes were not regulated by any of the ifnα subtypes (fig a) . these included cytokines and cytokine receptor genes such as il , ifngr , il r and tgfb ( fig a; s table) . we compared the ipa results for the ifnα subtypes versus ifnβ regulated genes. ifnβ induced more pathways (fig b) than the ifnα subtypes, such as 'th pathway', 'erk/mapk signaling' and 'regulation of actin motility'. these findings indicated that ifnβ induced a broader interferome than ifnα subtypes. moreover, compared to ifnα, ifnβ likely regulated more cellular pathways in primary gut cd + t cells. we recently reported that during chronic, untreated hiv- infection, ifn-i inducible antiretroviral genes apobec g, bst and mx , as well as ifnβ, but not ifnα, were expressed to significantly higher levels when compared to hiv- uninfected individuals [ ] . to expand on these findings, we performed rnaseq on these gut biopsies to more broadly investigate the table provides the demographic characteristics of this clinical cohort, which included pwh and hiv- uninfected individuals [ ] . on average, we processed . million sequence reads from colon biopsies per subject (table ) . filtered data were normalized using transcripts per million (tpm), trimmed mean of m values (tmm) [ , ] and deseq [ ] methods. based on relative log expression plots [ ] , the tmm method was most efficient at removing unwanted variation (s fig) . we next determined the expression levels in the colon biopsies in vivo of the 'core irgs' that were similarly regulated by the ifnα subtypes and ifnβ in gut cd + t cells in type i interferons in mucosal hiv- infection vitro. the majority ( %; n = ) of these core irgs that passed the rnaseq filter criteria were isgs (e.g., these genes were induced by the ifn-is tested, not downregulated). of these core isgs, ( %) were significantly altered in hiv- infected versus uninfected individuals at % fdr ( fig a) . the majority ( %) of these altered core isgs were upregulated during chronic hiv- infection. these included the antiretroviral genes apobec g, bst and mx , consistent with our previous report [ ] . genes linked to innate sensing, immunomodulation and cell death/proliferation, as well as negative feedback regulation of the ifn-i response, were also expressed at significantly higher levels in pwh (fig b and c , s table) . since ifnβ induced a broader interferome than all ifnα subtypes tested (fig a) , we evaluated whether isgs that were unique to ifnβ (ifnβ-specific isgs) were also induced in the gut during chronic hiv- infection. of the ifnβ-specific irgs, % (n = ) were isgs that passed the rnaseq filter criteria (fig c) . nearly a third of these ifnβ-specific isgs ( %; n = ) were significantly altered in pwh compared to hiv- uninfected controls (fig a) . only a few of the ifnβ-specific isgs were significantly upregulated in pwh (fig a) . by contrast, the vast majority (> % (n = ) of the altered ifnβ-specific isgs were downregulated during chronic hiv- infection in the gut (fig a) . these repressed ifnβ-specific isgs are involved in intracellular vesicle trafficking, transcriptional/translational regulation, protein ubiquitination and transport (fig b and c ; s table) . moreover, several known hiv- type i interferons in mucosal hiv- infection cofactors such as tsg , nup , and cul were downregulated [ ] [ ] [ ] . we again emphasize that all these genes were induced by ifnβ in gut cd t cells ex vivo (s table) . given that a great majority of these ifnβ-specific isgs were downregulated in pwh, we conclude that a significant inversion of ifnβ-specific isgs was observed during chronic hiv- infection in the gut. we next investigated potential mechanisms for how these ifnβ-specific isgs may have been downregulated in the gut in the presence of high ifnβ levels. two known negative feedback regulators, usp [ ] and unc b [ ] , were induced by all ifn-is tested ex vivo (s table) . gut ifnβ mrna levels positively correlated with usp and unc b transcripts (s table) . however, none of the ifnβ-specific isgs altered in pwh correlated with usp (s and s tables). by contrast, % of these ifnβ-specific isgs negatively correlated with unc b (s and s tables). thus, one potential mechanism for the inversion ifnβ-specific isgs in the gut during chronic hiv- infection may be the ifnβ-mediated induction of unc b . the clinical study described in table involved cohorts where paired blood (pbmcs/plasma) and colon pinch biopsies (up to per donor) were obtained. colon pinch biopsies (~ ) were type i interferons in mucosal hiv- infection pooled for same-day flow-based immunophenotyping [ , ] and the rest were frozen for later histology and transcriptomics. the study obtained comprehensive data including gut and pbmc ifnα and ifnβ transcript levels, plasma and gut viral loads, gut cd + t cell percentages (of cd + cells), myeloid activation (cd mfi in cd c+ myeloid dcs), blood cd + t cell counts, markers of microbial translocation (scd , lps, lta), inflammation (crp, il ), and epithelial barrier dysfunction (ifabp) ( table and s table) [ , , - ]. we investigated how individual altered core isgs (n = ) and ifnβ-specific isgs (n = ) correlated with these clinically relevant parameters using linear regression models, after adjusting the data for age and gender. we used a % fdr threshold for these associations. five clinically relevant parameters-gut ifnβ mrna, plasma lps, gut cd t cell frequencies, blood cd t cell counts and plasma il levels-correlated significantly with the altered core and ifnβ-specific isgs (fig and s table) . the expression of core and ifnβ-specific isgs significantly correlated with transcript levels of ifnβ (> %) rather than ifnα (< %) (s table) . interestingly, the directionality of the correlations was discordant between these gene sets: higher ifnβ levels correlated with higher expression of % of core isgs, whereas higher ifnβ levels were associated with lower expression of % of ifnβ-specific isgs ( fig a) . both core isgs and ifnβ-specific isgs were significantly associated with plasma lps levels, but again with discordant directionalities (fig b) . gut cd t cell counts (as a percentage of cd + cells) were more significantly associated with core-isgs ( % of genes negatively correlated) than ifnβ-specific isgs ( % of genes positively correlated) (fig c) . by contrast, blood cd t cell counts were more significantly associated with ifnβ-specific isgs ( % negatively correlated) than core isgs (fig d) . notably, the lower expression of a substantial fraction of ifnβ-specific isgs ( %) was associated with higher levels of plasma il . none of the core isgs correlated significantly with plasma il at the % fdr cut-off. the complete list of genes that correlated with the clinically relevant parameters (gut ifnβ mrna, plasma lps, gut cd t cell frequencies, blood cd t cell count and plasma il levels) are described in s table. we highlight several core isgs with the highest correlations in fig a, ] . nlrc and lag also positively correlated with plasma lps levels and inversely correlated with the percentage of cd + t cells in the gut (fig c and d ). since t cell exhaustion in persistent lcmv infection has been linked to the suppressive cytokine il [ ] , we evaluated if ifnβ mrna levels correlated with cytokine transcripts in the rnaseq dataset. notably, ifnβ mrna levels were associated with increased levels of il and il ra, which were in turn correlated with lag ( s a fig). ifnβ mrna levels also correlated with transcript levels of tnfα and ifnγ, but not il and tgfβ (s b fig) . these data suggest that increased ifnβ in the gut of chronic pwh may drive genes associated with sustained isg expression, antigen processing, t cell activation, inflammation and immune exhaustion. among the ifnβ-specific isgs (fig ; panels in the right half), higher ifnβ transcript levels negatively correlated with: ( ) eif h, a eukaryotic translational initiation factor (fig e) [ ]; ( ) smad , a key regulator of tgfβ [ ] , which in turn promotes mucosal barrier integrity ( fig f) ; ( ) vimp and sep , selenoproteins that regulate protein folding in the endoplasmic reticulum [ , ] that were linked to anti-inflammatory processes [ ] ; and ( ) two co-factors of hiv- vpr, nup and mus [ , ] . nup is a nuclear pore component and mus is an endonuclease; both are involved in maintaining genomic dna integrity [ , ] . reduced expression of vimp and sep were associated with lower gut cd t cell type i interferons in mucosal hiv- infection percentages (figs and g) . downregulated nup and mus were associated with higher plasma il levels (fig h and i) , lower gut cd t cell percentages (s table) and blood cd t cell counts (fig h and i) . thus, our analyses link elevated ifnβ levels in the gut of pwh to decreased protein translation and decreased protection against dna damage, protein misfolding and barrier dysfunction. fig a) and ifnβ-specific isgs (fig a) in gut biopsies from the clinical cohort were determined against (a) gut ifnβ transcripts, (b) plasma lps levels, (c) gut cd + t cell percentages, (d) blood cd t cell counts and (e) plasma il levels using linear regression models, controlling for age and gender. the clinical cohort included pwh (n = ) and matched hiv uninfected controls (n = ). correlations with fdr � % were considered significant; the proportion of those core and beta isgs are plotted as pie charts, with red, blue and gray depicting positive, negative and no significant correlations, respectively. the top genes in the relevant categories with � % representation are highlighted; a full list is available in s table. https://doi.org/ . /journal.ppat. .g two aspects of type i ifn biology that could influence its translational potential remain insufficiently addressed. first, there is an ongoing debate as to whether the biological effects of ifn-is are primarily due to quantitative differences in ifnar signaling capacity. for example, would administration of higher doses of weakly antiviral ifn-is (e.g., ifnα for hiv- infection) achieve the same in vivo effect as that of more potent ifn-is (e.g., ifnα )? second, the mechanisms governing how and why ifn-is become pathogenic during chronic hiv- infection remains unclear. which isgs are responsible for the discordant effects of ifn-is at distinct phases of infection with persistent viruses? we have undertaken an unbiased transcriptomics approach to gain deeper insights on these questions. our group and others reported diverse antiviral potencies of the ifnα subtypes and ifnβ that correlated with their ifnar binding properties. these data emphasize the contribution of quantitative differences in ifnar signaling in controlling acute viral infection [ ] [ ] [ ] [ ] ] . however, data from multiple in vivo studies also revealed that the ifnα subtypes may have expression levels were based on tmm-transformed counts. linear regression was performed in r statistical package, adjusting for age and gender. a full list of the proportion of core and ifnβ-specific genes that correlated with clinical parameters is presented in s table. https://doi.org/ . /journal.ppat. .g type i interferons in mucosal hiv- infection qualitative differences in mediating antiviral immunity (reviewed in [ ] ). in fact, ifnβ has a significantly higher binding affinity to ifnar than all ifnα subtypes [ , ] but did not exhibit higher isre activity and inhibitory activity against hiv- . recently, schaepler et al utilized saturating concentrations of various ifnα subtypes to inhibit hiv- infection in activated pbmcs in vitro. since canonical isgs were induced to similar levels at saturating ifn-i doses, the authors concluded that there were no qualitative differences between the ifnα subtypes [ ] . we argue that this conclusion is premature, as there are hundreds of isgs encompassing the interferome [ , ] . we postulate that if there were no qualitative differences, the interferomes of diverse ifn-is should have substantial overlap following stimulation of cells with ifn-is normalized for ifnar signaling strength. we utilized a luciferase reporter cell line linked to the isre of a canonical isg, isg , to normalize for ifnar signaling strength. the normalized ifn-is tested (ifnα , , , , and ifnβ) induced isg and other antiviral isgs to similar extents in gut cd + t cells as expected. interestingly, despite normalizing for quantitative differences in ifnar signaling strength, the overlap between the ifnα subtype 'interferomes' were not complete, ranging from to %. ifnα altered > genes not found in any of the other ifnα subtypes. ifnα altered > additional genes and weakly downregulated genes that were induced by other ifnα subtypes, raising the intriguing possibility that ifnα may modulate the overall ifn-i response. while it was possible that these ifnα-subtype specific genes were an artifact of the low numbers of donors used in this study, these genes were not close to statistical significance of being differentially expressed by the other ifnα subtypes, as majority had fdr values > %. the sample size we used for this work was also counterbalanced by utilizing a homogenous cell subpopulation (purified cd + t cells) treated in a controlled fashion in vitro that should decrease overall variability. in fact, > canonical isgs were captured by all ifns tested including our positive control, isg . interestingly, ifnβ altered nearly three times the number of genes compared to the individual ifnα subtypes we tested. we speculate that the higher binding affinity of ifnβ to the ifnar may contribute in part to the increased gene induction numbers, but it was notable that enhanced binding affinity did not track with isre activity. the broader interferome associated with ifnβ was also reported by other groups using pbmcs [ , ] , although it was unclear whether the doses were normalized for ifnar signaling strength. altogether, the incomplete overlap between the interferomes strongly suggests that there are qualitative differences between diverse ifn-is. the underlying molecular mechanisms for differential interferome induction remain unclear. one possibility is that subtle differences in ifnar binding may have triggered differential phosphorylation of diverse stats, jaks and mapks. this possibility is currently under investigation. we speculate that differences in interferome regulation may have consequences for the therapeutic use of ifn-is in vivo. clinical administration of ifnα and ifnβ in vivo were associated with a multitude of side-effects [ ] . one approach to potentially reduce toxicity issues is to utilize ifnα subtypes with a more 'restricted' gene regulation signature. ifnα is ten times more potent at inhibiting hiv- than ifnα , but did not induce as many genes as ifnα . thus, ifnα may be a potent anti-hiv- therapeutic with limited side-effects. however, ifnα did not seem to inhibit hiv- when administered in humanized mice as encoded plasmids via hydrodynamic injection [ ] . ifn-is with desirable immunomodulatory properties may also be useful in hiv- curative strategies [ ] , as reactivation of latent hiv- was insufficient to reduce the reservoir during antiretroviral therapy [ ] . ifn-is could be potent additive drugs for hiv- cure by activating immune cells with latent hiv- while stimulating immune responses that can kill infected cells and prevent subsequent infection through intrinsic restriction. ifnα strongly induced trail+ nk cells and apobec g-mediated hypermutation compared to ifnα in humanized mice [ ] and was predicted to induce pathways associated with immune function in gut cd + t cells compared to ifnα , , and . thus, ifnα could be a viable candidate to pursue for hiv- curative strategies. the current data could serve as a useful template to design transcriptome-wide studies that extend to the other ifnα subtypes not studied here, as well as other cell types (dcs, nk cells, cd + t cells, b cells) in various tissue compartments. such follow-up studies may aid in designing focused ifn-i biologicals for various clinical applications. our recent study suggested that ifnβ may play an important role during chronic hiv- immunopathogenesis in the gut [ ] . we observed downregulated ifnα, but elevated ifnβ levels in colon biopsies from pwh, while ifnβ was rarely detected in the pbmcs and plasma of these patients [ ] . since ifnβ induced a broader interferome than the individual ifnα subtypes, we determined how isgs induced by all ifn-is tested ('core isgs') versus isgs specifically induced by ifnβ ('ifnβ-specific isgs') were expressed in the gut biopsies following rnaseq. one limitation of our approach is that colon biopsies encompass other cell types that the interferomes based on gut cd + t cells may not capture. specifically, it is possible that the decreased expression of ifnβ-specific isgs in pwh relative to uninfected controls may be due to the significant loss cd + t cells in pwh. if this was the case, we should have also observed a decrease in core isg expression in pwh. however, over a hundred core isgs were upregulated in pwh, correlating significantly with gut ifnβ expression. alterations in ifnar expression in mucosal cd + t cells during hiv- infection may also contribute to our results, but our recent study have not detected significant changes in ifnar expression in mucosal immune cells in pwh [ ] . expanding the interferome analyses to other mucosal cell types may enable deconvolution of the bulk rnaseq data to specific cell types. consistent with our previous study [ ] , many canonical isgs that include antiviral genes remained upregulated in chronic hiv- infection in the gut. core isgs positively correlated with ifnβ rather than ifnα transcripts, suggesting that ifnβ drove these responses in the gut. interestingly, core isg expression positively correlated with plasma lps levels. previously, we showed that many isgs were upregulated in gut cd + t cells following co-incubation with prevotella stercorea, a gram-negative microbe present in colon tissue of pwh [ ] . these findings strengthen a link between microbial translocation, ifnβ, and elevated isg signatures in the gut during chronic hiv- infection. interestingly, we did not observe correlations between core isgs and the monocyte activation marker scd or myeloid dendritic cell activation. other markers such as scd [ , ] may need to be investigated in future work. irf , stat and stat (the components of isgf ) remained elevated in pwh, suggesting a mechanism for constitutive isg expression during chronic infection. notably, ifnβ expression in the gut correlated with gene markers of immune activation and inflammation (cd , psmb , nlrc , tnfa, ifng), and exhaustion (lag ). ifnβ-mediated induction of these immunomodulatory genes may account for how ifnβ contributes to pathogenesis during chronic infection. specifically, ifnβ levels in the gut were associated with il and il ra expression, which in turn correlated with lag levels, suggesting that ifnβ may drive immune exhaustion through the il pathway. it remains to be determined whether these core isgs are similarly regulated in the systemic circulation, where ifnα subtypes, and not ifnβ, are more prominent [ ] . further, the rationale for why the gut appears primed to express ifnβ remains unclear. a recent study noted that helix of ifnβ may have direct antimicrobial properties [ ] . efforts are ongoing to investigate links between ifnβ levels, ifnβspecific genes and the altered microbiome in pwh. could ifnβ have distinct biological effects that are likely not due to other ifn-is? in persistent lcmv infection of mice, neutralization of ifnβ, and not ifnα, accelerated virus clearance and improved t cell responses [ ] . in the present study, we observed that a substantial fraction of ifnβ-specific isgs, but not core isgs, correlated with plasma levels of the inflammatory cytokine il . a link between ifnβ and il has previously been reported [ ] . surprisingly, the ifnβ-specific isgs that correlated with il were downregulated during chronic hiv- infection in the gut. the magnitude of downregulation correlated with higher ifnβ levels, suggesting negative feedback control. among these negative feedback control genes, we identified unc b as a potential driver for the downregulation of ifnβ-specific isgs, possibly through the regulation of tlr signaling via endosomal trafficking pathways [ , ] . it is thought that constitutive expression of antiviral isgs may protect from virus infection [ , ] , but decreased protein translation (eif h) in pwh may minimize the contribution of antiviral isgs that are constitutively expressed. furthermore, our data suggest a potential link between ifnβ, decreased tgfβ signaling (smad ), increased unfolded protein response (vimp and sep ) and increased dna damage (nup and mus ) in chronic hiv- infection. these results raise the possibility that genes downregulated by ifnβ could have important consequences for mucosal hiv- pathogenesis. one possibility is that these altered ifnβ-specific genes (such as those that decreased protection from dna damage) may directly contribute to cd + t cell death, in line with previous studies linking ifn-is and cd + t cell depletion [ , ] . given the predicted pleiotropic effects of ifnβ, our data also raise concerns in utilizing ifnβ for treating chronic hepatitis b virus infections that became refractory to ifnα treatment [ , , ] . notably, ifnβ administered < days post-symptom onset may help resolve infection with the novel pandemic coronavirus, sars-cov- [ ] . by contrast, delayed ifnβ treatment in murine coronavirus models exacerbated immune pathology [ , ] . based on our studies as well as others, understanding the role of distinct ifns during the course of infection with diverse pathogenic viruses may yield important therapeutic insights. the mechanisms for the differential regulation of core versus ifnβ-specific isgs during chronic hiv- infection remain to be determined. studies in cell lines reveal that unphosphorylated stat , stat and irf can assemble into an unphosphorylated isgf (u-isgf ) complex that could sustain the expression of canonical isgs after a single stimulation with ifnβ [ , ] . constitutively expressed isgs regulated by u-isgf included antiviral genes such as bst , apobec g, mx that remained elevated during chronic hiv- infection. by contrast, isgs specifically regulated by phosphorylated isgf are more sensitive to negative feedback regulation. the isgf /u-isgf model would imply that core isgs are regulated by u-isgf , whereas the ifnβ-specific isgs are regulated by isgf . investigations on the phosphorylation status of stat , stat and irf in chronic hiv- infection are underway. in conclusion, we demonstrate that diverse ifn-is trigger non-overlapping interferomes in a single cell type, providing strong evidence for qualitative differences between the ifn-is. furthermore, conserved and qualitative differences in interferome induction correlated with clinically relevant markers of immunopathogenesis during chronic hiv- infection. further studies on the differences between the ifn-is and how these cytokines regulate distinct gene expression profiles in various tissue compartments could inform strategies to harness these biologicals and/or block these responses for hiv- control. the in vitro studies utilized disaggregated cells from macroscopically normal jejunum tissue that would otherwise be discarded. these tissues were obtained from patients undergoing elective surgery at the university of colorado hospital. the patients signed a release form for the unrestricted use of tissues for research following de-identification to laboratory personnel. the colorado multiple institutional review board approved the procedures and have given exempt status. colon pinch biopsies from pwh and age/gender-matched hiv-uninfected controls were obtained from archived samples from a completed, comirb-approved clinical study. this clinical study included pwh and hiv-uninfected controls, but archived colon pinch biopsies were available only for pwh and controls, respectively. study participants signed an informed consent. clinical parameters that include blood cd -t cell counts (cells/ μl), plasma viral load, tissue hiv rna (per cd t cell), tissue cd t cells (% viable cd + cells), il- (pg/ml), crp (μg/ml), ifabp (pg/ml), scd (u/ml), cd (ng/ml), lps (pg/ ml), lta (optical density), gut ifnα and ifnβ transcripts, were reported previously [ ]. ifn-is (pbl assay science, piscataway nj) were frozen into small aliquots for single-use. ilite type i ifn assay ready cells, purchased from svar life science ab (malmõ, sweden, cat# bm ) were seeded into a -well flat-bottomed cell culture plate and maintained in complete dmem (with % fbs and % penicillin-streptomycin and l-glutamine). various doses of recombinant ifn-is were added to corresponding wells. after h, the cells were lysed and luciferase activities were developed using bright-glo luciferase assay system (promega, madison, wi, cat# e ) according to manufacturer's instruction. luciferase activity was subsequently measured using a perkin-elmer victor x plate reader. % effective concentrations were computed using dose-response curve in prism . . hiv- bal (nih aids reagent program cat# ) was prepared in molt -ccr cells and titrated using an hiv- p elisa (advanced bioscience laboratories, rockville, md). ng p of hiv- bal / lpmcs (n = donors) was spinoculated in the presence or absence of titrated doses of ifnα subtypes and ifnβ (pbl assay science). the cells were harvested at d and analyzed by intracellular p flow cytometry as previously described [ , ] . % inhibitory concentrations and vres were calculated using a one-phase decay equation in prism . as previously described [ , ] . cd + t cells were purified from lpmcs (n = donors) by negative selection using easysep™ human cd + t cell isolation kit (stemcell, vancouver, bc, canada, cat# ) and followed by flow sorting to reach a purity greater than %. x of these purified cd + t cells were treated with mock, pg/ml ifnα , or an equivalent isre-activity of ifnα , , , and ifnβ. after h, rna was extracted using rneasy mini kit (qiagen, germany, cat# ). rnaseq libraries were constructed from ng rna using quantseq ' mrnaseq library prep kit (lexogen, austria, cat# . ). the rnaseq library quality was verified using bioanalyzer (agilent, santa clara, ca) before loaded into a hiseq by the genomics and microarray sequencing core facility at the university of colorado anschutz medical campus. the -well array contained primers for target genes isg , arhgef , lat and ahnak (s fig) , a reverse transcription control, a genomic dna control and a positive pcr control as well as the housekeeping gene gapdh (pph f). ninety-six-well custom rt profiler pcr arrays were performed according to the manufacturer's instructions (qiagen, valencia, type i interferons in mucosal hiv- infection california, usa) using the bio-rad cfx- real-time pcr instrument (bio-rad, hercules, california, usa) and bio-rad cfx manager software (ver. . ). cdna templates were mixed with ready-to-use rt qpcr master mixes and μl of the pcr component mix was aliquoted into each well containing predispensed gene-specific primer sets. each plate was loaded with cdna from individual samples. gene expression was normalized according to the average expression level of gapdh in the same sample. rna ( ng) from the colon pinch biopsies were used to prepare the rnaseq libraries. the rnaseq work flow of library construction, quality control, and the subsequent sequencing were similar to that of ifn-i treated gut cd + t cells as aforementioned. rnaseq data were downloaded as fastq files and their quality examined and visualized using fastqc (babraham institute, https://www.bioinformatics.babraham.ac.uk/projects/ fastqc). the removal of illumina sequencing adapters and the by-base quality screening was performed with cutadapt (https://cutadapt.readthedocs.io/en/stable). the quality screened fastq files were mapped to the current human genome assembly grch .p using hisat (johns hopkins university, https://ccb.jhu.edu/software/hisat /index.shtml), taking into account the ensembl id, gene symbol and gene length. raw gene expression counts were extracted using featurecounts from the subread package (walter+eliza hall institute of medical research, http://subread.sourceforge.net). rnaseq raw counts were obtained for gut cd t cells treated with ifn-is (ifnα , , , , and ifnβ) and mock from donors. gene counts were normalized using transcripts per million (tpm). principal component analyses necessitated removal of one donor as the transcriptome of the mock condition was extremely skewed relative to the other donors (s a fig). using edger differential expression analysis, ifn regulated genes (irgs) were defined based on a . -fold change (fc) cutoff relative to mock and a false-discovery rate (fdr) � %. the rnaseq raw counts data had gene-level read counts for hiv- -uninfected donors and pwh. lowly expressed genes which have average read counts less than per library were filtered out. as a result, out of genes were kept for further analysis and gene counts were normalized using the trimmed mean of m values (tmm) normalization method from edger (version . . ) in r (version . . ) with the "estimatedisp" function and its default settings. the tmm method calculates the scaling normalization factor for each sample by accounting for library size and observed counts, while under the assumption that the majority of genes are not de. in our case, the tmm method provided the best normalization results in terms of removing the unwanted variation, according to the relative log expression (rle) plots (s fig) . two-group differential expression (de) analysis was performed to test for differences between healthy controls and pwh, using normalized counts in edger with default settings and false discovery rate (fdr) of % to control for multiple testing. the de analysis was done in edger by the exact test using the "exacttest" function and its default settings for two-group design. the benjamini-hochberg procedure (bh step-up procedure) controlling the fdr was used as the multiple testing correction method in this study. the normalized rnaseq counts were further transformed by the variance stabilization and bias reduction method called "regularized log transformation" (rlog) from deseq (version . . ) [ ] . the transformed counts were then used as the outcome in a multivariate linear regression model. specifically, the linear regression models were fit in a gene by gene fashion to test the correlation between rna expression levels and immunopathogenic markers of chronic hiv- infection, with one marker included in the model at a time, while adjusting for age and gender. based on the results of de analysis, significantly altered genes from core-isgs and ifnβ-specific isgs were selected to test the associations with clinical parameters through the above model, separately. several immunopathogenic markers were used in this part, including gut ifnβ transcript levels, gut cd t cell percentages, blood cd t cell counts, plasma il levels and plasma lps levels. genes were considered as significantly associated with the corresponding clinical parameter with an fdr cutoff at %. in addition, the difference in proportions of significant genes in each gene list were tested with chi-squared test in r, as well as the difference in proportions of positive correlations of each gene list. all the plots were generated by ggplot (version . . ) package in r (version . . ). next generation sequencing data were deposited in the sequence read archive prjna (interferome dataset) and prjna (colon pinch biopsies). these were also deposited in the gene expression omnibus archive with accession numbers gse (interferome dataset) and gse (colon pinch biopsies). (left panels) ifnα -specific genes (n = ) were classified based on significant induction/suppression in ifnα -treated cells relative to mock at a % fdr cut-off. however, when these genes were evaluated in transcriptome datasets for ifnα , ifnα , ifnα and ifnα , most had fdr values > %, suggesting that these ifnα -specific genes were not even close to being statistically-significant in these other ifnα subtypes. (right panels) evaluation of ifnα -specific genes against gene datasets for ifnα , ifnα , ifnα and ifnα . bars correspond to the frequency of genes that fell within the fdr values noted in the x-axis. type i interferons in mucosal hiv- infection the interferons: characterization and application interferon alpha subtypes in hiv infection the significance of type-i interferons in the pathogenesis and therapy of human immunodeficiency virus infection the jak-stat pathway at twenty interferome: the database of interferon regulated genes interferon-alpha subtypes in an ex vivo model of acute hiv- infection: expression, potency and effector mechanisms interferon alpha subtype-specific suppression of hiv- infection in vivo gene therapy with plasmids encoding ifn-beta or ifn-alpha confers long-term resistance to hiv- in humanized mice dose-dependent differences in hiv inhibition by different interferon alpha subtypes while having overall similar biologic effects. msphere binding and activity of all human alpha interferon subtypes evolutionary genetic dissection of human interferons. the journal of experimental medicine interferon-alpha subtype activates nk cells and enables control of retroviral infection type i interferon differential therapy for erythroleukemia: specificity of stat activation anti-retroviral effects of type i ifn subtypes in vivo type i interferon responses in rhesus macaques prevent siv infection and slow disease progression reduced chronic lymphocyte activation following interferon alpha blockade during the acute phase of simian immunodeficiency virus infection in rhesus macaques blockade of chronic type i interferon signaling to control persistent lcmv infection persistent lcmv infection is controlled by blockade of type i interferon signaling interferons and interferon (ifn)-inducible protein during highly active anti-retroviral therapy (haart)-possible immunosuppressive role of ifn-alpha in hiv infection chronic cd + t-cell activation and depletion in human immunodeficiency virus type infection: type i interferon-mediated disruption of t-cell dynamics downregulation of robust acute type i interferon responses distinguishes nonpathogenic simian immunodeficiency virus (siv) infection of natural hosts from pathogenic siv infection of rhesus macaques an altered intestinal mucosal microbiome in hiv- infection is associated with mucosal and systemic immune activation and endotoxemia edger: a bioconductor package for differential expression analysis of digital gene expression data differential expression analysis of multifactor rna-seq experiments with respect to biological variation moderated estimation of fold change and dispersion for rna-seq data with deseq rle plots: visualizing unwanted variation in high dimensional data tsg and the vacuolar protein sorting pathway are essential for hiv- budding viral protein r regulates docking of the hiv- preintegration complex to the nuclear pore complex induction of apobec g ubiquitination and degradation by an hiv- vif-cul -scf complex usp -based negative feedback control is induced by type i and type iii interferons and specifically inactivates interferon alpha response unc b restricts systemic lethal inflammation by orchestrating toll-like receptor and trafficking gut dendritic cell activation links an altered colonic microbiome to mucosal and systemic t-cell activation in untreated hiv- infection brief report: inflammatory colonic innate lymphoid cells are increased during untreated hiv- infection and associated with markers of gut dysbiosis and mucosal immune activation low abundance of colonic butyrateproducing bacteria in hiv infection is associated with microbial translocation and immune activation enhancement of hiv- infection and intestinal cd + t cell depletion ex vivo by gut microbes altered during chronic hiv- infection elevated cd antigen expression on cd + t cells is a stronger marker for the risk of chronic hiv disease progression to aids and death in the multicenter aids cohort study than cd + cell count, soluble immune activation markers, or combinations of hla-dr and cd expression nlr family member nlrc is a transcriptional regulator of mhc class i genes roles, function and relevance of lag in hiv infection modulation of the helicase activity of eif a by eif b, eif h, and eif f tgf-beta signaling from receptors to smads selenoproteins: molecular pathways and physiological roles premature activation of the slx complex by vpr promotes g /m arrest and escape from innate immune sensing nucleoporin contributes to homologous recombination repair and post-replicative dna integrity the dna repair endonuclease mus facilitates fast dna replication in the absence of exogenous damage functional comparison of ifn-alpha subtypes reveals potent hbv suppression by a concerted action of ifn-alpha and -gamma signaling identification of genes differentially regulated by interferon alpha, beta, or gamma using oligonucleotide arrays ifn-beta induces greater antiproliferative and proapoptotic effects and increased p signaling compared with ifn-alpha in pbmcs of adult t-cell leukemia/lymphoma patients the interferons: years after their discovery, there is much more to learn stimulation of hiv- -specific cytolytic t lymphocytes facilitates elimination of latent viral reservoir after virus reactivation hiv infection does not alter interferon alpha/beta receptor expression on mucosal immune cells the transcriptome of hiv- infected intestinal cd + t cells exposed to enteric bacteria soluble cd made by monocyte/macrophages is a novel marker of hiv activity in early and chronic infection prior to and after anti-retroviral therapy. the journal of infectious diseases irf and unphosphorylated stat cooperate with nf-kappab to drive il expression the chaperone unc b regulates toll-like receptor stability independently of endosomal tlr transport unphosphorylated isgf drives constitutive expression of interferon-stimulated genes to protect against viral infections ifnbeta-dependent increases in stat , stat , and irf mediate resistance to viruses and dna damage cd + t-cell death induced by infectious and noninfectious hiv- : role of type interferon-dependent, trail/dr -mediated apoptosis type i interferon contributes to cd + t cell depletion induced by infection with hiv- in the human thymus development of a novel site-specific pegylated interferon beta for antiviral therapy of chronic hepatitis b virus interferon-beta and interferon-lambda signaling is not affected by interferon-induced refractoriness to interferon-alpha in vivo triple combination of interferon beta- b, lopinavir-ritonavir, and ribavirin in the treatment of patients admitted to hospital with covid- : an open-label, randomised, phase trial ifn-i response timing relative to virus replication determines mers coronavirus infection outcomes dysregulated type i interferon and inflammatory monocyte-macrophage responses cause lethal pneumonia in sars-cov-infected mice unphosphorylated stat prolongs the expression of interferon-induced immune regulatory genes microbial exposure alters hiv- -induced mucosal cd + t cell death pathways ex vivo we thank eric lee and christine purba for expert technical assistance with the lamina propria aggregate culture model, and cheyret wood for helpful statistical advice. we also thank the patients who agreed to use discarded tissue for research purposes and the clinical study participants. key: cord- -yr jwm authors: karnam, guruswamy; rygiel, tomasz p.; raaben, matthijs; grinwis, guy c. m.; coenjaerts, frank e.; ressing, maaike e.; rottier, peter j. m.; de haan, cornelis a. m.; meyaard, linde title: cd receptor controls sex-specific tlr responses to viral infection date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: yr jwm immunological checkpoints, such as the inhibitory cd receptor (cd r), play a dual role in balancing the immune system during microbial infection. on the one hand these inhibitory signals prevent excessive immune mediated pathology but on the other hand they may impair clearance of the pathogen. we studied the influence of the inhibitory cd -cd r axis on clearance and pathology in two different virus infection models. we find that lack of cd r signaling strongly enhances type i interferon (ifn) production and viral clearance and improves the outcome of mouse hepatitis corona virus (mhv) infection, particularly in female mice. mhv clearance is known to be dependent on toll like receptor (tlr )-mediated type i ifn production and sex differences in tlr responses previously have been reported for humans. we therefore hypothesize that cd r ligation suppresses tlr responses and that release of this inhibition enlarges sex differences in tlr signaling. this hypothesis is supported by our findings that in vivo administration of synthetic tlr ligand leads to enhanced type i ifn production, particularly in female cd (−/−) mice and that cd r ligation inhibits tlr signaling in vitro. in influenza a virus infection we show that viral clearance is determined by sex but not by cd r signaling. however, absence of cd r in influenza a virus infection results in enhanced lung neutrophil influx and pathology in females. thus, cd -cd r and sex are host factors that together determine the outcome of viral infection. our data predict a sex bias in both beneficial and pathological immune responses to virus infection upon therapeutic targeting of cd -cd r. to generate an appropriately controlled response during infections, the immune system is balanced by the action of activating and inhibitory receptors. lack of inhibition leads to excessive inflammation and autoimmunity and other severe disease symptoms. one of the receptors regulating this balance is cd receptor (cd r) [ ] . cd r was originally described as a myeloid receptor [ ] , being expressed on macrophages, granulocytes and dcs, but later we and others recognized that it is also expressed on t cells, b cells and nk cells [ , ] . the cd r intracellular domain is devoid of the classical immunoreceptor tyrosine-based inhibition motif (itim) present in most immune inhibitory receptors but it does have three tyrosine residues that can be phosphorylated, one of which is embedded in an npxy motif. cd r-downstream signaling is dependent on the recruitment of dok and rasgap [ ] . the signal that triggers cd r and results in delivery of an inhibitory intracellular signal to the cell is given by its ligand cd , which has a short intracellular tail devoid of any known signaling motifs. cd is expressed on thymocytes, activated t cells, b cells, dendritic cells (dcs), vascular endothelial cells, hair follicular cells, in the central nervous system and in the retina (reviewed in [ ] ). both in mice and humans, cd exclusively binds to the inhibitory cd r. in contrast to humans, the mouse cd r family contains several activating receptors, but these do not bind cd [ ] . cd / mice were first described to be more susceptible to autoimmune disorders [ ] . later its role in microbial infections was recognized. infection of cd / mice with the gram negative n. meningitides causes increased lethality, proinflammatory cytokine production and lymphocyte activation [ ] . we and others showed that in mouse influenza a virus infection cd deficiency aggravates immune pathology [ , ] . these studies were exclusively performed in female mice. they indicate that cd -cd r signaling controls the strength of the initial antimicrobial response and the return to homeostasis. we here studied the influence of cd -cd r blockade on clearance and pathology in two different virus infection models, coronavirus and influenza virus, in both male and female mice. mouse hepatitis coronavirus (mhv) is an accepted model for the most illustrious coronavirus (cov): severe acute respiratory syndrome (sars)-cov. host control of mhv infection is completely dependent on an immediate type i ifn response, initiated upon tlr triggering by viral rna. mice lacking this pathway show massive mhv replication and fatal infection within a few days [ , ] . as a model where a strong anti-viral response causes immune mediated pathology we studied influenza a virus infection in which immune pathology is known to be important for clinical outcome. we here report that lack of cd r signaling has a more profound effect on the beneficial but also on the pathological immune responses to viruses in female mice as compared to male mice, which can be attributed to the capacity of cd r to inhibit tlr responses. to determine the role of cd -cd r signaling in cov infection, we intraperitoneally inoculated male and female wild type (wt) and cd / mice with a recombinant mhv encoding luciferase (mhv-eflm). we monitored viral spread using bioluminescence imaging (bli) at day and after infection [ , ] . interestingly, at both time points we observed a decreased viral spread in female wt mice when compared to males. moreover, lack of cd resulted in a significantly lower level of viral replication in females ( figure a ,b and figure s a ,b,c). the viral rna load in the livers at day was assessed by quantitative pcr and confirmed the imaging results: wt female mice had significantly lower viral rna levels than wt male mice ( figure c) . again, cd -deficiency greatly decreased virus load in female mice. this was confirmed in histological liver sections stained with a monoclonal antibody against mhv (data not shown). the number of focal lesions in the liver, typical for mhv was also lower in female mice and again cd -deficiency had a significant effect on these lesions only in female mice ( figure d,e) . clearance of mhv critically depends on tlr -mediated type i ifn production by hematopoietic cells [ , ] . in wt mice, mhv infection resulted in detectable ifn-a production only in female mice ( figure a) . in cd / animals, all females and out of males produced detectable amounts of ifn-a and cd / female mice produced the highest amounts of ifn-a ( figure a ). ifn-a concentrations in serum inversely correlated with viral load at day (p = . ) (data not shown). thus, the combination of female sex and cd deficiency results in increased type i ifn production and decreased viral load and pathology upon mhv infection. enhanced tlr responses in female cd / mice sex differences in tlr responses have previously been reported for humans [ , ] . our observed sex difference in ifn-a production and viral clearance upon mhv infection in mice is likely due to a similar sex bias in tlr responses. we hypothesized that cd r signaling suppresses tlr responses in wt mice. release of this inhibition would further reveal the intrinsically higher tlr responses in females and result in more rapid viral clearance. to test this hypothesis, we first administered a tlr ligand in vivo. as described previously [ ] , intraperitoneal injection of a synthetic tlr ligand (imiquimod) leads to rapid release of type i ifn into the circulation. one-hour after injection we only detected significant amounts of ifn-a in the sera of female cd / mice, confirming the notion that cd r inhibits the intrinsic potential for a higher tlr response in females ( figure b ). we sacrificed the mice at hrs after ligand injection, at which time point no serum ifn-a was detectable (data not shown) but type i ifn mrna could be detected in the livers of these mice. again, females expressed more ifn-b mrna than males, with cd -deficient female mice displaying even more elevated ifn-b mrna levels ( figure c) . thus, release of cd -mediated inhibition leads to increased production of type i ifn in response to tlr ligands, particularly in female mice. we next tested whether cd r-mediated signaling directly inhibits signals transduced via tlr . we generated a chimeric construct containing a lair- receptor in which the intracellular tail was replaced by that of cd r. this allows for efficient cross-linking using anti-lair- antibodies to induce signaling via the cd r cytoplasmic tail. we transfected hek cells with plasmids encoding a lair- -cd r chimeric receptor, human tlr and a luciferase reporter under control of a nf-kb driven promoter. cross-linking of the chimeric receptor by anti-lair- antibody, but not by isotype-matched control antibody, resulted in robust inhibition of imiquimod-induced nf-kb activity ( figure a ). cd r contains three intracellular tyrosine residues. a chimeric lair- -cd r protein in which all three tyrosines are mutated to phenylalanine (fff) did not suppress tlr responses upon cross-linking, indicating that the observed inhibitory effect is indeed dependent on cd r-signaling ( figure a ). in a cell line with stable ectopic expression of tlr and transient expression of the nf-kb-reporter and the lair- -cd r constructs we also observed that tlr signaling was inhibited through cd r-mediated signaling. a similar effect was observed when luciferase expression was driven by an ifn-b promoter ( figure b) . thus, the enhanced type i ifn production and viral clearance of mhv in female cd / mice can be explained by the release of cd r-mediated inhibition of the intrinsically higher tlr responses in females. a strong anti-viral response can also cause immune mediated pathology that can be detrimental to the host. we therefore moved to a virus infection model in which immune pathology is known to immune responses need to be carefully orchestrated to prevent disease due to an overactive immune system. immunological checkpoints are provided by immune inhibitory receptors, which set a threshold for activation and dampen the immune system. in the case of a viral infection, this prevents pathology induced by the immune system, but on the other hand may prevent adequate removal of the virus. in this paper, we show that removal of such an immunological checkpoint in mice leads to rapid removal of corona virus, but also to more immuneinduced disease symptoms in case of influenza virus infection. we observe this predominantly in female mice. we demonstrate that this particular checkpoint inhibits anti-viral responses that are naturally stronger in females. release of this checkpoint enlarges these sex differences. our findings have major implications for therapeutic use of blockers of this pathway, which are currently in clinical trials for the treatment of cancer, as we predict that female patients will have a stronger response to such therapeutics. be important for clinical outcome. upon intranasal infection with influenza a virus we again observed a sex bias in the viral load, measured in the lungs at day post infection ( figure a ). female mice had lower viral loads compared to male mice, which was accompanied by enhanced ifn-a concentrations in the broncho-alveolar lavage (bal) fluid ( figure b ). however, as opposed to mhv infection, cd -deficiency did not enhance type i ifn production in influenza virus infection ( figure b ). confirming previous reports, we found a significantly enhanced body weight loss in female cd / mice compared to wt females ( figure c ) [ , ] . although male cd / mice lost more weight than wt males, the weight loss started later and the difference was not as prominent ( figure d ). this may indicate that lack of cd results in a more severe pathology in females. we observed an increased level of cellular infiltration in lung tissue of females ( figure s a ). therefore we determined lung cellular influx by differential cell count in bal fluid ( figure s b-d) . the total number of cells in bal fluid was higher in all female groups but differences were not significant ( figure s b ). the number of lymphocytes was increased in females of both genotypes ( figure s d ). at day after infection, the number of lymphocytes was increased in females of both wt and cd / groups. at day , the number of neutrophils in the bal fluid was decreased in all groups, still the wt females displayed elevated numbers over males. importantly, lack of cd resulted in significantly higher neutrophil counts in females but not in males ( figure e ). as an additional parameter of lung damage we measured the total protein content in the bal fluid. lack of cd resulted in increased protein levels in the bal fluid of both males and females, especially at day after infection ( figure f ). overall these data indicate that female mice experience increased lung pathology upon influenza a virus infection, which is aggravated by the lack of the cd r-regulatory pathway. in agreement with the increased neutrophil counts we measured elevated levels of kc (il- ) in the bal fluid ( figure g ). il- concentrations were increased in all females at and days after infection, which was further enhanced by the lack of cd only at day ( figure h ). tnf-a was hardly detectable, but significantly increased levels were measured in cd / female mice at day after infection ( figure i ). thus, from two different viral infection models we can conclude that sex has a profound effect on type i ifn production and viral clearance. this study is the first to report a significantly enhanced viral clearance in female mice due to a sex bias in tlr responses. sex differences in tlr induced type i ifn production have previously been reported for humans [ , ] and our data show that in mice this has a strong impact on the course of a viral infection. the mechanism for this is not understood. on the one hand, incomplete inactivation of the tlr gene, on the x chromosome, resulting in higher tlr expression in females has been proposed [ , ] . in our experiments, tlr mrna expression was equal in male and female mice ( figure s ). this is consistent with a prior report in which no evidence for escape from x-inactivation of the tlr gene in humans was found [ ] . there are conflicting reports concerning the influence of sex hormones on tlr responses [ , ] . alternatively, sex-dependent epigenetic mechanisms may contribute [ ] . we demonstrate direct inhibition of tlr signaling through cd r. previously, cd r-mediated inhibition of lpsinduced cytokine production was reported [ , , ] . this suggests that cd r affects proximal events in the tlr signaling pathway. cd r is a unique inhibitory receptor, since its intracellular tail does not contain itims. cd r does contain three intracellular tyrosine residues. mutation of all three tyrosine completely abrogates its inhibitory function [ ] . the most distal tyrosine is located in an npxy motif, to which the adaptor molecule dok is recruited [ ] . dok activates rasgap and knockdown of these proteins diminishes the inhibitory action of cd r [ ] . however, the down-stream targets for cd r mediated inhibition are not yet identified. upon influenza a virus infection, cd -deficiency strongly enhances neutrophil influx into the lungs of female mice possibly leading to pathology, but it does not affect viral clearance and type i ifn production. this implies that, for influenza virus, the sexbiased type i ifn production and viral clearance are not regulated by cd r, while the events leading to increased neutrophil recruitment and lung pathology are. neutrophil responses to influenza virus infection were shown to be dependent on tlr [ ] . since neutrophils express cd r, the strongly increased neutrophil influx in female cd / mice is in line with our finding that cd r inhibits sex-biased tlr responses. in contrast to mhv infection, clearance of influenza a virus is not dependent on plasmacytoid dendritic cells [ ] . although influenza rna triggers tlr [ ] , the main source of type i ifn is the infected respiratory epithelium [ ] . these cells do not express cd r and hence are not influenced by cd -deficiency, explaining the lack of effect of cd -deficiency on type i ifn production. there is emerging evidence that tumor cells employ immunological checkpoints for their benefit. as a result of this, inhibitory immune pathways have become therapeutic targets to strengthen anti-tumor responses and develop (adjuvant) therapeutic strategies in cancer treatment. the successful application of anti-ctla (cytotoxic t-lymphocyte antigen ) in melanoma is followed up with blocking agents for other checkpoints, among which the cd -cd r immune inhibitory pathway. strong evidence for a role for cd in tumor progression comes from studies in patients. expression of cd is an independent prognostic factor for multiple myeloma and acute myeloid leukemia predicting worse overall survival of these patients [ , ] . a clinical trial with a blocking anti-cd antibody aims to enhance anti-tumor responses towards cd -expressing malignancies (clinical-trials.gov identifier: nct ). on the basis of our data, one of the predicted side effects would be severe immune pathology to infections. our finding that the combination of lack of cd r signaling and female sex has such a profound impact on the control of virus infection as well as on immune pathology raises some important issues. we are the first to demonstrate a strong sex bias in type i ifn production and viral clearance in mice utilizing two different models of virus infection. this is of importance for scientists studying these widely used models and may result in a completely different interpretation of data obtained, depending on the sex of the mice used. moreover, sex biased clinical responses to virus infections have been reported in humans [ ] . for influenza a virus (h n ) a significantly enhanced case-fatality rate was found in women [ ] . in agreement with these findings we now show increased lung damage, enhanced neutrophil influx and elevated il- , il- levels in bal fluid of female mice upon influenza a virus infection. a few reports discuss the possibility of a sex bias in severity of sars-cov infection [ , ] . also for hiv- infection, sex-related differences have been well-established [ , ] . our results underscore the importance of the issue of sex bias in scientific research, clinical trails, and vaccine studies, previously raised by others [ , ] . particularly, cd blocking antibodies are currently entering clinical trials for cancer treatment. our data point to a possible pathological outcome of e.g. influenza virus infection in women as a result of cd blocking therapies. wild-type c bl/ j mice and cd / mice, which were made and maintained on a full c bl/ j background [ ] , were bred at the specified pathogen free (spf) unit at the utrecht university central animal laboratory and used between and weeks of age. mice were injected intraperitoneally with tcid of mhv strain a expressing the firefly luciferase (fl) reporter gene (mhv-eflm) [ ] in ml pbs. intranasal infection with . tcid of influenza strain a/hk/ / was performed as described [ ] . mice were monitored once every hours for symptoms of illness. in additional experiments we injected the mice intraperitoneally with the tlr agonist imiquimod (invivogen; mg in ml pbs). the utrecht university ethical committee for animal experimentation approved the animal study protocols, in accordance with the advice of the central committee on animal experimentation ( januari ) and the dutch law on animal experimentation (art. a). after mhv-eflm injection (day ), mice were imaged at day and day as described previously [ ] with minor modifications. briefly, mice were anaesthetized with isoflurane and subsequently injected with ml of the fl substrate d-luciferin (synchem figure . stimulation of cd r directly inhibits tlr mediated nfkb activity. a hek t cells were transiently transfected with tlr , nf-kb luciferase reporter and lair- -cd r chimera or signaling-defective lair- -cd r-fff constructs. twenty-four hours later cells were stimulated with control or anti-lair- antibody. forty-eight hours after transfection cells were stimulated with the tlr ligand imiquimod ( . mg/ml). seventytwo hours after transfection cells were harvested, luciferase activity, and total protein content were determined. protein normalized, luciferase activity from independent experiments is shown. b hek t cells with stable expression of tlr were transiently transfected with a nf-kb luciferase reporter construct or an ifnb luciferase reporter construct and a lair- -cd r chimera. tlr stimulation and cd r crosslinking was performed as in (a). protein normalized with luciferase activity from independent experiments is shown. mean sem is shown. statistical significance was calculated with mann-whitney test. ns = not significant. * = p, . . doi: . /journal.ppat. .g laborgemeinschaft ohg) dissolved in pbs ( mg/kg). mice were positioned to the ventral side in a specially designed box and placed onto the stage inside the light-tight photon imager (biospace laboratory). five mice were imaged simultaneously exactly min after the injection of d-luciferin. the biolumines-cence signals were acquired with photovision software (biospace laboratory) over a -min interval and are expressed as integrated light intensity (photons/min). a low-intensity visible light image was generated and used to create overlay (heatmap) images for each individual animal. tissue homogenization and isolation of total rna whole livers or left lungs were dissected from the mice. the tissues were processed in lysing matrix d tubes (mp biomedical), containing ml of pbs, using a fastprep instrument (mp biomedical). the tissues were homogenized at g for sec and immediately placed on ice. subsequently, the homogenates were centrifuged at g for minutes at uc and supernatants were harvested and stored at uc. total rna was isolated from the homogenates using the trizol reagent (invitrogen) according to manufacturer's instructions. gene expression levels of ifn-a, ifn-b , and tlr were measured by quantitative pcr using lightcycler rna master hydrolysis probes in combination with a lightcycler system (both from roche applied science), according to the manufacturer's instructions. the housekeeping gene gapdh was used as a reference in all experiments, and expression of this gene was found relatively constant among samples. the amounts of mhv rna were determined by quantitative rt-pcr using primers and probe directed against the n gene of mhv-a [ ] . for the influenza rna quantification the primers mapping to the influenza a nucleoprotein (n) gene were used. amplification and detection were performed with an abi prism system. samples were controlled for the presence of possible inhibitors of the amplification reaction by internal control (murine encephalomyocarditis virus dna). bronchoalveolar lavage (bal) fluid was obtained by flushing the lungs two times with ml pbs using a canula inserted into the trachea, yielding around . ml bal fluid. pelleted cells from bal fluid were counted and cytospins were prepared and stained with may-grunwald/giemsa and neutrophils were scored on the basis of morphology (dade behring, switzerland). bal fluids were kept on ice or stored at uc until further processing. bal fluid was centrifuged, and ml of aliquot was used to determine the protein concentration with a bca kit (pierce) according to the manufacturer's instructions. to measure the interferon concentration in the sera, blood was sampled from naïve mice or four days after mhv infection or one hour after imiquimod treatment. sera were separated by spinning the blood at g for livers of mhv-infected mice were sampled, fixed in % neutral buffered formalin, and embedded in paraffin. seven mm liver sections were stained with hematoxylin and eosin. total liver sections were examined by light microscopy and foci of hepatocellular necrosis and inflammation were scored in a semiquantitative manner. hek t cells were transiently co-transfected with: human tlr (kindly provided by rogier sanders, amc, amsterdam, the netherlands), and nf-kb-reporter or ifna-reporter constructs, kindly provided by dr paul moynagh (national university of ireland). a chimeric construct containing the extra cellular region of human lair- (amino acids - ) fused with the transmembrane and intracellular rat cd r (rcd r) (amino acids - ) was cloned into pcdna . /zeo (invitrogen, breda, the netherlands). a tyrosine (y) to phenylalanine (f) mutant of tyrosines , , and in the intracellular rcd r tail were generated with pcr-based mutagenesis. the mutant was cloned into the same vector and all sequences were confirmed by automated dna sequencing. the lair-cd r plasmid was co-transfected with the tlr and reporter constructs. twentyfour hours after transfection cells were trypsinized and seeded in -well plates coated with ug/ml anti-lair- monoclonal antibody (clone a ). forty-eight hrs after transfection cells were stimulated with imiquimod mg/ml (invivogen) in pbs. on the next day, cells were lysed with passive lysis buffer (promega), luciferase activity was measured on a luminometer (berthold technologies centro lb ), and data were analyzed with microwin software. total protein content was determined with a pierce bca protein assay (thermo scientific). all luciferase values were normalized to protein concentration. alternatively, we used hek cells stably expressing human tlr (invivogen). significance was calculated with mann-whitney test using graphpad prism software. online supplemental material figure s shows bli measurement in naïve mice and viral spread of mhv at day after infection. figure s depicts the quantification of lung pathology and differential cell count in the bal fluid in influenza a virus infected mice. figure s is a quantitative analysis of tlr mrna in male and female mice. the nuclear lung surface area was used as a benchmark of the inflammatory response, its color range selections were measured thrice. the relative nuclear surface area (nuclear surface area/lung surface area * ) of the lung sections was used as a measure of the tissue response to exposure to the influenza virus (b) total cell count in bal fluid. quantification of monocyte (c) and lymphocyte (d) numbers in bal fluid by differential cell count. in all panels mean sem is shown, statistical significance was calculated with mann-whitney test. * = p, . , ** = p, . . (doc) figure s no sex difference in expression of tlr mrna. four days after mhv injection mice were sacrificed, rna was isolated from livers and tlr mrna expression was quantified by qpcr in male and female wt and cd / mice. mean sem is shown. (doc) cd and its receptors as targets for immunoregulation the leukocyte/ neuron cell surface antigen ox binds to a ligand on macrophages characterization of the cd receptor family in mice and humans and their interactions with cd the inhibitory cd r is differentially expressed on human and mouse t and b lymphocytes essential roles for dok and rasgap in cd receptor-mediated regulation of human myeloid cells cd and membrane protein interactions in the control of myeloid cells recombinant cd protein does not bind activating proteins closely related to cd receptor downregulation of the macrophage lineage through interaction with ox (cd ) immune inhibitory ligand cd induction by tlrs and nlrs limits macrophage activation to protect the host from meningococcal septicemia a critical function for cd in lung immune homeostasis and the severity of influenza infection lack of cd enhances pathological t cell responses during influenza infection control of coronavirus infection through plasmacytoid dendritic-cell-derived type i interferon hematopoietic cell-derived interferon controls viral replication and virusinduced disease non-invasive imaging of mouse hepatitis coronavirus infection reveals determinants of viral replication and spread in vivo the proteasome inhibitor velcade enhances rather than reduces disease in mouse hepatitis coronavirus-infected mice tlr ligands induce higher ifn-alpha production in females sex differences in the toll-like receptor-mediated response of plasmacytoid dendritic cells to hiv- small anti-viral compounds activate immune cells via the tlr myd -dependent signaling pathway the xs and y of immune responses to viral vaccines x-inactivation profile reveals extensive variability in x-linked gene expression in females mice lacking cd r show absence of suppression of lipopolysaccharide-induced tumor necrosis factor-alpha and mixed leukocyte culture responses by cd long term potentiation is impaired in membrane glycoprotein cd -deficient mice: a role for toll-like receptor activation identification of tyrosine residues crucial for cd r-mediated inhibition of mast cell activation molecular mechanisms of cd inhibition of mast cell activation toll-like receptor-mediated activation of neutrophils by influenza a virus clearance of influenza virus from the lung depends on migratory langerin+cd b-but not plasmacytoid dendritic cells innate antiviral responses by means of tlr -mediated recognition of single-stranded rna differential type i interferon induction by respiratory syncytial virus and influenza a virus in vivo cd as a prognostic factor in acute myeloid leukaemia cd is a new prognostic factor in multiple myeloma the impact of sex, gender and pregnancy on h n disease the epidemiological and molecular aspects of influenza h n viruses at the human-animal interface in egypt sars in singapore-predictors of disease severity do men have a higher case fatality rate of severe acute respiratory syndrome than women do? the x-files in immunity: sex-based differences predispose immune responses sex differences in hiv- viral load and progression to aids sex bias in trials and treatment must end cyclooxygenase activity is important for efficient replication of mouse hepatitis virus at an early stage of infection we thank hans clevers, lewis lanier, louis bont, josé borghans and erik hack for critical discussions and review of versions of the manuscript. we also thank eva rijkers, rogier sanders and paul moynagh for providing plasmids, guus rimmelzwaan for influenza a virus, julia drylewicz for advice on statistics and alan rigter, robert de vries and marco viveen for their technical assistance. conceived and designed the experiments: gk tpr mr pjmr camh lm. performed the experiments: gk tpr mr gcmg fec. analyzed the data: gk tpr mr pjmr camh lm. contributed reagents/ materials/analysis tools: mer fec. wrote the paper: gk tpr mr camh lm. key: cord- - rit h authors: chinchio, eleonora; crotta, matteo; romeo, claudia; drewe, julian a.; guitian, javier; ferrari, nicola title: invasive alien species and disease risk: an open challenge in public and animal health date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: rit h nan protecting public and animal health. for example, empirical research on ias pathogens, which would be needed to assess the risk of infectious disease emergence, is skewed toward a few species (e.g., vector species like the tiger mosquito aedes albopictus) or toward selected pathogens known to harm biodiversity conservation, while a global vision of ias-associated health threats is still not available [ , [ ] [ ] [ ] . in this context, it is urgent to raise awareness in people working in the fields of animal and public health of the need to consider ias as a health threat. to this aim, we provide here an overview of how animal ias may affect local disease dynamics both directly and indirectly, i.e., acting as pathogen hosts or disrupting the recipient ecosystem structure, through real-case examples from the ecological literature, and, in the last paragraph, we propose future initiatives aimed at improving our capacity for targeted actions toward the ias most likely to threaten human and animal health, calling for an increased involvement of people working in the fields of animal and public health in a new invasion epidemiology field. ias may host pathogens that are absent in the area of release and cause their establishment and subsequent spillover to local species, possibly resulting in an increase of disease risk for humans, domestic animals, and native wildlife. the north-american raccoon procyon lotor, for example, introduced to central europe baylisascaris procyonis [ ] , a nematode causing larva migrans syndromes potentially inducing severe central nervous system disease in humans ( fig a) . introduction to europe of north-american crayfishes procambarus clarkii infected with the fungus aphanomyces astaci caused huge economic losses to fisheries, being the pathogen lethal to native crayfishes [ ] . similarly, squirrelpox virus, introduced to the united kingdom along with the american eastern gray squirrel sciurus carolinensis, is significantly contributing to the increased mortality of native red squirrels sciurus vulgaris [ ] . however, while pathogen co-introductions occur over a wide range of parasite and host taxa [ ] , some pathogens are lost during the invasion process [ ] : for example, there is no evidence for poxvirus in italian gray squirrel populations [ ] . pathogen loss may be due to the absence of the pathogen in the individuals of the founding populations or to its inability to survive to translocation or establish in the area of release. the outcome depends on several factors related to the ias (e.g., founding population origin), the pathogens (e.g., host specificity), and the area where the species is released (e.g., environmental conditions, presence, and density of local hosts) [ ] . as shown by a study on ectoparasites of introduced birds, factors related to transmission efficiency, such as the number of host introduced and host longevity, are likely to play a major role [ ] . an increase of local disease risk may also occur if the introduced ias is susceptible to, and able to transmit, local pathogens. pathogens acquired by ias may be amplified and possibly spill back to humans and local species [ ] . a case in point is the australian brushtail possum, trichosurus vulpecula, in new zealand ( fig b) . invasive possums probably became infected with mycobacterium bovis, the causal agent of tuberculosis in cattle, from wild deer, after the beginning of commercial deer hunting in . currently, they are the most important maintenance host for bovine tuberculosis, supporting higher transmission rates compared to local species and, being sympatric with cattle, providing interface for transmission between livestock and forest residents [ ] . another case is represented by invasive raccoon dogs nyctereutes procyonoides, which may amplify rabies circulation in eastern europe or cause its reemergence in currently rabies-free countries [ ] . ias competence for pathogen transmission plays a major role in defining the outcome of pathogen acquisition, and, as the possum-tuberculosis case exemplifies, it is the result of both ias-pathogen interaction (e.g., ias susceptibility, period of communicability, and pathogen excretion rate) and ias behavioral patterns (e.g., habitat, home range extension, and intraand interspecific contact rates). based on ias competence, the acquisition of a local pathogen may even lead to the reduction of disease risk (the so-called dilution effect [ ] ) or to no consequences at all. for example, in ireland, the invasive bank vole myodes glareolus has been found to divert fleas from the native wood mice apodemus sylvaticus, which is a more competent host for bartonella spp. [ ] . however, the identification of the contexts in which a dilution effect may occur is still highly debated in ecology, as it strongly depends on local host species diversity and on the interactions occurring between the species involved in the transmission cycle [ ] . introduced species may disrupt local infection dynamics also indirectly, i.e., nonacting as pathogen hosts but through competitive and trophic interactions with native species or modification of local habitats, thus altering the abundance and/or contact rates among local host species, parasite infective stages, or vectors. in southern florida, the invasive python python bivittatus caused the decrease of several mammal species, inducing the local mosquito vector of zoonotic everglades virus to feed almost exclusively on the virus' main reservoir host, the hispid cotton rat sigmodon hispidus, potentially leading to an increase in pathogen circulation (fig c) [ ] . an example of habitat alteration is given by the activity of invasive feral pigs sus scrofa on the island of hawaii: they create wallows and cavities in tree fern trunks improving habitat suitability for mosquito vectors for avian malaria plasmodium relictum [ ] , one of the main threats to native hawaiian forest birds' conservation. again, ias indirect effects on local infection dynamics are highly context dependent, and mechanisms presented so far may act in concert, producing unpredictable outcomes. in scotland and northern england, for example, the invasive gray squirrel has been found to harbor several local strains of borrelia burgdorferi [ ] . however, in those areas, gray squirrels are also causing the decline of another competent host for b. burgdorferi, the red squirrel, and the effect of these concurring mechanisms on human lyme disease risk remains unknown [ ] . during the last centuries, more than , ias introduction events have been recorded worldwide, and this number still presents an increasing trend [ ] . in such context, the identification of those species deserving priority attention, based on their actual and potential impacts, is essential to support decision-making [ ] . several tools to inform preventive and management actions on animal ias, including horizon scanning protocols, risk assessments, and impact assessments, have been developed in the last years (see [ ] for a recent review), but the majority of them focus on environmental impacts, not specifically considering disease emergence risks in humans and local animal populations [ , ] . some authors have called for a greater attention on the potential health risks posed by biological invasions [ ] [ ] [ ] ] , highlighting the need for a better integration between biological and health sciences, surveillance actions, and coordinated policies. we support their appeal, arguing that an increased awareness of people working in the fields of animal and public health on the risks concerning biological invasions and their consequent involvement in the invasion biology field is the first step toward a complementary invasion epidemiology field. such field would be integrated with invasion ecology, but more specifically aimed at the prevention of the emergence of diseases in human and animal populations consequent to ias introduction and establishment. to this aim, we propose some initiatives that should be addressed by future research work. a first major constraint in addressing the issue of disease emergence connected to ias is given by the lack of comprehensive data on pathogens affecting ias. in this sense, we recommend the gathering in ad hoc databases of all the available information on ias pathogens affecting human and animal health, including their geographical distribution and prevalence in ias populations, in both native and introduced ranges. it would also be advisable to improve our understanding of the key epidemiological events and factors driving the emergence of infectious diseases following ias establishment, for example, through ex-post analyses on the already established ias. in particular, as the emergence process of a disease is composed of several stages (introduction in a new area/host population, establishment, and spread) [ , , ] , the key factors involved in the process and related to ias biology, pathogenic features, and the biotic and abiotic components of the area of release should be identified for each of these stages. we also suggest urgently directing research efforts at developing transparent and flexible tools able to prioritize ias based on the risk of transmitting pathogens with the potential to impact the health of humans, production animals, and native wildlife. such tools could be based on the framework of the world organisation for animal health (oie)/international union for conservation of nature (iucn) for wildlife disease risk analysis and readapted to account for the main mechanisms through which alien species may affect local health, in particular the introduction of new pathogens and the acquisition and spread of local ones. the lack of data on ias pathogens is certainly an obstacle in underpinning in-depth risk assessments [ ] , in particular, quantitative ones. however, a simple and transparent qualitative disease risk assessment procedure would enable the prioritization of empirical research needed to cover these knowledge gaps, while at the same time guiding local health administrators in the allocation of resources for management and preventive actions toward ias. the issue related to irregular data availability could be partially overcome, as a first step, by eliciting opinions from experts. finally, awareness and action will be influenced by, and need to consider, the wider public perspective, not just researchers and institutions. initiatives aimed at sensitizing citizens about the health threats of ias will be needed to promote responsible behaviors when crossing borders and to improve the general public attitude toward ias control and eradication programs. all the suggested initiatives, to be successful, necessitate a stronger connection between ecologists, biologists, and other people working in the fields of animal and public health and beyond. only through wider collaboration and dialogue will the potential health impacts of biological invasions be fully appreciated and, perhaps, ameliorated. emerging infectious diseases of wildlife-threats to biodiversity and human health global trends in emerging infectious diseases alien species as a driver of recent extinctions invasive species database impact of the invasive brown marmorated stink bug in north america and europe: history, biology, ecology, and management aquatic invasive species and emerging infectious disease threats: a one health perspective the spread of invasive species and infectious disease as drivers of ecosystem change invasive species challenge the global response to emerging diseases invading with biological weapons: the importance of disease-mediated invasions is invasion success explained by the enemy release hypothesis? indirect effects of parasites in invasions parasites as drivers and passengers of human-mediated biological invasions co-invaders: the effects of alien parasites on native hosts parasites lost-do invaders miss the boat or drown on arrival? natural history of host-parasite interactions the effects of invasion on parasite dynamics and communities wildlife diseases: from individuals to ecosystems horizon scanning for invasive alien species with the potential to threaten biodiversity and human health on a mediterranean island alien species and public health impacts in europe: a literature review alien pathogens on the horizon: opportunities for predicting their threat to wildlife first detection of baylisascaris procyonis in wild raccoons (procyon lotor) from leipzig biological invaders in inland waters: profiles, distribution, and threats ecological replacement of native red squirrels by invasive greys driven by disease introduced species and their missing parasites invasions and conservation: no evidence of squirrelpox virus in grey squirrels introduced to italy parasite spillback: a neglected concept in invasion ecology? epidemiology and control of mycobacterium bovis infection in brushtail possums (trichosurus vulpecula), the primary wildlife host of bovine tuberculosis in new zealand rabies in northeastern europe-the threat from invasive raccoon dogs effects of species diversity on disease risk disruption of a host-parasite system following the introduction of an exotic host species mammal decline, linked to invasive burmese python, shifts host use of vector mosquito towards reservoir hosts of a zoonotic disease managing disease an invasive mammal (the gray squirrel, sciurus carolinensis) commonly hosts diverse and atypical genotypes of the zoonotic pathogen borrelia burgdorferi sensu lato no saturation in the accumulation of alien species worldwide prioritizing species, pathways, and sites to achieve conservation targets for biological invasion developing a framework of minimum standards for the risk assessment of alien species a comparison of impact and risk assessment methods based on the imo guidelines and eu invasive alien species risk assessment frameworks review of risk assessment systems of ias in europe and introducing the german-austrian black list information system (gablis) novel organisms: comparing invasive species, gmos, and emerging pathogens parasites and biological invasions: parallels, interactions, and control key: cord- -mqnqjn c authors: roberts, anjeanette; deming, damon; paddock, christopher d; cheng, aaron; yount, boyd; vogel, leatrice; herman, brian d; sheahan, tim; heise, mark; genrich, gillian l; zaki, sherif r; baric, ralph; subbarao, kanta title: a mouse-adapted sars-coronavirus causes disease and mortality in balb/c mice date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: mqnqjn c no single animal model for severe acute respiratory syndrome (sars) reproduces all aspects of the human disease. young inbred mice support sars-coronavirus (sars-cov) replication in the respiratory tract and are available in sufficient numbers for statistical evaluation. they are relatively inexpensive and easily accessible, but their use in sars research is limited because they do not develop illness following infection. older ( - to -mo-old) balb/c mice develop clinical illness and pneumonitis, but they can be hard to procure, and immune senescence complicates pathogenesis studies. we adapted the sars-cov (urbani strain) by serial passage in the respiratory tract of young balb/c mice. fifteen passages resulted in a virus (ma ) that is lethal for mice following intranasal inoculation. lethality is preceded by rapid and high titer viral replication in lungs, viremia, and dissemination of virus to extrapulmonary sites accompanied by lymphopenia, neutrophilia, and pathological changes in the lungs. abundant viral antigen is extensively distributed in bronchial epithelial cells and alveolar pneumocytes, and necrotic cellular debris is present in airways and alveoli, with only mild and focal pneumonitis. these observations suggest that mice infected with ma die from an overwhelming viral infection with extensive, virally mediated destruction of pneumocytes and ciliated epithelial cells. the ma virus has six coding mutations associated with adaptation and increased virulence; when introduced into a recombinant sars-cov, these mutations result in a highly virulent and lethal virus (rma ), duplicating the phenotype of the biologically derived ma virus. intranasal inoculation with ma reproduces many aspects of disease seen in severe human cases of sars. the availability of the ma virus will enhance the use of the mouse model for sars because infection with ma causes morbidity, mortality, and pulmonary pathology. this virus will be of value as a stringent challenge in evaluation of the efficacy of vaccines and antivirals. the occurrence in late and early of cases of severe acute respiratory syndrome (sars) in southeast china quickly drew international attention as the disease sickened more than , people and spread to more than countries within six months. since the identification of the etiological agent, the sars-coronavirus (sars-cov), in , development and characterization of animal models for evaluation of prophylaxis and treatment strategies have been of great interest. although sars-cov has not been associated with a subsequent widespread outbreak since - , the potential for such an outbreak remains. identification of a sars-like coronavirus in chinese horseshoe bats (rhinolophus species) that are indigenous across southeast asia suggests that they may represent a natural reservoir from which viruses may be introduced into the human population [ ] . the course of infection in animal models is abbreviated compared with the course of sars in humans; however, many aspects of sars-cov-associated disease are reproducible in animal models, including age-dependent susceptibility, re-covery of sars-cov from respiratory tissues and secretions, infection of type i and type ii pneumocytes and bronchial epithelial cells, detection of viral genome in blood and extrapulmonary tissues, and pulmonary pathology (including pneumonitis, edema, necrotic debris, and hyaline membrane formation) [ , ] . clinical symptoms have been reported in some species, but the findings are not entirely reproducible in outbred species. variability in clinical symptoms seen in outbred species may result from additional factors, including infection with co-pathogens, stress, existence of sub-species of test animals, and use of different virus strains. this variation can be problematic in studies of pathogenesis and vaccine efficacy unless a large enough number of animals are included in each treatment group [ ] . the ideal animal model would demonstrate viral replication in respiratory tissues, histopathologic evidence of respiratory disease, and consistent clinical signs of disease, including mortality. a small animal model in which all of these aspects of virus-associated disease are seen would be desirable because reproducible data can be generated in inbred animals, and larger numbers of animals can be included for statistical analysis of biological outcomes. to generate a model that satisfies these criteria, we have serially passaged sars-cov in the respiratory tract of young balb/c mice, resulting in a lethal virus that causes dosedependent weight loss and mortality associated with higher viral titers in the respiratory tract than are seen with the wildtype virus and with histopathologic findings of severe pulmonary disease. the characteristics of this lethal mouseadapted sars-cov, (ma ), are reported here. adaptation of sars-cov (urbani) was achieved by serial passage through lungs of balb/c mice as previously described for influenza a and influenza b viruses [ , ] . lightly anesthetized mice were inoculated intranasally with % tissue culture infectious dose (tcid ) per mouse of sars-cov (urbani). two to three days post-infection (d.p.i.), when peak viral titers are observed, lungs were harvested from infected mice and clarified homogenates were used as inocula for continued serial passage via intranasal inoculation in mice. to screen for virulence in mice, groups of young naïve mice (n ¼ - ) were inoculated with lung homogenates collected at passage , , and (p , p , and p , respectively), weighed daily, and observed twice daily for signs of morbidity or mortality. deaths were not observed following inoculation with p or p ; however, increased morbidity, as indicated by weight loss, was noted in p inoculated mice at day post-infection (p.i.) (unpublished data). mortality was observed in p -inoculated mice; % of p -inoculated mice died or displayed extreme morbidity and were euthanized by d.p.i. mortality was not observed in mice infected with intermediate passages (p -p ), and accompanying morbidity was not measured (unpublished data). supernatant from lung homogenates at p contained a heterogeneous virus pool as evident upon sequence analysis of cdna fragments generated by reverse transcriptase pcr (rt-pcr) amplification from purified viral rna (vrna). dual peaks were observed on sequence chromatograms at several nucleotide residues, indicating mixed populations in the virus pool. to obtain a clonal population, p virus was biologically cloned by three rounds of terminal dilution in vero cells. five clones were screened for lethality in -to wk-old balb/c mice; these clones caused mortality from % to %. one clone, designated ma , resulted in % mortality within d. balb/c mice aged to wk, mo, and mo were all susceptible to ma infection, with severe morbidity or death occurring within to d following intranasal inoculation. in order to identify the mutations in ma associated with the lethal phenotype, the sequence of ma was compared with that of sars-cov (urbani). the sequence of the initial sars-cov (p ) virus was identical to the published sars-cov (urbani) sequence, and six nucleotide substitutions were identified in the ma genome compared with that of sars-cov (urbani). all six substitutions were predicted to cause amino acid substitutions. these six mutations were localized to open reading frame (orf) ab (four mutations) and orfs s and m (one each) of sars-cov ( figure ). independent analysis conducted by the institute for genomic research (rockville, maryland, united states), under a national institute of allergy and infectious diseases (niaid) contract, confirmed the same six mutations in the ma sequence compared with those in the sars-cov (urbani) sequence. to confirm that the six mutations identified in the ma virus were responsible for lethality in mice, the mutations were introduced into cdna clones from which recombinant sars-covs were recovered. sequence analysis confirmed that recombinant viruses contained the appropriate mutation sets that were derived from the ma virus. in addition to generating a recombinant virus that included the six mutations (rma ), two additional recombinants were generated containing either the two mutations in the structural protein genes or the four mutations in the orf ab (rma sm and rma orf ab , respectively). the three recombinant viruses demonstrated similar kinetics and levels of viral replication compared with that severe acute respiratory syndrome (sars) is a severe, sometimes fatal respiratory disease caused by a coronavirus (sars-cov). in order to study the disease and evaluate vaccines and antiviral drugs, animal models that mimic the disease are necessary. however, no single animal model for sars reproduces all aspects of the disease as it affects humans. sars-cov replicates in the lungs of young mice, but they do not show signs of illness. adaptation of sars-cov by serial passage in the lungs of mice resulted in a virus (ma ) that is lethal for young mice following intranasal inoculation. lethality is preceded by rapid and high titer viral replication in lungs, viremia, and dissemination of virus to extrapulmonary sites accompanied by hematological changes and pathological changes in the lungs. mice infected with ma virus die from an overwhelming viral infection with extensive, virally mediated destruction of pneumocytes, and ciliated epithelial cells. the ma virus has six coding mutations in its genome, which, when introduced into a recombinant sars-cov, confer lethality. the ma virus will enhance the use of the mouse model for sars because infection with this virus in mice reproduces many aspects of severe human disease, including morbidity, mortality, and pulmonary pathology. of sars-cov infectious clone (icsars-cov), a wild-type recombinant sars-cov (urbani) generated from cdnas, the biologically derived ma virus, and sars-cov (urbani) in in vitro single-cycle growth analyses in vero e cells. at a multiplicity of infection (moi) of . , viruses reached peak replication ( . - . pfu/ml) at ; h.p.i., with a slight delay in peak titers for rma orf ab ( figure s ). northern blot analysis of rna from infected vero e cells indicated that genomic vrna and viral mrna and all eight sub-genomic mrnas were present in similar ratios for the recombinant viruses and ma virus as for sars-cov (urbani) ( figure a ). the level of expression and mobility of the structural proteins (s and n) and an accessory protein (orf a, also called x ) of the recombinant viruses and ma virus were comparable to those of sars-cov (urbani), as determined by western blot analysis ( figure b ). although s, n, and x are somewhat reduced in the lane ( figure b ) from icsars-covinfected cultures, this is associated with reduced amounts of total protein added to the well, rather than with any specific reduction in x expression. thus, the recombinant viruses (rma sm , rma orf ab , rma , and icsars-cov) and the ma virus demonstrate no defects in replication or in rna or protein expression compared with sars-cov (urbani) in vero e cells. groups of naïve mice were inoculated with serial -fold dilutions of ma virus, and the dose-dependent weight loss and lethality observed following infection is summarized in table . mice receiving a dose ! . tcid of ma virus died or lost more than % of their initial body weight between days and p.i. mice experiencing weight loss in excess of % initial body weight are euthanized in accordance with our animal study protocol. the % lethal dose (ld ) was . tcid . at an intermediate, non-lethal dose ( . /mouse), mice lost . % . % of initial body weight by day p.i. at a lower dose, weight loss was not significant ( . % . % by day p.i.) similarly, groups of naïve mice were inoculated with serial -fold dilutions of rma , rma orf ab , or rma sm , and followed daily for signs of morbidity (indicated by weight loss) and mortality. mortality was observed only in rma inoculated mice at doses equal to or in excess of . tcid /mouse ( table ). the ld for rma of . tcid is similar to the ld observed for the ma virus. consistent with observations following infection with ma virus, mice inoculated with low concentrations of rma ( . - . tcid /mouse) had little to no significant weight loss, but an intermediate dose of rma ( . tcid /mouse) resulted in significant weight loss but no mortality. thus rma recapitulated the phenotype of the ma virus in mice. inoculation with the highest doses of rma sm and rma orf ab ( . tcid /mouse) did not result in mortality (table ) , and only rma orf ab -infected mice demonstrated significant morbidity as indicated by weight loss. the rapid lethality observed following administration of ma virus (or its recombinant clone rma ) to balb/c mice could result from changes in tissue tropism with or without viremia, increased viral load and subsequent necrosis in pulmonary or extrapulmonary tissues, failure of innate or early adaptive immune responses, immunopathology in pulmonary or extrapulmonary tissues, or a combination of these or other factors. in order to evaluate whether changes in tissue tropism or levels of viral replication could contribute to the lethal phenotype of the ma virus, viral titers in lungs, spleen, liver, and brain of balb/c mice were determined at various time points following intranasal inoculation with sars-cov (urbani) or ma . mice were inoculated intranasally with sars-cov (urbani) ( tcid /mouse, a dose that results in peak viral titers in lungs of mice d.p.i.) or with lethal ( . tcid /mouse) or sub-lethal ( . tcid /mouse) doses of ma virus. at various time points p.i., mice were sacrificed and tissues harvested for subsequent processing. data from replicate experiments were combined. high titers of virus were detected in the lungs of mice through day p.i. (figure ). one day after inoculation, the amount of sars-cov (urbani) in lungs was ; tcid /g tissue. following administration of a lethal dose, ma virus was found to replicate to significantly higher titers of ; tcid /g tissue within h (p ¼ . ; figure ). in this study and in previous studies (k. subbarao, a. roberts, l. vogel, e. lamirande, et al., unpublished data), peak virus titers (; . tcid /g tissue) occur at day following inoculation with sars-cov (urbani). in comparison, by day p.i., viral titers in ma -inoculated mice reached, or persisted at, titers of ; tcid /g tissue, regardless of inoculating dose ( figure ). furthermore, in mice inoculated with a sublethal dose of ma , virus was detected at . tcid /g lung on day p.i. by comparison, in sars-cov (urbani)-infected mice, virus was detected at titers of . tcid /g lung on day p.i. by d.p.i., virus was no longer consistently detected in mice inoculated with sars-cov (urbani) or with sub-lethal doses of ma virus ( figure ). following inoculation with a lethal dose of ma virus, virus was also recovered from spleen, brain, and liver from day through day p.i. at titers ranging from . to . tcid /g (brain) and from . to . tcid /g (spleen) ( table ) . virus was detected more frequently in the liver and at higher titers ( . - . tcid /g) than in brain or spleen. virus was not recovered from any of these organs following inoculation with sars-cov (urbani), except in one mouse, sacrificed d.p.i., in which virus was detected in the spleen at a titer just above the limit of detection (table ). although the levels of viral replication detected in these tissues in ma -infected mice are modest, the number of mice in which virus was detected is remarkable, and both the frequency and the titers are significantly higher than those observed in sars-cov (urbani)-infected mice. in a separate experiment, infectious virus was also detected in sera of mice infected with ma virus or recombinant sars-covs on days and p.i. data from this experiment suggest that the presence of virus in serum may be facilitated by the mutations in the s and m genes, since virus was detected more frequently and at higher titers in sera from mice infected with ma , rma , or rma sm than in sera from mice infected with icsars-cov or ma orf ab ( figure s ). in addition to recovery of infectious virus from these tissues, total rna was also isolated from whole blood, brain, kidney, liver, intestine, spleen, thymus, heart, and lungs of balb/c mice after infection. although quantitative virology holds biological relevance since it indicates viral burden by measuring infectious virus, rt-pcr of vrna or sub-genomic mrnas may be more sensitive assays for the presence of virus. due to the replication strategy of sars-cov, primers that specifically amplify sub-genomic mrnas allow detection of products of viral transcription. furthermore, amplification of virus-specific mrnas allows distinction from vrna that may represent non-viable virus [ ] . rt-pcr amplification of genomic, antigenomic, and sub-genomic mrna-specific sequences was employed to confirm the presence of sars-cov nucleic acid or viral transcription products in different tissues. groups of four mice were inoculated with a lethal dose of ma virus or with sars-cov (urbani) ( tcid /mouse). mice inoculated with ma virus did not survive past day p.i. sars-cov (urbani)-infected mice were followed through day p.i. vrna, and viral mrna were amplified from lungs of ma -infected mice on days - p.i. in contrast, vrna was amplified from sars-cov (urbani)-infected mice through day p.i., but mrna, indicating viral transcription, was consistently amplified only through day p.i. mrna was detected as late as day p.i. by rt-pcr in one of four of the sars-cov (urbani)-infected mice (tables and ) . we were also able to amplify vrna and mrna from blood, brain, thymus, heart, and spleen of ma -infected mice (tables and ). viral mrna was consistently detected by rt-pcr in the tissues of ma -inoculated mice from day p.i. through day p.i. and in the thymus and heart of ma infected mice as early as day p.i. in contrast, vrna and viral mrna were detected transiently in very few sars-cov (urbani)-infected mice and only in the blood, spleen, and thymus. vrna and viral mrna were not detected by rt-pcr analysis in liver, intestines, or kidneys from ma or sars-cov (urbani)-infected mice. these tissues may contain factors that inhibit rt-pcr amplification of sars-cov rnas, since virus was isolated from liver homogenates of ma -infected mice but was not detected by rt-pcr, and viral mrna was detected in intestines by in situ hybridization (unpublished data). in summary, virus or viral-specific rna was detected in blood, lung, thymus, brain, spleen, liver, and heart from almost all ma -infected mice. in contrast, virus or viral-specific rna was detected in the lungs, and only sporadically in blood, spleen, and thymus from sars-cov (urbani)-infected mice (tables - ) . the lungs of mice infected with sars-cov (urbani) showed a rapid progression from focal, mild, perivascular, and peribronchiolar mononuclear inflammatory cell infiltrates to a diffuse, but transient, interstitial pneumonitis on day p.i. ( figure a , c, e, and g). occasional ciliated columnar epithelial cells of the bronchioles and alveolar pneumocytes stained for viral antigen by immunohistochemistry (ihc) ( figure a , c, e, and g). no viral antigens were detected at day p.i., and most mice at this time point showed no significant pulmonary histopathology (unpublished data). in comparison, the pulmonary pathology of mice infected with ma virus showed a rapid progression of inflammatory changes ( figure b , d, f, and h), but with more extensive damage to bronchiolar and alveolar epithelial cells ( figure a and b). these changes were especially evident in pneumocytes, and many detached, pyknotic, and necrotic cells were identified in alveolar spaces ( figure a ). inflammatory infiltrates, although consistently identified, were generally similar in intensity to those observed in the lungs of sars-cov (urbani)-infected mice ( figure ) . however, the distribution and amount of viral antigens identified by ihc was far greater in the lungs of ma -infected mice than in the lungs of mice infected with sars-cov (urbani) ( figure ). intracellular sars-cov antigens were extensively and abundantly distributed in bronchiolar epithelium, alveolar pneumocytes, and in necrotic debris within the alveoli and bronchiole lumens of ma -infected mice ( figure c and d) . no significant histopathologic changes were identified in extrapulmonary organs, including the liver, spleen, thymus, or brain, of mice infected with sars-cov (urbani) or ma virus. furthermore, sars-cov antigens were not detected in any of these tissues by ihc staining. infection of young balb/c mice with . tcid /mouse of ma virus resulted in elevated levels of the liver enzyme alkaline phosphatase (alp) in sera collected on days - p.i. alp levels were significantly higher in sera of ma inoculated mice than they were in pre-infection sera from the same mice. serum alp levels in ma -inoculated mice were also significantly higher than those of mice inoculated with . tcid /mouse of sars-cov (p ¼ . ; table s ). levels of other liver enzymes, including aspartate aminotransferase, alanine aminotransferase, gamma glutamyl transferase, creatine kinase, urea nitrogen, total bilirubin, and albumin, were not significantly altered following inoculation with ma virus (unpublished data). although moderately elevated levels of creatinine were observed in ma inoculated mice, no significant differences were seen between ma -and sars-cov-inoculated mice, suggesting that this was not associated with the lethal phenotype of the ma virus. furthermore, significant lymphopenia and neutrophilia were observed following infection with sars-cov and ma virus. although some samples were lost due to coagulation of blood, the alterations in the lymphocyte and neutrophil counts were more severe following infection with ma virus than they were following sars-cov infection (table s ) . in order to determine whether ma virus can be used as a more stringent challenge in evaluating vaccines and antiviral therapy designed for sars-cov than could the non-lethal sars-cov (urbani), we inoculated eight mice with ll of sars-cov (urbani) at tcid /mouse and mock-immunized an additional eight mice. four weeks after inoculation, the mice were bled for determination of sars-cov-specific serum neutralizing antibodies and challenged with . tcid of ma virus. mice were followed daily for signs of (figure ). serial passage of sars-cov (urbani) in the lungs of balb/ c mice resulted in a mouse-adapted sars-cov (ma virus) that is lethal for young ( -to -wk-old) balb/c mice. the virulence and lethality of the ma virus result from six mutations in the sars-cov genome that occurred within passages through balb/c mice. introduction of these six mutations into a recombinant sars-cov infectious clone, rma , conferred a lethal phenotype on the virus for young balb/c mice. five of these six mutations (not including the t a mutation occurring in the non-structural protein [nsp] of orf ab) occur within nsp (two mutations), nsp , s, and m. these genes have been reported as ones where sequence evolution occurred during adaptation of sars-cov in humans [ ] . the mutations in nsp , nsp (h y and e a within the main protease, cl pro ), and nsp (a v within the helicase protein) do not occur within any known functional domains and do not alter known cleavage sites utilized in the processing of the orf ab polyprotein. furthermore, the mutations in the ma virus do not occur at any specific amino acid positions identified by the chinese sars molecular epidemiology consortium [ ] . only the mutation in s (y h) occurs within a known functional domain, the receptor-binding motif (rbm). other reports have indicated that mutations within the rbm may account for increased affinity of the virus for its cellular receptor angiotensin converting enzyme [ ] . however, the y h mutation in ma virus does not occur at previously identified sites of the rbm and angiotensin converting enzyme interaction, and preliminary findings suggest that the y h mutation does not increase binding of the sars-cov (tor ) rbd to murine angiotensin-converting enzyme [ ] . reports of adaptation of an influenza a virus for increased virulence in mice indicate that multiple gene products may interact or contribute independently to virulence. in one adaptation of influenza a/fm/ / (fm-ma), findings indicated that four viral gene segments contributed to increased virulence [ ] , but additional analyses indicated that mutations occurring in at least two and likely three gene products acted synergistically to account for the increased virulence [ ] [ ] [ ] . recombinant icsars-cov produced mild pneumonia identified by x-ray changes in macaques, similar to the clinical disease noted with wild-type sars-cov (urbani) [ ] . consistent with these findings, recombinant sars-cov bearing the six novel mouse-adapted mutations, rma , recapitulated a fatal respiratory disease phenotype in mice, demonstrating the ability of the sars molecular clones to capture complex disease phenotypes. we generated two other recombinant sars-covs, rma sm (encoding the two mutations in the s and m genes) and rma orf ab (encoding the four mutations in orf ab). neither of these was lethal ( table ) in balb/c mice, indicating that sars-cov adaptation for balb/c mice involves mutations in at least two and possibly three genes (s þ orf ab, m þ orf ab, or s þ m þ orf ab). although recombinant viruses bearing two or four of the mutations were not lethal, each had a different phenotype than that of sars-cov (urbani). it is possible that the mutations in s and m contribute to increased viremia ( figure s ) and that the mutations in orf ab contribute to increased pathogenicity indicated by weight loss (table ) . both quantitative virology and ihc analysis of the lungs of ma -infected mice indicate extraordinarily high levels of viral replication in pulmonary tissues as early as h.p.i., and the level of replication remains high through day p.i. unlike sars-cov-infected mice, in which viral antigen is detected by ihc staining at modest levels in bronchial epithelium and in alveolar pneumocytes on days and p.i., the bronchial epithelium of ma -infected mice is replete with viral antigen at day p.i., as are alveolar pneumocytes on days and p.i. by day p.i., viral antigen is rarely detected in the in this model, day to day p.i. seems to be a critical time for the outcome of sars-cov infection. sars-cov (urbani)-infected mice demonstrate a pronounced but transient interstitial inflammation at day p.i. that is absent in ma infected mice. this transient inflammation is not associated with significant weight loss but coincides with the beginning of viral clearance from the lungs. in contrast, by day p.i., ma virus-infected mice lose weight, and necrotic cellular debris begins to fill the bronchioles and alveoli. by day p.i., ma -infected mice have lost in excess of % of their initial body weight, and several die without other overt clinical signs such as ataxia, paralysis, hunched posture, etc. viral titers in lungs of ma -infected mice are up to , -fold higher than those in sars-cov-infected mice by h.p.i. and remain higher than peak levels in sars-cov-infected mice through day p.i. in situ hybridization further confirms the intense and persistent signal of ma (and rma ) vrna in lung tissues at day p.i. when vrna from sars-cov (urbani) and the non-lethal rma sm and rma orf ab viruses are less abundant ( figure s ). objective data related to respiratory distress such as plethysmography and measurement of blood gases could not be collected because of practical and logistical constraints on experiments carried out in an animal biosafety level laboratory. however, our findings indicate that ma virus-infected mice die as a result of overwhelming pulmonary viral infection and destruction of bronchiolar epithelial cells and alveolar pneumocytes. viral load in sars cases was an important determinant of severe disease and death [ ] , but the mechanism of disease leading to fatal outcome in human cases of sars may be different than that observed in ma -infected balb/c mice. the mechanism of death following sars-cov infection in humans, and particularly the relative contribution of virusinduced damage and immunopathology, are not fully under- four weeks after immunization, mice were challenged intranasally with ll ma virus ( . tcid /mouse), weighed daily, and observed twice daily for morbidity and mortality. surviving mice that lost in excess of % initial body weight were euthanized. symbols represent mean values for sars-cov-immunized mice (triangles) and mock-immunized mice (circles). error bars indicate standard error. doi: . /journal.ppat. .g stood. the histopathology documented in the mice infected with the ma virus includes a rapid progression and extensive damage to bronchiolar and alveolar epithelial cells ( figure a and b ), but it does not show some features such as alveolar edema and hyaline membranes that were reported in many sars-cov-infected patients, or in aged mice infected with the urbani strain of sars-cov [ ] . this can be explained by the relative virulence of the ma strain, and the time p.i. when the lungs were evaluated. mice infected with the ma virus developed severe morbidity and died within to d following infection and did not survive long enough to show progression of the diffuse alveolar damage seen in aged mice or in human patients, who survive for a relatively prolonged length of time before succumbing to complications of the acute infection. in aged mice following sars-cov infection, histopathologic changes indicative of progressive diffuse alveolar damage, including proteinaceous deposits around alveolar walls and intraalveolar edema, are seen beginning on day [ ] . the rapid progression of infection and the extensive and persistent pulmonary replication of the virus are accompanied by viremia and detection of virus in extrapulmonary sites, suggesting that other factors may contribute to the increased pathogenicity of the ma virus. a prolonged viremic state or a secondary viremia is seen in mice infected with ma and rma that is not observed following infection with the recombinant sars-covs (urbani), rma sm , or rma orf ab . the ma virus model captures viremia and multi-organ involvement noted in human sars patients [ ] . however, as in all sars cases, the primary site of infection is the lung. perfusion of tissues harvested from mice in these experiments was not performed, and therefore, we cannot be certain that the detection of vrna in various extrapulmonary sites in ma -inoculated mice is not due to virus carried to and remaining in the organs and blood. however, on days through p.i., ma -specific mrnas in various tissues are detected at a higher frequency and signal intensity than in whole blood, and the presence of ma vrna in extrapulmonary tissues is detected by in situ hybridization. furthermore, infectious virus was detected on day p.i. in homogenates of extrapulmonary tissues when rt-pcr of rna from blood did not amplify any viral specific products (tables - ) . taken together, these observations support a hypothesis that virus is present and replicating at low levels in extrapulmonary tissues. in balb/c mice infected with ma virus or sars-cov, significant lymphopenia and neutrophilia were observed compared with pre-infection levels. ma virus-infected mice had significantly greater lymphopenia and neutrophilia than sars-cov-infected mice, reflecting hematological evidence of increased disease severity following ma virus infection. ma virus-infected mice also had significantly higher levels of alp than uninfected or sars-cov-infected mice. elevated levels of alp, an enzyme found in all tissues, but especially concentrated in the liver, may indicate cell destruction in the liver, intestines, or other tissues. reports of elevated alp levels in sars patients are rare [ , ] , but neutrophilia and lymphopenia have been reported frequently in human cases of sars [ ] [ ] [ ] [ ] [ ] [ ] [ ] . although steroid treatment and secondary bacterial infections may be speculated to be the cause of neutrophilia, some patients with sars had neutrophilia prior to initiation of steroid treatment and in the absence of bacterial infections [ ] . additionally, elevated absolute neutrophil counts at presentation were independent indicators of severe outcome of sars in human cases [ , , ] . the definitive causes of lymphopenia, neutrophilia, and elevated alp levels have not been identified in human cases of sars or in this mouse model. the mouse-adapted sars-cov ma virus will be a valuable tool in evaluating sars-cov vaccines and antiviral therapy. quantitative virology was the only outcome measure available in young balb/c mice challenged with sars-cov (urbani). because the ma virus replicates rapidly to high titer and is lethal for young balb/c mice, this virus provides a more stringent challenge than the sars-cov (urbani) virus in evaluating the efficacy of therapeutic interventions or prevention strategies. prophylaxis that is able to rescue ma -infected mice from a lethal outcome must lower viral burden in lungs very rapidly (e.g., within the first h). prophylaxis that accomplishes such reduction in the burden of ma virus may also reduce the severity of immunopathology that follows. a reduction of immunopathology was demonstrated in a sars-cov hamster model when a monoclonal antibody specific to sars-cov spike protein administered the day after infection was able to arrest a further rise in viral titer in the lungs [ ] . if similar protection can be demonstrated against challenge with ma virus in balb/c mice, the efficacy of intervention will be well proven. infection of young balb/c mice with the ma virus provides a model that is small and accessible and that can be evaluated extensively at an immunological level. finally, and most importantly, ma virus infection of young balb/c mice provides many elements that replicate observations in acute (and chronic) cases of sars infection in humans, including sequence changes during adaptation in several genes (nsp , nsp , s, and m); viral replication and histopathological changes in lungs of infected animals; viremia; detection of vrna in extrapulmonary sites, including the intestines; clinical indicators of illness, including mortality; and changes in blood counts, including lymphopenia and neutrophilia. the availability of a molecular clone of the ma virus, and the ability to generate additional sars-cov recombinant viruses that recapitulate the in vivo phenotype, will allow for detailed probing of the mechanisms mediating sars-cov pathogenesis and acute lung damage. all animal and viral experiments were conducted in biosafety level laboratories or animal facilities, and all personnel wore personal protective equipment, including tyvek suits and hoods and positive pressure hepa-filtered air respirators. all animal protocols employed in these studies have been approved by niaid's animal care and use committee. serial passage of sars-cov in mice. a dose of % tcid of sars-cov (urbani) was administered intranasally to a lightly anesthetized, -wk-old female balb/c mouse in a total volume of ll [ ] . two days after inoculation, the mouse was euthanized, and its lungs were removed and homogenized with an ex-gen omni glh homogenizer (omni international, http://www.omni-inc.com) as a % w/v suspension in leibovitz's l medium (invitrogen, http:// www.invitrogen.com), supplemented with the following antibiotics: . mg/l piperacillin (sigma, http://www.sigmaaldrich.com), . mg/l gentamicin (invitrogen), and mg/l amphotericin b (quality biological, http://www.qualitybiological.com). the lung homogenate was clarified by low-speed centrifugation at , rpm ( g) for min, and the supernatant was administered intranasally to three naïve mice. the process of intranasal inoculation of three female balb/c mice with pooled, clarified supernatants of % lung homogenates collected to d.p.i. was repeated times. identification of lethal phenotype and biological cloning of a lethal virus. the supernatant from p lung homogenates was subjected to three rounds of terminal dilution on vero cells. then, -fold serial dilutions were added to cells in -well plates (one dilution/plate). two to three days later, when cytopathic effect was evident in no more than nine wells per plate, supernatants from infected wells were collected, diluted, and transferred to fresh monolayers of vero cells. after the third round of terminal dilution, ll of five independent clones were screened for lethality in four -wk-old female balb/c mice. mortality associated with these five clones was between % and %. one of these five clones (ma ) that caused % lethality within d was expanded by two more passages in vero cells and used in further studies. virus replication in the respiratory tract of mice. young ( -to wk-old), female balb/c mice were lightly anesthetized and inoculated intranasally with ll of serially diluted virus. for sars-cov (urbani), a dose of tcid /mouse was administered unless noted otherwise. for lethal doses of ma virus, the dose administered was equal to . tcid /mouse; for sub-lethal doses, the dose administered was equal to . tcid /mouse. at various time points p.i., mice were euthanized and tissues collected for analyses. for determination of viral titers, tissues were homogenized to a final %w/v suspension in leibovitz's l medium supplemented with antibiotics. tissue homogenates were clarified by low-speed centrifugation, and virus titers were determined in vero cells on -and -well plates as previously described [ ] . virus titers are expressed as tcid /g of tissue with a lower limit of detection of . tcid /g. purification of vrna. tissues homogenates were clarified by lowspeed centrifugation; supernatants were transferred to eppendorf tubes (http://www.eppendorf.com) and further clarified by centrifugation at , g for min. vrna was extracted from ll of supernatant using the qiaamp viral rna mini kit (qiagen, http:// www .qiagen.com) eluted in ll of water, quantified by measurement of optical density at nm on a nanodrop nd- spectrophotometer (nanodrop technologies, http://www.nanodrop.com), and stored at À c. rt-pcr and sequence analysis. first-strand cdnas were generated by reverse transcription from rna purified from clarified cell supernatants of vero cells infected with either sars-cov ma , sars-cov (urbani) (p ), or from the clarified supernatants of p lung homogenates using reagents from the brilliant qrt-pcr plus core reagent kit (stratagene, http://www.stratagene.com). in brief, ll of vrna was heat denatured at c for min and quenched on ice before addition of x core rt buffer, . lg random primers, . lmol dntp mixture, and u stratascript reverse transcriptase for a -ll reaction mixture. reverse transcription was performed with an initial incubation of c for min, followed by a -min incubation at c and a -min denaturation at c. first-strand cdnas were amplified in overlapping pcr products spanning the entire genome. three microliters of each cdna reaction were amplified by pcr using the advantage-hf pcr kit (bd biosciences, http://www.bdbiosciences.com) or herculase enhanced dna polymerase (stratagene) as per manufacturers' protocols. fifteen pairs of primers were used to generate the overlapping pcr products (tables s and s ). amplification products were visualized on agarose gels and purified by use of the qiaquick gel extraction kit (qiagen). pcr products were sequenced in both forward and reverse directions, using forward and reverse primers (table s ) . automated sequencing was performed utilizing the bigdye terminator version . cycle sequencing kit (applied biosystems) as per manufacturer's instructions on an abi prism dna analyzer (applied biosystems). sequences were assembled and analyzed with vector nti and auto assembler dna sequence assembly software (abi prism; applied biosystems). when a mutation was identified in comparison with the sars-cov (urbani) published sequence, independent rt-pcr reactions were run and subsequent rt-pcr products were sequenced through the region containing the putative mutation to confirm the mutation. detection of sars-cov by rt-pcr. total rna was isolated from various tissues or whole blood and purified using an rneasy mini kit (qiagen) with an on-column dnase digestion (rnase-free dnase set; qiagen), as per manufacturer's protocol. approximately . g of tissue, cut into pieces no larger than . cm on any one side, was collected into ml of rnalater (ambion, http://www.ambion.com) and stored at room temperature for h and subsequently at c until processed. any unused tissue remaining after one month was moved to À c. approximately one-half or . - . g of tissue was homogenized with a disposable probe (omni international) in ll of rlt buffer (rneasy mini kit; qiagen), supplemented with % (v/v) b-mercaptoethanol (sigma). rlt suspensions were transferred to . -ml eppendorf microcentrifuge tubes and centrifuged at c for min at , g. rna was purified further as per manufacturer's protocol. rna from blood (; . ml per sample) was purified using a qiaamp rna blood mini kit (qiagen) as per manufacturer's protocol with the additional dnase on-column digestion. rnas were quantified and ng were reverse transcribed and amplified as described above. in brief, total rna was heat denatured at c for min and quenched on ice before addition of x first strand buffer, u rnase block, . lg random primers, . lmol dntp mixture, u stratascript reverse transcriptase, and water for a -ll reaction. reverse transcription was performed with an initial incubation at c for min, followed by a -min incubation at c and a -min denaturation at c. three microliters from each rt reaction were subsequently used in a -ll pcr with sars-covspecific primers for amplification of a -bp sequence of the nucleocapsid gene (sars-cov forward ( -ggtgacggcaaaatgaaagagc- ), sars-cov reverse ( -ggagaatttcccctactg- )). b microglobulin (b m) primers were used in separate reactions as rna quantity and pcr controls (b m forward ( -atgggaagccgaacatactg- ), b m reverse ( -cagtctcagtgggggtgaat- )). pcr reactions were performed for a total of cycles with x advantage cdna polymerase (clontech, http://www.clontech.com), . lmol dntp mixture (stratagene), and x cdna pcr reaction buffer (clontech) or opti-prime x buffer (stratagene) supplemented with . lmol mgcl (quality biological) for rna amplification of b m and sars-cov, respectively (table s ) . construction of sars-cov cdna plasmids for reconstructing the mouse-adapted recombinant virus rma . six codon changes ( t.a, a.c, c.t, a.g, t.c, and e.k) identified in ma compared with the published sars-cov (urbani) sequence, were inserted into cdna clones and used to construct and rescue infectious recombinant clones of sars-cov (urbani) as described previously [ ] . in brief, for each mutation two overlapping amplicons were generated by pcr, joined at primerintroduced bsmbi sites, and ligated into a cdna of icsars-cov using two unique restriction sites that flanked the mutation of interest. pcr reactions were performed with expand long taq (roche applied sciences, http://www.roche-applied-science.com) in cycles of c for s, c for s, and extensions at c for min using the following primers on plasmids encoding portions of the icsars-cov genome: for the mutation at nucleotide position , primer pairs - ( -catgt cattt gcaca gcag) and - c ( -attag gtctc atggc acac) and - t.a ( -agacc taatt atacc attaa ag) and - c ( -caagc acaag aatgc gtgc) were used. a second pcr amplification using primers - and - c produced a pcr product that was digested with the appropriate restriction enzymes (bglii, mfe ) and ligated into the icsars-cov cdna plasmid. similarly, the mutation was constructed using primer pairs - and - c ( -ctttc aaagc agcac acata tc) and - a.c ( -gaaag cgctg ctgca gaatg) and - c, followed by amplification with primers - and - c. this pcr product was digested with the appropriate restriction enzymes (bglii and mfe ) and ligated into the icsars-cov cdna plasmid. the mutation was similarly constructed using the primer pairs -m r and -sars d mu (À) ( -nnncg tctcg ttcca gttct gcgta aattg tacct gtacc) and sars dmu (þ) ( -nnncg tctct ggaac cacct tgtag gtttg) and d (À) ( -ccctg tagac gacat cagtac). the two resulting amplicons were joined following digestion (with bsmbi) and purification (qiaquick pcr purification kit; qiagen). the product dna was digested with bamhi and acli and inserted into an icsars-cov cdna. the mutation was constructed using primers - ( -catcctaatcaggagtatgc) and - c ( -caccta-c a g c c t g c a a g a c ) a n d p r i m e r s - c .t ( -gctgtaggtgtttgtgtattg) and - c (ctttatat-caacgctgaggtg). primers - and - c were used to amplify the appropriate fragment that was purified and digested with pflmi and bbvci, and ligated into an icsars-cov cdna. the mutation was constructed using primer pairs # ( -agagg aactg ctgta atgtc tc) and smas mus(À) ( -nnncg tctct atgat tacca gttga agtag catc) and smas smus(þ) ( -nnncg tctca tcataa ttata aatat aggta tctta gacat gg) and sars e (À) ( -ctagc acaaa tgcca gctcc). the two resulting amplicons were joined following digestion (with bsmbi) and purification and ligation. the full-length product was digested and inserted into an icsars-cov cdna utilizing restriction endonuclease sites agei and sali. the mutation was constructed using primer sets # ( -tgatcctctgcaacctgagc) and smas mum(À) ( -nnncgtctcacggtaatagtaccgttgtctgc) and smas mum(þ) ( -nnncgtctctaccgttgagaagcttaaacaactcc) and sars x (À) ( -nnnnnttaattaattaatttgttcgtt-tatttaaaacaaca). resulting amplicons were ligated following digestion with bsmbi. the mutation-containing product was digested and inserted into an icsars-cov cdna at swai and ndei restriction sites. in all cases, the final plasmids and the mutations in the plaquepurified viruses were verified by rt-pcr and sequencing. assembly of full-length cdnas and recovery of recombinant viruses. the full-length cdnas of wild-type sars-cov (icsars-cov) or mouse-adapted sars-cov recombinants were produced as previously described [ ] . rna transcripts from full-length cdnas were added to ll of the vero e cell suspension ( . ) in an electroporation cuvette and four electrical pulses of v at lf were given with a gene pulser ii electroporator (bio-rad, http://www. bio-rad.com) similar to protocols previously described [ , ] . the presence of full-length cdnas and transcripts was verified by separation on agarose gels and visualization by uv light. the transfected vero e cells were seeded in a -cm flask and incubated at c for d. viruses were plaque purified in vero e cells. in vitro growth of recombinant viruses. sars-cov (urbani), icsars-cov (urbani infectious clone), rma (icsars-cov with all six mutations found in ma ), rma orf ab (icsars-cov with the four mutations found in the replicase genes of ma orf ab), and rma sm (icsars-cov with the two mutations found in the structural genes of ma s and m) viruses were propagated on vero e cells in eagle's mem supplemented with % fetal calf serum, kanamycin ( . lg/ml), and gentamicin ( . lg/ml) at c in a humidified co incubator. cultures of vero e cells were infected in duplicate at an moi of . for h. cell monolayers were washed twice with ml of pbs and overlaid with complete mem. supernatants were collected at various times p.i. and virus was quantified by plaque assay on vero e cells in mm dishes. plaques were visualized by neutral red staining and counted at h. northern blot analysis. cultures of vero e cells were inoculated with sars-cov viruses at an moi of and incubated for h at c. at . h.p.i., intracellular rna was isolated using trizol reagent (invitrogen) as directed by the manufacturer, and . lg of total mrna was treated with glyoxal and separated on agarose gels using northernmax-gly according to the manufacturer's directions (ambion). the rna was transferred to brightstar-plus membrane (ambion) for . h and then cross-linked to the membrane by uv light. the blot was prehybridized and probed with an n gene-specific oligodeoxynucleotide probe ( -cttgact gccgcct ctgct b t b ccct b ct b gc b - ), where biotinylated nucleotides are designated with a subscript b. blots were hybridized overnight, and washed with low and high stringency buffers as recommended by the manufacturer. filters were incubated with strepavidin-ap, washed, and then incubated with chemiluminescent substrate cdp-star (ambion). the blots were overlaid with film and developed. western blot analysis. ten hours p.i. with sars-cov (urbani), ma (the biologically derived clone), icsars-cov, rma , rma or-f ab, or rma sm cells were washed once in pbs and lysed in buffer containing mm tris-hcl (ph . ), mm nacl, . % deoxycholine, % nonidet-p- , and . % sodium dodecyl sulphate (sds). supernatants clarified of nuclei were added to an equal volume of mm edta/ . % sds, resulting in a final sds concentration of . %, and samples were heat inactivated for min at c prior to transfer to biosafety level . after transfer to biosafety level , samples were again heat inactivated for min at c before use. equivalent sample volumes were loaded onto . % ready gels (bio-rad) and transferred to a pvdf membrane (bio-rad). for detecting sars-cov antigens, blots were probed with polyclonal mouse antisera directed against venezuelan equine encephalitis virus replicon particles (vrps) that expressed the sars-cov orf a (vrp-orf a), s (vrp-s), or n (vrp-n) proteins diluted : for sars-cov orf a and : for s and n antisera. dilutions were done in % blotto in pbs/ . % tween and developed using ecl chemiluminescence reagents (amersham, http://www.amershambiosciences.com). in situ hybridization. paraffin-embedded sections, -mm thick, were probed with s utp-labeled riboprobes complementary to the n gene of sars-cov (urbani) or the sindbis virus genome, as a negative control, using previously described methods [ ] . in brief, following treatment to prevent nonspecific probe binding, the tissues were incubated overnight with either probe at cpm/ll in hybridization buffer at c. the slides were then washed, dehydrated, and coated with nbt emulsion (eastman kodak, http:// www.kodak.com), and incubated at À c for d prior to development. positive signal, as determined by silver grain deposition, was evaluated by light microscopy. statistics. log-transformed virus titers were compared in a mann-whitney u test, and statistical significance was assigned to differences with p-values , . . histopathology and immunohistochemistry. lungs, liver, spleen, thymus, and brain were obtained from individual mice euthanized at various time points, fixed in % neutral buffered formalin, and processed for histopathologic and ihc examination as described [ , ] . tissue sections ( lm) were stained with hematoxylin and eosin and by using an immunoalkaline phosphatase technique with a hyperimmune rabbit anti-sars-cov nucleocapsid protein antibody at a dilution of / . figure s . i. lungs and sera were collected for viral titration. virus titers are expressed as log pfu/g lung (circles) or log pfu/ml serum (squares) from individual mice; bars represent the geometric mean for the group. limits of detection for viral titers from lung and sera are pfu/ml and pfu/ml, respectively. found at doi: . /journal.ppat. .sg ( kb pdf). figure s . in situ hybridization of sars-cov rna in lungs of balb/c mice balb/c mice were infected with sar-cov (urbani), rma sm , rma orf ab , rma , or ma virus. lungs were harvested on days and p.i., and in situ hybridization was performed as described in materials and methods. no specific signal was observed with a control riboprobe at any time point, while sars-cov-n specific signal was readily apparent within the lungs for all five viruses at day p.i. in contrast, at day p.i., abundant sars-cov-specific in situ signal was observed in lungs of mice infected with the lethal ma virus or the lethal recombinant rma , but not in lungs of mice infected with the non-lethal sub-clones or sars-cov (urbani). found at doi: . /journal.ppat. .sg ( . mb pdf). the genbank (http://www.ncbi.nlm.nih.gov/genbank) accession numbers for the viruses and sequences discussed in this paper are mouse-adapted sars-cov ma sequence (dq ) and sars-cov (urbani) (ay ). author contributions. ar performed passage of the sars-cov through balb/c mice, and all other experimental work except generation of recombinant viruses and in situ analysis of lungs. ar generated related figures, materials and methods, figure legends, and interpretation of findings, and wrote and revised the primary manuscript. dd contributed generation and characterization of recombinant clones, generation of related figures, recording of data, and data interpretation. dd made written contributions to figure legends, materials and methods, and review of primary manuscript. cdp performed all histopathological processing and evaluation of sars-cov-and ma -infected tissues, interpretation of findings, generation of related figures, materials and methods, and figure legends, as well as review of primary manuscript. ac performed partial sequencing of ma genome, evaluation of mouse tissues for sars-cov mrnas and vrnas by rt/pcr, generated supporting tables s -s , and wrote related materials and methods, and reviewed the primary manuscript. by contributed to the generation and characterization of recombinant clones, generation of figures, recording of data, and data interpretation. by contributed to the writing of materials and methods and the review of primary manuscript. lv performed passage of the sars-cov through balb/c mice and all other experimental work except generation of recombinant viruses and in situ analysis of lungs. lv's written contributions include data recording, generation of materials and methods, and review and editing of primary manuscript. bdh performed partial sequencing of sars-cov, p , and ma genomes, and primary manuscript review and editing. ts contributed preparation of sars-cov-infected and recombinant virus-infected tissues for in situ hybridization analysis. ts contributed to the writing of materials and methods and the review of primary manuscript. mh contributed experimental design and evaluation of sars-covinfected and recombinant virus-infected tissues by in situ hybridization techniques. mh made written contributions that include generation of materials and methods, the figure s legend, and a review of primary manuscript. glg performed all preparation of sars-cov-and ma -infected tissues for histopathological processing and evaluation, generation of related figures, materials and methods, figure legends, and interpretation of findings, as well as review of the primary manuscript. srz contributed review of all histopathological evaluation of sars-cov-and ma -infected tissues, generation of related figures, materials and methods, figure legends, and interpretation of findings, as well as review of the primary manuscript. rb contributed experimental design, characterization of recombinant clones, generation of related data and figures, and data evaluation and interpretation. rb made written contributions to figure legends, materials and methods, and the drafting and review of the primary manuscript. ks provided data interpretation and review and editing of the manuscript. funding. this research was supported in part by the intramural research program of the us national institutes of health (nih), niaid, and in part by nih/niaid grants ai and ai . competing interests. the authors have declared that no competing interests exist. bats are natural reservoirs of sars-like coronaviruses animal models and antibody assays for evaluating candidate sars vaccines: summary of a technical meeting - is there an ideal animal model for sars? increased virulence of a mouse-adapted variant of influenza a/fm/ / virus is controlled by mutations in genome segments , , , and a single amino acid change in the c-terminal domain of the matrix protein m of influenza b virus confers mouse adaptation and virulence mechanisms and enzymes involved in sars coronavirus genome expression molecular evolution of the sars coronavirus during the course of the sars epidemic in china receptor and viral determinants of sars-coronavirus adaptation to human ace structure of sars coronavirus spike receptor-binding domain complexed with receptor genetic analysis of mouse-adapted influenza a virus identifies roles for the na, pb , and pb genes in virulence the influenza virus variant a/fm/ / -ma possesses single amino acid replacements in the hemagglutinin, controlling virulence, and in the matrix protein, controlling virulence as well as growth mutations in the hemagglutinin and matrix genes of a virulent influenza virus variant, a/fm/ / -ma, control different stages in pathogenesis cynomolgus macaque as an animal model for severe acute respiratory syndrome initial viral load and the outcomes of sars aged balb/c mice as a model for increased severity of severe acute respiratory syndrome in elderly humans fatal severe acute respiratory syndrome is associated with multiorgan involvement by coronavirus clinical significance of hepatic derangement in severe acute respiratory syndrome viral loads in clinical specimens and sars manifestations a prediction rule for clinical diagnosis of severe acute respiratory syndrome pathogenesis of severe acute respiratory syndrome a major outbreak of severe acute respiratory syndrome in hong kong difference and significance of t-lymphocyte subsets in differential diagnosis between severe acute respiratory syndrome and common atypical pneumonia clinical and laboratory features of severe acute respiratory syndrome vis-a-vis onset of fever coronavirus as a possible cause of severe acute respiratory syndrome haematological manifestations in patients with severe acute respiratory syndrome: retrospective analysis temporal patterns of hepatic dysfunction and disease severity in patients with sars severe acute respiratory syndrome among children severe acute respiratory distress syndrome (sars): a critical care perspective therapy with a severe acute respiratory syndrome-associated coronavirusneutralizing human monoclonal antibody reduces disease severity and viral burden in golden syrian hamsters prior infection and passive transfer of neutralizing antibody prevent replication of severe acute respiratory syndrome coronavirus in the respiratory tract of mice severe acute respiratory syndrome coronavirus group-specific open reading frames encode nonessential functions for replication in cell cultures and mice sars cov replication and pathogenesis in human airway epithelial cultures reverse genetics with a full-length infectious cdna of severe acute respiratory syndrome coronavirus a single amino acid change in nsp attenuates neurovirulence of the sindbis-group alphavirus s.a.ar replication of sars coronavirus administered into the respiratory tract of african green, rhesus and cynomolgus monkeys immunohistochemical, in situ hybridization, and ultrastructural localization of sars-associated coronavirus in a fatal case of severe acute respiratory syndrome in taiwan characterization of a novel coronavirus associated with severe acute respiratory syndrome we would like to thank jadon jackson for his expertise and assistance in all animal studies carried out at niaid. we would like to thank maria giovanni (microbial genomics, niaid) and elodie ghedin (the institute for genomic research) for contributions in confirming ma mutations. we would like to thank other members of the subbarao lab for their support. key: cord- - nlid c authors: guo, wen-ping; lin, xian-dan; wang, wen; tian, jun-hua; cong, mei-li; zhang, hai-lin; wang, miao-ruo; zhou, run-hong; wang, jian-bo; li, ming-hui; xu, jianguo; holmes, edward c.; zhang, yong-zhen title: phylogeny and origins of hantaviruses harbored by bats, insectivores, and rodents date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: nlid c hantaviruses are among the most important zoonotic pathogens of humans and the subject of heightened global attention. despite the importance of hantaviruses for public health, there is no consensus on their evolutionary history and especially the frequency of virus-host co-divergence versus cross-species virus transmission. documenting the extent of hantavirus biodiversity, and particularly their range of mammalian hosts, is critical to resolving this issue. here, we describe four novel hantaviruses (huangpi virus, lianghe virus, longquan virus, and yakeshi virus) sampled from bats and shrews in china, and which are distinct from other known hantaviruses. huangpi virus was found in pipistrellus abramus, lianghe virus in anourosorex squamipes, longquan virus in rhinolophus affinis, rhinolophus sinicus, and rhinolophus monoceros, and yakeshi virus in sorex isodon, respectively. a phylogenetic analysis of the available diversity of hantaviruses reveals the existence of four phylogroups that infect a range of mammalian hosts, as well as the occurrence of ancient reassortment events between the phylogroups. notably, the phylogenetic histories of the viruses are not always congruent with those of their hosts, suggesting that cross-species transmission has played a major role during hantavirus evolution and at all taxonomic levels, although we also noted some evidence for virus-host co-divergence. our phylogenetic analysis also suggests that hantaviruses might have first appeared in chiroptera (bats) or soricomorpha (moles and shrews), before emerging in rodent species. overall, these data indicate that bats are likely to be important natural reservoir hosts of hantaviruses. emerging infectious diseases have a substantial and ongoing impact on public health and agricultural production [ ] [ ] [ ] . over half of the currently recognized pathogens are zoonotic, and nearly all of the most important human pathogens are either zoonotic or originated as zoonoses before adapting to human transmission [ , ] . hence, wildlife species play a key role in disease emergence by providing a ''zoonotic pool'' from which previously unknown pathogens may emerge [ ] . a major goal of infectious disease research is therefore to characterize those unknown pathogens circulating in animal host reservoirs before they emerge in human populations [ , ] . hantaviruses (genus hantavirus, family bunyaviridae) are the etiological agent(s) of hemorrhagic fever with renal syndrome (hfrs) and hantavirus pulmonary syndrome (hps) in humans [ ] . unlike the other genera of bunyaviridae, hantaviruses are not known to be transmitted by arthropods, and instead are harbored by small mammals, particularly rodents [ ] . the first hantavirus (thottapalayam virus (tpmv)), was isolated from the asian house shrew (suncus murinus) in india in [ ] , but it had not been classified as a bunyavirus until [ ] . all hantaviruses found subsequently and until were from muroidea (i.e. 'mouse-like') rodents. to date, only rodent-borne viruses have been shown to cause human diseases, namely hfrs in eurasia and hps in the americas [ ] . as the phylogeny of the rodent-borne hantaviruses appears to be largely congruent with that of subfamily muridae and family cricetidae of muroidea, hantaviruses are often considered to have co-diverged with their rodents hosts over time-scales of millions of years [ ] [ ] [ ] [ ] . since , at least new species of hantaviruses have been identified in soricomorpha insectivores (shrews and moles) worldwide [ , ] . recently, tpmv was also found in china, nepal, and vietnam [ ] [ ] [ ] , and is thought to have had an early evolutionary divergence from rodent-borne hantaviruses [ , ] . more recently, hantavirus rna sequences have been detected in bats from western africa [ , ] . the presence of newly described hantaviruses in insectivores and bats has challenged the conventional view that hantaviruses originated from rodents, and suggests there may be additional unrecognized hantaviruses circulating in a wide range of animal hosts. furthermore, that the viruses sampled from rodents and insectivores (soricomorpha) do not form strict monophyletic groups [ , , ] indicates that host jumping has also occurred during the evolutionary history of these viruses. as a consequence, the respective roles of virus-host co-divergence and cross-species virus transmission are more complex than previously envisioned, although determining the relative frequency of these two processes is critical for understanding the evolutionary and biogeographic processes that have produced the current diversity of hantaviruses and their potential for future emergence. in this study, we describe four novel hantavirus sequences detected in bats and shrews collected in china. with these data we then explore key aspects of hantavirus evolution, particularly the frequency of cross-species virus transmission. a total of bats of eight different species were captured in longquan city and wenzhou city, zhejiang province in the spring of ( figure and table ). similarly, bats representing eight species were captured in hubei province in the spring of . a total of insectivores (representing two species -anourosorex squamipes and suncus murinus) were captured in lianghe county, yunnan province in the spring of and autumn of . in , two shrews (from the species sorex isodon and suncus murinus) were collected from yakeshi city, inner mongolia autonomous region. rt-pcr was performed to detect hantaviral rna based on the l segment sequences. in bats, pcr products of the expected size were amplified from six rhinolophus affinis, three rhinolophus sinicus, one rhinolophus monoceros collected from longquan, and one pipistrellus abramus from huangpi. in insectivores, expected size products were generated from one sorex isodon from yakeshi and nine anourosorex squamipes from lianghe. these sequences most closely resembled those of other hantaviruses (table s ) (see below). to characterize the novel hantaviruses found in this study, sequences of the complete s and m segments were recovered from the rna positive bat and shrew samples described above. key features of these sequences are described in detail in table and figure s . clearly, the viruses from bats and shrews are distinct from each other and from other known hantaviruses, representing four novel species of hantavirus (see below). we therefore named these new viruses as huangpi virus (hupv), longquan virus (lquv), lianghe virus (lhev), and yakeshi virus (yksv), and which were found in p. abramus, rhinolophus spp. (r. affinis, r. sinicus, and r. monoceros), a. squamipes, and in s. isodon, respectively. hupv, lquv, and yksv exhibit # . % sequence similarity in the n, gpc and l proteins from all known hantaviruses (tables s , s ). in contrast, lhev is clearly related to cao bang virus (cbnv) also identified in anourosorex squamipes in vietnam [ ] in sequences of the n (# . % similarity), gpc (# . %) and l (# . %) proteins. however, lhev is different from cbnv in the gpc protein, exhibiting more than the % amino acid difference required for hantavirus species demarcation [ ] . to determine the phylogenetic relationships among the novel hantaviruses described here and those described previously, phylogenetic trees based on s and m segment sequences were inferred using three methods. the bayesian and maximum likelihood (ml) trees were rooted in the way suggested by the (molecular clock-rooted) mcc tree. the ml trees based on the m or s segment sequences produced very similar topologies ( figure ). in the s segment tree (figure a ), all known hantaviruses including the viruses identified in bats and insectivores could be placed into four well supported 'phylogroups'. the first phylogroup only comprised viruses from insectivore (soricidae) species and included the asian viruses tpmv and imjin virus (mjnv) sampled from the ussuri white-toothed shrew (crocidura lasiura) in south korea [ ] . notably, this phylogroup occupied a basal position with respect to the remaining viruses. the second phylogroup comprised hupv and lquv found in bats in this study and which were closely related each other, along with the more divergent nova virus (nvav) identified in the european common mole (talpa europaea) in hungary [ ] . phylogroup iii contained all other known soricomorpha-associated viruses, including lhev and yksv found in this study, as well as a distinct clade of murinae-borne (i.e. rodent) viruses. finally, the fourth phylogroup included viruses sampled from the arvicolinae, neotominae, and sigmodontinae subfamilies of rodents, although these did not form three clearly distinct monophyletic groups in the s segment, along with the reassortant rkpv sampled from an insectivore (see below). importantly, the topologies of ml and bayesian trees estimated using amino acid sequences of n (encoded by the s segment) and gpc (encoded by the m segment) hantaviruses are important human pathogens, occasionally emerging from animal reservoirs. however, both the biodiversity of hantaviruses in nature, as well as the frequency with which they have jumped species barriers in the past, are unclear. here, we describe four novel hantaviruses (huangpi virus, lianghe virus, longquan virus, and yakeshi virus) that were sampled from bats and shrews in china. these viruses are different from known hantaviruses, with each representing a novel species. an evolutionary analysis of all known hantaviruses including the novel viruses described here reveals the existence of four distinct phylogenetic groups of viruses that infect a range of mammalian hosts, and which have sometimes exchanged genes through segment reassortment. our analysis also suggests that hantaviruses might have first appeared in bats or insectivores, before spreading to rodents, even though rodents are currently the best documented hosts of hantaviruses. because the phylogenetic trees of the hantaviruses do not always match those of their mammalian hosts, we conclude that both host-jumping and co-divergence have played important roles in hantavirus evolution. overall, our study shows that bats are likely to be important natural reservoir hosts of hantaviruses from which novel hantaviruses may emerge in the future. proteins were consistent with those of the trees based on the nucleotide sequences, indicating that site saturation has not adversely affected our phylogenetic inference ( figure s a -f). although a closer phylogenetic relationship between the first and second phylogroups were observed in the bayesian tree ( figures s b and s e ), these two phylogroups still occupied basal positions. the most striking difference between the s and m segment trees was that phylogroup ii (i.e. lquv and nvav) were basal in the m segment tree with relatively strong statistical support ( figure b ) (although it is important to note that we were unable to amplify the m segment sequence from hupv), while the soricidaeassociated viruses of phylogroup i occupied the basal position in the s segment tree and with much stronger support ( figure a ). this different phylogenetic pattern was also apparent in the relevant amino acid trees of the n and gpc proteins ( figure s a -f). such phylogenetic incongruence is strongly suggestive of reassortment among hantaviruses of phylogroups i and ii, and which might have occurred during the evolution of hantaviruses carried by bats and insectivores as these phylogroups are currently only associated with these mammalian species. irrespective of this history of reassortment it is clear that there have been multiple cross-species transmission events in the evolutionary history of the hantaviruses with, for example, those viruses sampled soricomorpha forming a paraphyletic group, as do those from bats shown in the l tree. in both the m and s segment trees yksv (a member of the soricomorpha clade of phylogroup iii) showed a close phylogenetic relationship with qiandao lake virus (qdlv) sampled from sorex cylindricauda in china (gu ), kenkeme virus (kkmv) collected from the flat-skulled shrew (sorex roboratus) in the far eastern asian region of russia [ ] , seewis virus (swsv) from the eurasian common shrew (sorex araneus) in switzerland [ ] , and asama virus (asav) from the japanese shrew mole in japan (urotrichus talpoides) [ ] . hence, this well-supported subgroup contained four viruses from asia and one from europe. also of note was that all lhev sequences exhibited a close relationship with cbnv and jeju virus (jjuv) sampled from the asian lesser white-toothed shrew (crocidura shantungensis) in south korea [ ] , bats rhinolophus affinis rhinolophus hipposideros armiger - / / jemez springs virus (jmsv) from the dusky shrew (sorex monticolus) [ ] and oxbow virus (oxbv) from the american shrew mole (neurotrichus gibbsii) [ ] , with the latter two viruses both found in the usa. kang et al. [ ] found that rkpv sampled from a s. aquaticus mole in the usa shared a closer relationship with viruses harbored by cricetidae rodents than with soricomorpha-borne hantaviruses, a topology confirmed by our analysis. interestingly, in the s segment tree rkpv was most closely related to another novel hantavirus (luxv) identified in the yunnan red-backed vole (e. miletus) in china [ ] . more notable was that both viruses were more closely related to sigmodontinae/neotominae-borne hantaviruses in the s segment tree but with arvicolinae-borne hantaviruses in the m segment tree, suggesting that both luxv and rkpv were generated by a common reassortant event (figures a- b) . a rather different picture of the evolutionary history of hantaviruses was observed in the phylogenies of l segment sequences. in particular, these trees provided evidence for five phylogroups, as viruses from phylogroup ii could be subdivided into a subgroup containing hpuv, mouyassué virus (mouv) detected in bat from cote d'ivoire [ ] , nvav, and altai virus (eu ) sampled from a soricidae shrew in the neighboring area of russia with china, and a subgroup containing the lquv and mgb virus sampled from bats in sierra leone [ ] (phylogroup v, figure c ). however, this novel subdivision of phylogroups was not supported strongly. the clustering patterns of other viruses were similar to those in the s and m segment trees ( figure s a-b) , although lquv and mgb virus grouped with tpmv and mjnv in the bayesian tree ( figure s b ). finally, and in contrast what is seen in the l nucleotide sequence phylogenies, mgb virus shared a closer relationship with tpmv and mjnv than hupv and lquv in the l amino acid tree ( figure s g -i). our phylogenetic analysis also provided insights into the geographic distribution of these viruses. all those s segment phylogroup i viruses identified so far are from asia (soricidae, figure ), while phylogroup ii viruses have been recovered from both asia and europe (talpidae and chiroptera). in the l gene tree the two viruses found in african bats were closely related to hpuv and lquv found in bats from china, respectively ( figure c ). with respect to phylogroup iii, viruses of soricomorpha clade have been mainly found in asia, with a few from europe, north america, and africa. with the exception of sangassou virus (sangv) found in the wood mice from guinea [ ] , almost all viruses of the murinae clade are from asia and europe. finally, for phylogroup iv, most of the arvicolinae clade viruses have been identified in asia and europe, with a few sampled from north american animals. in contrast, almost all viruses of the sigmodontinae clade are from the new world, and the lineage comprising luxv from china and rpkv found in usa occupied a basal position in this clade. overall, those hantaviruses sampled from asian mammalian species exhibit the greatest genetic diversity and tend to fall at basal positions on the phylogenetic trees. this tentatively suggests that hantaviruses may have an asian origin, although this will need to be confirmed on a far larger sample of taxa. co-divergence and cross-species transmission in hantavirus evolution we inferred ml and mcc trees of mitochondrial cytochrome b (mt-cyt b) gene sequences among the known mammalian hosts (chiroptera, soricomorpha, and rodentia) of the hantaviruses. both trees had very similar topologies. specifically, using ornithorhynchus anatinus as an outgroup, the rooted phylogenetic trees based on mt-cyt b gene sequences including the sequences obtained in this study (table s ) resulted in a clear phylogenetic division between those viruses sampled from rodentia, chiroptera and soricomorpha, with each forming a monophyletic group as expected (figure ). in agreement with previous studies [ ] , soricomorpha showed a closer relationship with chiroptera than with rodentia. within rodentia, the murinae subfamily and cricetidae family formed two monophyletic groups. the cricetidae were further subdivided into the subfamilies neotominae, arvicolinae and sigmodontinae. based on this single-locus study, neotominae, which was once considered an exclusively north american subset of the south american sigmodontinae, was more closely related to arvicolinae than sigmodontinae. however, studies based on multiple nuclear loci place the neotiminae as a distinct sister subfamily with the sigmodontinae [ ] . we used treemap . to test the strength of congruence between the viral s, m, and s+m segment trees with that of the host mt-cyt b gene ( figure , figure s ; table s ). notably, the viral phylogenies inferred using the s segment sequences were not always consistent with their hosts' phylogeny as measured by both ce (p = . . ) and nce (p = . . ) frequencies, with multiple deep and more recent topological differences, and hence an indication of relatively frequent host jumping ( figure ). this analysis also indicated that cross-species transmission events had occurred at four levels during hantavirus evolution ( figure , table ): inter-species within a genus (e.g. htnv and asv, dobv and saav), inter-genus within a family (e.g. dbsv and htnv), inter-family within an order (e.g. oxbv and jmsv, asav and swsv), and even inter-order (e.g. nvav and lquv; luxv and rkpv). in addition, some viruses exhibited a phylogenetic pattern that reflected their geographic origins rather than the phylogeny of their hosts -such as viruses oxbv and jmsv, dobv and saav within the soricomorpha and murinae clades of phylogroup iii (table s ) -such that the likelihood of host jumping in part reflects geographic proximity. however, in other instances there were clear matches between the virus and host phylogenies. most notably, there was significant congruence between phylogenies of the two clades of phylogroup iv and their rodent hosts -arvicolinae (ce (p = . . ) and nce (p = . . )) and sigmodontinae (ce (p = . . ) and nce (p = . . )) -indicating that these rodent hantaviruses may have a long history in their primary hosts, likely co-diverging with their hosts in some cases. we describe four novel hantavirus sequences from bats and insectivores captured in china. the hantavirus harbored by three rhinolophus bats and one carried by the sorex isodon shrew exhibited # . % amino acid similarity in the n, gpc and l protein sequences with any recognized hantaviruses, while the hantavirus carried by one pipistrellus bat shared # . % amino acid similarity in both the n and l protein sequences with known hantaviruses. the hantavirus found in anourosorex squamipes (shrew) from lianghe (yunnan province) was most closely related to cbnv also identified in anourosorex squamipes in vietnam, but with quite different n (. . % amino acid), l (. . %), and gpc (. . %) amino acid sequences. interestingly, the mt-cyt b gene differences between anourosorex squamipes in yunnan of china and vietnam are . %, compatible with the existence of the two subspecies of anourosorex squamipes. according to the criteria for species demarcation in the genus hantavirus proposed by the international committee on taxonomy of viruses [ ] , these four hantaviruses are sufficiently genetically distinct that they should be recognized as distinct species. accordingly, we propose naming these four novel hantaviruses as huangpi virus (hupv), lianghe virus (lhev) longquan virus (lquv), and yakeshi virus (yksv), reflecting their geographic origins. in addition, as lhev has not been isolated, such that two-way cross neutralization tests cannot be performed, further studies are needed to clarify whether lhev is a novel species or simply a variant of cbnv. finally, the identification of lquv in three rhinolophus bats also means that hantaviruses may spread relatively easily among different species of bats. although rodents are considered the primary hosts of hantaviruses [ ] , the increasing number of viruses found in insectivore species (shrews and moles) over the past five years has raised an important question mark over the host range and origin of hantaviruses. indeed, the first hantavirus (tpmv) was isolated from shrews in india in [ ] . our work further suggests that bats are likely to be important hosts for hantaviruses. bats (order chiroptera) have been shown to be sources of a broad variety of emerging pathogens, including coronaviruses, filoviruses, henipaviruses, and lyssaviruses [ ] . recently, partial hantaviral sequences were found in one slit-faced bat (nycteris hispida) and two banana pipistrelles (neoromicia nanus) in west africa [ , ] . we document two novel hantaviruses in rhinolophus bats (r. affinis, r. sinicus, r. monoceros) and p. abramus. consequently, these data, together with other recent studies [ , ] , demonstrate that bats in china and africa are hosts of hantaviruses and thereby constitute a potential sylvatic mammalian reservoir of hantaviruses. as their global distribution, abundance, ability to fly long distances, often large population densities, and sociality favor the efficient maintenance, evolution, and spread of viruses, it is clear that further study is needed to elucidate the potential importance of bats as hantavirus hosts. indeed, it seems likely that additional hantaviruses will be isolated from bats, and especially from insectivorous bats as all four bat species in which hantavirus sequences were detected in this study are insectivorous. moreover, because they consistently occupy basal phylogenetic positions, these phylogenetic data suggest that the ancestor of the extant hantaviruses might have first appeared in chiroptera and/or soricomorpha, although this will need to be confirmed on a larger sample of mammalian taxa. one notable feature of our phylogenetic analysis was the basal position of phylogroup i viruses in the s segment tree but of phylogroup ii viruses in the m segment. such deep phylogenetic incongruence is strongly suggestive of an ancient reassortment event. in the s segment tree, hupv and lquv share a closer relationship with nvav identified in talpa europaea [ ] , as does lquv in the m segment tree and hpuv in the s segment tree, suggesting that these viruses share common ancestry. in addition, hantaviruses identified in bats in africa are closely related to nvav or tpmv [ , ] . within phylogroup iv, rkpv identified in a mole is closely related to luxv from the yunnan red-backed vole in china. these viruses also share a history of reassortment since they occupy the basal positions within the arvicolinae clade in the m segment tree but with the sigmodontinae clade in the s segment tree. the current geographic distribution of hantaviruses in large part reflects that of their host species [ , ] . if hantaviruses have indeed been associated with mammalian species for millions of years, then it is possible that these geographic distributions are long established. the oldest eutherian is juramaia sinensis (an insectivore) found in china, at an estimated million years ago [ ] . it was recently suggested that both euarchontoglires and laurasiatheria, excluding chiroptera, originated in eurasia [ ] . geographic reconstructions further suggest that bats originated in the laurasian land masses, with an asian origin for the suborder yinpterochiroptera and a most likely asian/european origin for the suborder yangochiroptera [ ] . within the insectivores, talpidae occupies the basal position within the soricomorpha [ , ] , and both molecular clock dating and the fossil record suggest a eurasian origin of the soricidae [ ] [ ] [ ] . as the hantaviruses sampled from asian mammals are genetically very diverse and tend to occupy basal positions in the phylogenetic trees, these data tentatively support an asian origin for hantaviruses, although this will need to be assessed on a more geographically diverse sample. hantaviruses have traditionally been considered to have codiverged (including co-speciation) with their rodent hosts on timescales of millions of years [ ] [ ] [ ] [ ] , and some evidence for such codivergence was apparent here. in particular, rodent hantaviruses clustered according to whether their hosts were members of the murinae subfamily and cricetidae family. indeed, the close phylogenetic relationships among some hantavirus taxa across large geographic areas, and which infect related hosts, supports the occurrence of long-term virus-host co-divergence [ ] . hence, rodent hantaviruses might have a long history in their primary hosts, and which in part explains their biodiversity [ ] . despite this, more examples of incongruence between the gene trees of hantaviruses and their hosts are being identified, suggesting that some of the congruence between the two might have arisen from preferential host switching and local adaptation [ ] . indeed, it was recently shown that cross-species transmission has even played a role in shaping the genetic diversity of the currently known murinae-associated hantaviruses [ ] . in accord with this, the current study provides evidence for cross-species transmission events at the family, genus, and species levels. in particular, that viruses from both the chiroptera and soricomorpha form paraphyletic groups in all our analyses strongly suggests that ancestral hantaviruses jumped between mammalian orders. as a consequence, it is clear that cross-species virus transmission as well as the geographic dispersal of chiroptera, soricomorpha and rodentia, has also contributed to the high biodiversity and near global distribution of those hantaviruses known today, although the time-scale of these host jumping events remains uncertain. this study was reviewed and approved by the ethics committee of the national institute for communicable disease control and prevention of the chinese cdc. all animals were treated in strict according to the guidelines for the laboratory animal use and care from the chinese cdc and the rules for the implementation of laboratory animal medicine ( ) from the ministry of health, china, under the protocols approved by the national institute for communicable disease control and prevention. all surgery was performed under ether anesthesia, and all efforts were made to minimize suffering. (figure ). according to protocols described previously [ ] , insectivore animals were trapped in cages using fried foods as bait in the inner mongolia autonomous region in or in yunnan province in the autumns of and . all animals kept were alive after capture. they were initially identified by morphological examination according to the criteria for bats described by wang [ ] and for insectivores by chen [ ] , and further confirmed by sequence analysis of the mt-cyt b gene. all animals were anesthetized with ether before surgery, and all efforts were made to minimize suffering. tissue samples of heart, liver, spleen, lung, kidney and brain were collected from bats and insectivores for detecting hantaviruses. total dna was extracted using the dneasy blood & tissue kit (qiagen) from tissue samples of bats or insectivores according to the manufacturer's protocol. the mitochondrial (mt)-cyt b gene ( bp) was amplified by pcr with the primer pair for bats [ ] and one for insectivores [ ] . total rna was extracted from tissue samples using trizol reagent (invitrogen, carlsbad, ca) according to the manufacturer's instructions. cdna was prepared with amv reverse transcriptase (promega, beijing) with the primer p [ ] . hantaviral rna was detected by rt-pcr as described previously [ , ] . primers designed based on the conserved regions of known complete s and m segment sequences from hantaviruses were used to amplify the entire s and m segments. in the amplification of the terminus of unknown hantaviruses, an adaptor plus p was used as a primer in the synthesis of cdna. semi-pcr was used to amplify the terminus with the adaptor as the forward primer and two specific reverse primers. semi-pcr was also used to amplify the terminus with two specific forward primers and an adaptor plus modified p ( -tagtagtr-gacwcc- ) [ ] as the reverse primer. primer sequences used in this study are provided in table s . the rt-pcr products were separated by agarose gel and further purified using the agarose gel dna purification kit (takara, dalian, china). amplicons less than bp were sequenced from both directions. amplicons greater than bp were cloned into pmd -t vector (takara, dalian, china). sequencing was performed using the abi-prism dye termination sequencing kit and abi -a genetic analyzer. at least three clones were sequenced. one to three sequences of the entire open reading frame (orf) were randomly chosen from each hantavirus species for phylogenetic analysis. the rdp program [ ] was used to examine potential intra-segment recombination in the viral sequences, although no recombinant sequences were identified (although we do find evidence for segment reassortment -see below). identical sequences were excluded from this study. both animal mt-cyt b gene and viral genome sequences were aligned using the clustalw method implemented in the lasergene program, version (dnastar, inc., madison, wi). poorly figure , figure s ; aligned positions and divergent regions of the alignment, and which could negatively affect phylogenetic analysis, were removed using gblocks [ ] . the following data set sizes were used in the final analysis: hantavirus s segment = sequences, bp; m segment = sequences, bp; l segment = sequences, bp, partial l segment = , bp; mt-cyt b gene = sequences, bp. phylogenetic trees were estimated using the maximum likelihood (ml) method available at the raxml blackbox web-server [ ] . the best-fit evolutionary model was determined using jmodeltest version . [ ] , and found to be the general time reversible (gtr) with a gamma-distribution model of among site rate heterogeneity and a proportion of invariant sites (gtr+c+i). phylogenetic trees were also inferred using the bayesian method implemented in mrbayes v . . [ ] . the same evolutionary model was employed as described above. for this analysis, three hot and one cold markov chain monte carlo (mcmc) chains were used, sampling every generations and with a % burnin. the effective sample size (ess) of all parameters was larger than indicating that parameter convergence had occurred. a (molecular clock) rooted tree of these sequences was inferred using the bayesian mcmc method available in the beast v . . package [ ] . the same evolutionary model was employed as described above. we also incorporated a relaxed (uncorrelated lognormal) molecular clock, with an extended bayesian skyline tree prior. two independent runs were undertaken sampling every , generations. each run was continued until ess . was achieved, with the output analyzed in tracer v . . treeannotator was used to generate a maximum cade credibility (mcc) tree with a burn-in of % of the sampled trees. because the mcc tree is automatically rooted on the assumption of a molecular clock it enables determination of which viral lineages are most likely to be basal. accordingly, the basal lineage estimated by the mcc tree was used as an outgroup to root the phylogenetic trees inferred under the ml and bayesian phylogenetic analyses. in addition, because the high levels of sequence divergence across the hantaviruses, we also inferred phylogenetic trees based on the amino acid sequences of the l protein, n protein (encoded by the s segment), and gpc protein (encoded by the m segment) using the ml approach available in the phyml program [ ] . the lg amino acid substitution model was used for both the l and gpc proteins, and while the flu model was used for the n protein. finally, a phylogenetic tree for the host mt-cyt b sequences tree was estimated using the ml and beast (mcc tree) methods, again employing the gtr+c+i substitution model as estimated by jmodeltest. in the case of the beast analysis a relaxed (uncorrelated lognormal) molecular clock was used along with the yule model as a coalescent prior. to determine the degree of congruence between the phylogenies of hantaviruses and their hosts we used tree-map ( . b) [ ] , although such analyses are complicated by uncertainties in the virus or host trees. a tanglegram was generated by matching each hantavirus species to their associated host(s). specifically, nodes of the viral mcc tree were mapped onto the related nodes of the host (mcc) tree. significance testing was undertaken by generating viral trees with randomized branches and mapping these random trees onto the fixed host tree. we then evaluated the proportion of these reconciliations with the same or fewer non-co-divergence events (nces), or the same or more codivergence events (ces), compared to the ''real'' viral tree. if the p value is greater than . , we can reject the null hypothesis that the level of congruence is no more than that expected between randomly generated trees. due to computational limitations in treemap [ ] , we reduced the complexity of the host and virus phylogenies as much as possible before performing the full reconciliation analysis. thus, for viruses-host matches, we divided all hantaviruses and their hosts into four groups: (i) bats and insectivores and their viruses (rockport virus (rkpv) was removed because it was a reassortant virus), (ii) murinae and their viruses, (iii) arvicolinae and their viruses (luxi virus (luxv) was removed because it was a reassortant virus), and (iv) sigmodontinae and their viruses. for the full reconciliation analysis, three species (virus or host) representing each of four groups (bats and insectivores, murinae, arvicolinae, and sigmodontinae) were used to compare the host and the virus phylogenies (and including scalopus aquaticus and rkpv, eothenomys miletus and luxv). cross-species transmission events were then inferred by comparing the topologies of the virus and host phylogenies. specifically, we considered the degree of congruence between the viral s, m, and s+m segment trees with that of the host mt-cyt b gene tree ( figure , figure s ; table s ). importantly, any viruses or hosts exhibiting phylogenetic uncertainty were excluded from the analysis. emerging infectious diseases of wildlife-threats to biodiversity and human health the challenge of emerging and reemerging infectious diseases emergence and pandemic potential of swine-origin h n influenza virus origins of major human infectious diseases epidemic dynamics at the human-animal interface public health. pathogen surveillance in animals a distinct lineage of influenza a virus from bats a global perspective on hantavirus ecology, epidemiology, and disease virus taxonomy: th report of the international committee on taxonomy of viruses thottapalayam virus: a presumptive arbovirus isolated from a shrew in india electron microscopic and antigenic studies of uncharacterized viruses. ii. evidence suggesting the placement of viruses in the family bunyaviridae evolutionary diversification of protein-coding genes of hantaviruses virus evolution and genetic diversity of hantaviruses and their rodent hosts molecular evolution of puumala hantavirus genetic analysis of the diversity and origin of hantaviruses in peromyscus leucopus mice in north america divergent ancestral lineages of newfound hantaviruses harbored by phylogenetically related crocidurine shrew species in korea a new subtype of thottapalayam virus carried by the asian house shrew (suncus murinus) in china short report: genetic diversity of thottapalayam virus, a hantavirus harbored by the asian house shrew (suncus murinus) in nepal studies on hantavirus infection in small mammals captured in southern and central highland area of vietnam thottapalayam virus: a prototype shrewborne hantavirus thottapalayam virus is genetically distant to the rodent-borne hantaviruses, consistent with its isolation from the asian house shrew (suncus murinus) divergent lineage of a novel hantavirus in the banana pipistrelle (neoromicia nanus) in côte d'ivoire hantavirus in bat evolutionary insights from a genetically divergent hantavirus harbored by the european common mole (talpa europaea) hantavirus evolution in relation to its rodent and insectivore hosts: no evidence for codivergence newfound hantavirus in chinese mole shrew characterization of imjin virus, a newly isolated hantavirus from the ussuri white-toothed shrew (crocidura lasiura) novel hantavirus in the flat-skulled shrew (sorex roboratus) seewis virus, a genetically distinct hantavirus in the eurasian common shrew molecular phylogeny of a newfound hantavirus in the japanese shrew mole (urotrichus talpoides) phylogenetically distinct hantaviruses in the masked shrew (sorex cinereus) and dusky shrew (sorex monticolus) in the united states host switch during evolution of a genetically distinct hantavirus in the american shrew mole (neurotrichus gibbsii) shared ancestry between a newfound mole-borne hantavirus and hantaviruses harbored by cricetid rodents a novel hantavirus detected in yunnan red-backed vole (eothenomys miletus) in china hantavirus in african wood mouse molecular phylogenetics and the origins of placental mammals phylogeny and divergence-date estimates of rapid radiations in muroid rodents based on multiple nuclear genes bats: important reservoir hosts of emerging viruses migration of norway rats resulted in the worldwide distribution of seoul hantavirus today jurassic eutherian mammal and divergence of marsupials and placentals the historical biogeography of mammalia a molecular phylogeny for bats illuminates biogeography and the fossil record molecular phylogenetics of shrews (mammalia: soricidae) reveal timing of transcontinental colonizations fossil history of shrews in europe fossil history of shrews in asia cross-species transmission in the speciation of the currently known murinae-associated hantaviruses methods for trapping and sampling small mammals for virologic testing a complete checklist of mammal species and subspecies in china -a taxonomic and geographic reference classification and identification of medical animals. beijing: the institute of epidemiology and microbiology, chinese academy of preventive medicine phylogenetics of small horseshoe bats from east asian based on mitochondrial and sequence variation molecular phylogeny and biogenography of oriental voles: genus eothenomys (muridae, mammalia) coding strategy of the s genome segment of hantaan virus rdp : a flexible and fast computer program for analyzing recombination improvement of phylogenies after removing divergent and ambiguously aligned blocks from protein sequence alignments a rapid bootstrap algorithm for the raxml web servers jmodeltest: phylogenetic model averaging mrbayes: bayesian inference of phylogenetic trees beast: bayesian evolutionary analysis by sampling trees new algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of phyml . a cophylogenetic perspective of rna-virus evolution an integer linear programming formulation of the cophylogeny reconstruction problem key: cord- -fh ic kp authors: hatesuer, bastian; bertram, stephanie; mehnert, nora; bahgat, mahmoud m.; nelson, peter s.; pöhlman, stefan; schughart, klaus title: tmprss is essential for influenza h n virus pathogenesis in mice date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: fh ic kp annual influenza epidemics and occasional pandemics pose a severe threat to human health. host cell factors required for viral spread but not for cellular survival are attractive targets for novel approaches to antiviral intervention. the cleavage activation of the influenza virus hemagglutinin (ha) by host cell proteases is essential for viral infectivity. however, it is unknown which proteases activate influenza viruses in mammals. several candidates have been identified in cell culture studies, leading to the concept that influenza viruses can employ multiple enzymes to ensure their cleavage activation in the host. here, we show that deletion of a single ha-activating protease gene, tmprss , in mice inhibits spread of mono-basic h n influenza viruses, including the pandemic swine influenza virus. lung pathology was strongly reduced and mutant mice were protected from weight loss, death and impairment of lung function. also, after infection with mono-basic h n influenza a virus body weight loss and survival was less severe in tmprss mutant compared to wild type mice. as expected, tmprss -deficient mice were not protected from viral spread and pathology after infection with multi-basic h n influenza a virus. in conclusion, these results identify tmprss as a host cell factor essential for viral spread and pathogenesis of mono-basic h n and h n influenza a viruses. annual influenza epidemics and unpredictable pandemics pose a severe threat to human health, exemplified by the estimated - million deaths caused by the pandemic. current therapy targets viral proteins, neuraminidase and m , but is hampered by development of resistance [ ] , due to the high mutation rate of the virus. novel antiviral strategies are urgently required and invariable host cell factors essential for viral spread are attractive targets. the cleavage of the influenza virus hemagglutinin (ha) by host cell proteases is essential for viral infectivity [ , ] . the ha proteins of highly pathogenic avian influenza viruses harbor multiple basic amino acids at their cleavage site and are activated by furin [ ] . in contrast, low pathogenic avian and human influenza viruses contain a mono-basic cleavage site in their ha proteins. several studies showed that multiple secreted proteases can activate human influenza viruses for infection of cell lines (see [ , ] for examples and [ ] for a review). however, the analysis of cultured human respiratory epithelium demonstrated that influenza virus cleavage activation is a cell-associated process and no evidence for a role of secreted proteases was obtained [ ] . subsequently, the type ii transmembrane serine protease (ttsp) family member tmprss , a membrane associated protease, was shown to activate ha proteins of diverse human influenza viruses in cell culture [ , , , ] . in addition, tmprss was found to be expressed in human respiratory epithelium positive for alpha , linked sialic acid [ ] . however, the role of tmprss in influenza virus spread and pathogenesis in the infected host has not yet been studied. therefore, we investigated if tmprss contributes to influenza virus replication and pathogenesis in experimentally infected mice. we focused our analysis on viruses of the h n (including the pandemic influenza virus) and h n subtypes, since viruses of these subtypes are presently circulating in the human population. our study shows that deletion of tmprss in knock-out mice strongly limits virus spread and lung pathology after h n influenza a virus infection. the deletion of tmprss also reduces body weight loss and mortality after h n infection but to a much lower degree than for h n infected mice. to assess the role of tmprss during influenza virus infection in vivo, we used mice carrying a deletion of the tmprss gene [ ] . non-infected tmprss knock-out mice did not show a phenotype in the absence of infection, as described previously [ ] and rt-pcr analysis of kidney tissue confirmed the absence of full length tmprss transcripts. upon intranasal infection of mice with mouseadapted pr m (a/puertorico/ / h n münster variant, [ ] ), wild type mice lost weight significantly after infection and % of infected mice died, whereas tmprss knock-out mice did not exhibit body weight loss and showed no signs of disease (figs. a; fig. s ). the same results were obtained after infection with a human isolate of the pandemic ha (a/hamburg/ / h n ) virus ( figure b) . also, after infection with a lethal dose of the more virulent pr f virus isolate (a/puertorico/ / h n freiburg variant) all wild-type mice died within seven days post infection whereas no knock-out mice showed symptoms of disease ( figure c ). similar results were observed for blood oxygen saturation levels which provide a measurement for lung function. whereas wild type mice exhibited a significant drop in peripheral blood oxygen saturation that peaked at day post infection (p.i.) with pr m virus, tmprss / mutant mice showed only a very mild change ( figure ). histological analyses of infected lungs revealed a similar onset of the influenza infection on day post infection in tmprss / and wild type mice with infected epithelial cells in the bronchiole ( figure a ,b,e,f). however, at day p.i. virus was spreading into the alveolar regions of wild type mice whereas it was only found in bronchioles in tmprss / mice (figure c ,d and g,h, respectively). furthermore, infected wild type mice showed a strong increase in lung infiltrates and also in the number of infected cells whereas in tmprss knock-out mice a much lower number of infiltrating cells and infected cells were observed (figure k,l and o,p, respectively). thus, the absence of tmprss largely protects animals from virus spread and virus induced pathogenesis. next, we assessed if protection from pathogenesis was due to reduced viral spread. after infection with pr m, we could detect infectious viral particles in the lung in both homozygous tmprss / and wild type mice. however, the number of infectious particles was close to background at day p.i. in tmprss / mice and was markedly reduced at days and p.i. compared to wild type mice ( figure ). influenza-specific antibodies were readily detectable in sera of tmprss knock-out mice ( figure s ) after infection with pr m and pr f virus demonstrating that the inoculated virus was able to infect lung cells and elicit a humoral immune response. in conclusion, these results show that tmprss is critical for efficient spread and pathogenesis of epidemic and pandemic h n influenza viruses in vivo. tmprss is required for ha cleavage in mouse lungs next, we sought to obtain direct evidence for the lack of proteolytic cleavage of ha in tmprss deficient mice. for this, broncho-alveolar lavages (bal) of infected mice were collected and the proteolytic processing of the ha precursor protein ha was analyzed. after infection with a high dose of pr m virus, processed ha as well as non-processed ha protein were detected in infected wild type mice whereas only ha protein was found in homozygous tmprss mutant mice ( figure ). these results demonstrate that tmprss is essential for efficient ha cleavage activation in mice. tmprss -deficient mice were also protected from pathogenesis after infection with h n virus but to a lesser extent at present, influenza viruses of the ha subtypes h and h are circulating in humans. therefore, we investigated if a h virus was also dependent on expression of a functional tmprss gene. after infection with a low dose ( focus forming units (ffu)) of a mouse-adapted h n virus (a/hk/ / [ ] ), body weight loss is less severe and survival is increased in tmprss knock-out compared to wild type mice ( figure a , b). after infection with a higher dose ( ffu) of h n virus, mortality was significantly lower in tmprss / mice compared to wild type mice ( figure a ). however, no significant differences were observed in the amount of infectious particles at day to p.i. ( figure b ). thus, activity of tmprss is required for the processing of both h n and h n but h n viruses may be cleaved by other proteases in addition to tmprss . tmprss is not required for spread and pathogenesis of a virus with a multi-basic cleavage site in cell culture, tmprss is dispensable for cleavage activation of viruses with a multi-basic cleavage site [ ] . thus, if the resistance of tmprss knock-out mice to h n infection was indeed due to lack of ha processing, a virus with a multi-basic cleavage site should spread efficiently and cause disease. to investigate this, we infected mice with sc m (mouse-adapted a/ seal/massachusetts/ / , h n ) influenza virus which contains a multi-basic ha cleavage site. mortality and body weight loss in infected tmprss / mice were not significantly different compared to wild type and tmprss +/ infected mice ( figure ), suggesting that the presence of a multi-basic cleavage site renders viral spread and pathogenesis independent of tmprss expression. finally, we investigated if murine tmprss was able to activate ha proteins of h n and h n viruses. the co-expression of protease and the respective ha proteins of pr m, ha , and h n viruses in transfected cells facilitated ha cleavage of all viruses ( figure s a ). additionally, expression of tmprss in this cell culture system allowed the spread of pr m and ha viruses in a trypsin-independent fashion ( figure s b ). in contrast, spread of mouse-adapted sc m virus, containing a multi-basic cleavage seasonal influenza epidemics and pandemics represent a serious health threat to the human population. resistance to presently available anti-viral drugs is frequently observed. therefore, identification of new targets for anti-viral therapy is an urgent need. host proteases are required for processing of the virus hemagglutinin and may thus represent a suitable target for intervention. here, we report that the deletion of a single host protease gene, tmprss , in mice protects the host against viral spread in infected lungs. only very mild pathogenesis was observed in tmprss mutant mice after infection with h n virus and less severe pathogenesis was observed after infection with h n virus. thus, our results suggest that the host protease tmprss may be a prime target for antiviral intervention. tmprss knock-out influenza resistance figure . tmprss is essential for spread and pathogenesis of h n influenza viruses. eight to eleven weeks old female mice were infected with ffu mouse-adapted pr m (h n ; a), ffu ha (ph n , b), ffu mouse-adapted pr f (h n ; c). body weight loss was monitored until day p.i. mice with a weight loss of more than % of the starting bodyweight were euthanized and recorded as dead. weight loss data represent mean values +/ sem. note that only data of surviving mice are presented (e.g. about % of infected mice died after infection with pr m, see fig. s ). body weight loss was significantly different between wild type and homozygous mutant mice at day p.i. (p, . for pr m infected mice, and p, . for ha infected mice, using the non-parametric mann whitney u test) and between heterozygous and homozygous mutant mice (pr m infected mice, p, . using the mann whitney u test). doi: . /journal.ppat. .g tmprss knock-out influenza resistance plos pathogens | www.plospathogens.org site did not depend on tmprss expression [ ] . thus, murine tmprss , like its human homologue [ , ] can activate ha. cleavage activation of influenza virus ha by host cell proteases is essential for viral infectivity [ , ] . however, the nature of the proteases required for the cleavage activation of viruses with a mono-basic ha cleavage site in the infected host organism remains unclear. at least eight candidate enzymes from different protease families have been suggested based on cell culture studies [ ] , leading to the concept that redundant proteolytic enzymes activate influenza viruses in the host. here, we show for the first time that the deletion of a single protease, tmprss , in mice largely abrogates viral spread and protects animals from severe pathology and death after h n and, to a lower extent, h n influenza virus infection. after infection of tmprss / mice with h n virus, no processing of the ha precursor protein ha was observed in bal. however, an initial increase in viral titers was measured from day to day p.i. whereas at later times p.i. viral titers rapidly decreased. this initial increase in viral titers is expected because the virus used for infections had been produced in embryonated chicken eggs. it therefore carries an activated ha allowing it to enter cells and replicate [ , ] . in addition, it is conceivable that other proteases besides tmprss may facilitate low levels of h cleavage and allow limited viral spread which is rapidly cleared once the antiviral immune responses have been activated. however, the cleavage activation by such alternative enzymes must be very inefficient since viral titers in h n infected tmprss / mice were markedly reduced compared to wild type animals and no weight loss was observed. surprisingly, h n virus which also carries a mono-basic cleavage site in the ha, was able to replicate in tmprss / mice. however, body weight loss and mortality were significantly reduced in knock-out mice compared to wild type mice in a dose-dependent manner. on the other hand, viral load was not significantly lower in mutant mice after infection with ffu. the amino acid sequence in the ha loop which is recognized by proteases differs between ha subtypes h and h (figure ) . a recent study demonstrated that different proteases, including tmprss , tmprss d (hat) and even trypsin, cleave ha from different subtypes and variants with varying efficiency [ ] . in addition, st (matriptase) has the capability to cleave ha of particular h subtype strains but only minimal cleavage was observed for h and h [ ] . furthermore, klk and (kallikrein related-peptidase and ) have been described as host proteases that are capable of cleavage activation of viral h , h and h ha in vitro [ ] . thus, h n appears to be processed to some extent by tmprss , resulting in the reduced pathology in tmprss knock-out mice but also other proteases are able to cleave h hemagglutinin in vivo. finally, it is noteworthy that tmprss / mutant mice were not protected from infection with an h n virus which contains a multi-basic ha cleavage site and can be activated by ubiquitously expressed proteases [ ] . our findings may have potential for the development of future influenza virus therapeutics. broad spectrum protease inhibitors have been shown to inhibit influenza virus in cell culture and in vivo [ , , , , ] but unwanted side effects are a major concern. the results reported here suggest that targeting a single protease, tmprss , may be sufficient to achieve a notable therapeutic benefit against h n influenza viruses and possibly other subtypes. blocking of tmprss may not be associated with severe unwanted side effects, since tmprss / mice are healthy and do not show any phenotypic alterations in the absence of an infection [ ] . furthermore, tmprss inhibitors might exert activity against diverse respiratory infections. for example, human metapneumovirus [ ] , the emerging mers-coronavirus [ , ] and sars-coronavirus [ , , ] can also be activated by tmprss in cell culture and might use this protease to support their spread in the infected host. it should, however, be noted that our findings in the mouse model system require validation in humans. infectious virus particles were determined in lung homogenates. viral load was higher in infected wild type mice compared to infected homozygous mutant mice at days , and p.i. individual values, mean and sem are presented. detection limit of the assay is at infectious particles per lung indicated by the blue line. day p.i. n = for tmprss / , n = for tmprss +/+ , day p.i. n = for tmprss / , n = for tmprss +/+ , day p.i. n = for tmprss / , n = for tmprss +/+ . doi: . /journal.ppat. .g figure . the hemagglutinin of h n pr m influenza virus is not processed in tmprss knock-out mice. bal from infected wild type and tmprss / male mice was harvested at day after infection with ffu pr m and viral particles were concentrated by centrifugation through a sucrose cushion. as control, bal from noninfected wild type and tmprss / was analyzed. each sample was loaded undiluted (first lane), and in two dilutions (second lane : . , third lane : ). the virus-containing pellets were then analyzed for ha cleavage by western blots. as loading control, the stripped membranes were incubated with anti-influenza a virus antibody confirming that equal amounts of proteins were loaded for respective undiluted and diluted samples. [ ] ) and from thorsten wolff, robert-koch-institute, berlin (ha ). the different virulence of pr m and pr f virus isolates has been described before [ ] . sc m (h n ) was originally derived from the seal, adapted to the mouse by serial passages in the mouse lung [ ] , and the laboratory strain which was used here was generated by reverse genetics using the plasmid rescue system [ ] . virus figure . tmprss knock-out mice show reduced body weight loss and mortality after infection with low dose h n influenza a virus infections. eight to eleven weeks old female mice were infected with ffu mouse-adapted h n influenza virus by intra-nasal application and bodyweight (a) and survival (b) was monitored until day p.i. in addition to mice that were found dead, mice with a weight loss of more than % of the starting body weight were euthanized and recorded as dead. homozygous tmprss knock-out mice lost significantly less weight than wild type (e.g. p = , at day , and p = , at day , using mwu test) mice and showed reduced mortality compared to wild type mice, although this difference was not significant (using the log rank test). doi: . /journal.ppat. .g tmprss knock-out influenza resistance stocks of pr were prepared by infection of -day-old embryonated chicken eggs and for ha on mdck cells as described [ ] . mutant tmprss / mice were on a mixed c bl/ j - background [ ] . animals were maintained under specific pathogen free conditions at the animal facility of the hzi in braunschweig. heterozygous mutant mice were interbred and wild type, heterozygous and homozygous mutant mice were genotyped by pcr analysis and then used for infections. genotyping of tmprss alleles was carried out using a three primer strategy (p tgtgcccttggacagatgactc , p ggactacagatatgaggtgttc , p aggcca-gaggccacttgtgtag ) that allows to distinguish between wild type (yielding a bp product) and knock-out (yielding a bp product) alleles, respectively. expression plasmids encoding human and mouse proteases tmprss were published earlier [ , ] . previously described expression plasmids for pr m [ ] were used as templates for amplification of the respective coding regions using oligonucleotides pr -ha- acc: gggggtac-caccatgaaggcaaacctactggtcctg, pr -ha- nhe: gggcgctagctcagatgcatattctgcactg. the resulting pcr products were inserted into plasmid pcaggs using the acc i and nhei sites. for cloning of ha ha protein, expression plasmid kindly provided by prof. klenk [ ] was used as templates for amplification of the coding region using figure . tmprss knock-out mice show reduced mortality after infection with high dose h n influenza a virus infections. eight to eleven weeks old female mice were infected with ffu mouse-adapted h n influenza virus by intra-nasal application and survival (a) was monitored until day p.i. in addition to mice that were found dead, mice with a weight loss of more than % of the starting body weight were euthanized and recorded as dead. infectious viral particles were determined in lung homogenates (b). individual values, mean and sem are presented. detection limit of the assay is at infectious particles per lung indicated by the blue line. homozygous tmprss knock-out mice showed significantly reduced mortality compared to wild type and heterozygote mice (p, . and p = . , respectively, using the log rank test). viral load was not significantly different in infected wild type mice compared to infected homozygous mutant mice at days to p.i. doi: . /journal.ppat. .g tmprss knock-out influenza resistance plos pathogens | www.plospathogens.org oligonucleotides swi -ha- eco: gggaattcaccatgaag-gcaatactagtagttctgc and swi -ha- xho: gggc-tcgagttaaatacatattctacactgtagag. the resulting pcr products were inserted into plasmid pcaggs using ecori and xhoi. for infection experiments, female mice at the age of - weeks were anesthetized by intra-peritoneal injection of ketamin-xylazine solution in sterile nacl ( mg/ml ketamine, invesa arzneimittel gmbh, freiburg; % xylazine, bayer health-care, leverkusen) with a dose adjusted to the individual body weight. infection was performed by intranasal application of virus solution in ml of sterile phosphate-buffered saline. subsequently survival and body weight loss were monitored until day p.i. in addition to mice that were found dead, mice with a weight loss of more than % of the starting body weight were euthanized and recorded as dead. viral load in infected lungs was determined on mdck ii (madin-darby canine kidney ii) cells using the ffu assay as described [ ] . detection limit of the assay is at infectious particles/lung. thus, for samples where no virus was detected, the data points were set to ffu/lung. for analysis of ha in viral particles, broncho-alveolar lavages (bals) of wild type and tmprss knock-out mice infected with ffu pr m were harvested at day p.i., centrifuged at full speed and supernatant were loaded on a % sucrose cushion for concentration of viral particles for - h at full speed and uc. the pelleted viral particles were lysed in sds loading buffer. i. in addition to mice that were found dead, mice with a weight loss of more than % of the starting bodyweight were euthanized and recorded as dead. no significant differences were observed in survival between heterozygous and homozygous mutant mice after h n infections (using the log rank test for immuno-blotting, the lysates were separated by sds gel electrophoresis, blotted on a nitrocellulose membrane and ha was detected by staining with a rabbit anti-pr ha antibody (sino biological) at a dilution of : , followed by incubation with a horseradish peroxidase (hrp)-coupled anti-rabbit antibody (dianova) at a dilution of : . . as loading control, membranes were stripped (stripping buffer: m tris-hcl (ph , ), % sds, mm ß-mercaptoethanol) for min at uc, washed h with dh o and stained for na by using an anti-influenza a virus goat serum (millipore) at a dilution of : followed by incubation with a horseradish peroxidase (hrp)-coupled antigoat antibody (dianova) at a dilution of : . . bands were visualized by using a commercially available kit ecl prime western blotting detection reagents (amersham). eight to twelve weeks old mice were infected intranasally with ffu pr m and the amount of oxygen saturation was determined by the mouseoxh system (starr life science corp.) over a period of days. for oxygen measurements mice were anesthetized using an isoflurane inhalator. lungs were prepared and immersion-fixed for hours in % buffered formaldehyde solution (ph . ), dehydrated in a series of graded ethanol and embedded in paraffin. sections ( . mm) were cut from five evenly distributed levels of the paraffin blocks and stained with haematoxylin and eosin. for immunohistochemical studies, sections were stained with a polyclonal primary antibody (against influenza a h n virions; virostat) overnight at uc and subsequently tissue sections were incubated for min with secondary antibody (rabbit anti-goat-biotin; kpl; gaithersburg, madison, usa) and counterstained with haematoxylin. t cells were transiently transfected with expression plasmids encoding human or mouse tmprss or control transfected with empty plasmid. at h post transfection, cells were infected with either of the influenza viruses pr m, ha or sc m at a multiplicity of infection (moi) of . after h incubation at uc, virus was removed and fresh mem medium (supplemented with . % bsa and mg/ml tpck-trypsin or pbs) was added to the cells. at h p.i., supernatants were harvested, cleared from debris by centrifugation for min at . rpm and stored at uc until quantification of infectious virus particles by focus formation assay (ffu) as described previously [ ] . figure s tmprss is essential for h n influenza virus pathogenesis. eight to eleven weeks old female mice were infected with ffu mouse-adapted pr m (h n ). survival was monitored until day p.i. in addition to mice that were found dead, mice with a weight loss of more than % of the starting bodyweight were euthanized and recorded as dead. all tmprss knock-out mice survived the infections whereas about % of wild type or tmprss heterozygous mice died. (tif) figure s surviving tmprss / mice mount antibodies against viral proteins after infection with h n influenza a virus. after infection, blood from surviving mice was collected by heart puncture. sera were diluted : and an elisa was performed using plates that were coated with . ffu pr m virus. for detection of virus specific igg, peroxidaselabeled anti-mouse igg (kpl, gaithersburg, madison, usa) was used as a secondary antibody and visualization of the reaction was carried out using a peroxidase specific substrate. absorbance at nm is shown. as control, sera from non-infected mice were analyzed. sera from surviving wild type and homozygous tmprss mutant mice infected with pr m (a) or pr f (b) were analyzed days after infection for influenza-specific igg antibodies. individual values, mean and sem are presented. (tif) figure s murine tmprss activates diverse h n influenza viruses by cleavage of the hemagglutinin. the indicated ha proteins (pr m, ha and h n ) and proteases (human or mouse tmprss ) were transiently co-expressed in t cells, the cells were treated with pbs or trypsin, and ha cleavage was detected by western blotting (a). protease transfected t cells were infected with the indicated viruses at a multiplicity of infection of in the presence or absence of trypsin. antiviral resistance during the influenza a h n pandemic: public health, laboratory, and clinical perspectives activation of influenza a viruses by trypsin treatment enhancement of the infectivity of influenza a and b viruses by proteolytic cleavage of the hemagglutinin polypeptide influenza virus hemagglutinin with multibasic cleavage site is activated by furin, a subtilisin-like endoprotease isolation and characterization of a novel trypsin-like protease found in rat bronchiolar epithelial clara cells. a possible activator of the viral fusion glycoprotein miniplasmin found in the epithelial cells of bronchioles triggers infection by broadspectrum influenza a viruses and sendai virus novel insights into proteolytic cleavage of influenza virus hemagglutinin cleavage of influenza a virus hemagglutinin in human respiratory epithelium is cell associated and sensitive to exogenous antiproteases inhibition of influenza virus infection in human airway cell cultures by an antisense peptideconjugated morpholino oligomer targeting the hemagglutinin-activating protease tmprss tmprss and tmprss facilitate trypsin-independent spread of influenza virus in caco- cells proteolytic activation of influenza viruses by serine proteases tmprss and hat from human airway epithelium characterization of lentiviral pseudotypes with influenza h n hemagglutinin and their performance in neutralization assays influenza and sars-coronavirus activating proteases tmprss and hat are expressed at multiple sites in human respiratory and gastrointestinal tracts phenotypic analysis of mice lacking the tmprss -encoded protease pathogenicity of different pr influenza a virus variants in mice is determined by both viral and host factors natural, genetically determined resistance toward influenza virus in hemopoietic mouse chimeras. role of mononuclear phagocytes proteolytic activation of the influenza virus hemagglutinin proteases essential for human influenza virus entry into cells and their inhibitors as potential therapeutic agents an endoprotease homologous to the blood clotting factor x as a determinant of viral tropism in chick embryo isolation of factor xa from chick embryo as the amniotic endoprotease responsible for paramyxovirus activation influenza ha subtypes demonstrate divergent phenotypes for cleavage activation and ph of fusion: implications for host range and adaptation cleavage activation of the human-adapted influenza virus subtypes by matriptase reveals both subtype-and strain-specificity cleavage activation of human-adapted influenza virus subtypes by kallikrein-related peptidases and aprotinin and similar protease inhibitors as drugs against influenza inhibition of lung serine proteases in mice: a potentially new approach to control influenza infection suppression of influenza virus replication in infected mice by protease inhibitors high protection of animals lethally infected with influenza virus by aprotinin-rimantadine combination aprotinin aerosol treatment of influenza and paramyxovirus bronchopneumonia of mice efficient multiplication of human metapneumovirus in vero cells expressing the transmembrane serine protease tmprss the spike protein of the emerging betacoronavirus emc uses a novel coronavirus receptor for entry, can be activated by tmprss , and is targeted by neutralizing antibodies middle east respiratory syndrome coronavirus (mers-cov) infection mediated by the transmembrane serine protease tmprss evidence that tmprss activates the severe acute respiratory syndrome coronavirus spike protein for membrane fusion and reduces viral control by the humoral immune response a transmembrane serine protease is linked to the severe acute respiratory syndrome coronavirus receptor and activates virus entry efficient activation of the severe acute respiratory syndrome coronavirus spike protein by the transmembrane protease tmprss the viral polymerase mediates adaptation of an avian influenza virus to a mammalian host characterization of the influenza a virus gene pool in avian species in southern china: was h n a derivative or a precursor of h n ? the mouse as model system to study host-pathogen interactions in influenza a infections replication fitness determines high virulence of influenza a virus in mice carrying functional mx resistance gene characterization of the neuraminidase of the h n / pandemic influenza virus we would like to thank the animal caretakers at the central animal facilities at the hzi for maintaining the mice for this study, karin lammert, lukas gusdek and jared lucas for excellent technical assistance, michael winkler for ha and pr f ha expression plasmid, and peter stä heli, stefan ludwig and thorsten wolff for providing the original stocks of influenza a viruses. at h p.i., viral spread was quantified by focus formation assay (b). virus release into the medium is presented as means sd and was confirmed in at least two independent experiments. (tif) key: cord- -jhetyd t authors: kovalev, nikolay; pogany, judit; nagy, peter d. title: a co-opted dead-box rna helicase enhances tombusvirus plus-strand synthesis date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: jhetyd t replication of plus-strand rna viruses depends on recruited host factors that aid several critical steps during replication. in this paper, we show that an essential translation factor, ded p dead-box rna helicase of yeast, directly affects replication of tomato bushy stunt virus (tbsv). to separate the role of ded p in viral protein translation from its putative replication function, we utilized a cell-free tbsv replication assay and recombinant ded p. the in vitro data show that ded p plays a role in enhancing plus-strand synthesis by the viral replicase. we also find that ded p is a component of the tombusvirus replicase complex and ded p binds to the ′-end of the viral minus-stranded rna. the data obtained with wt and atpase deficient ded p mutants support the model that ded p unwinds local structures at the ′-end of the tbsv (−)rna, rendering the rna compatible for initiation of (+)-strand synthesis. interestingly, we find that ded p and glyceraldehyde- -phosphate dehydrogenase (gapdh), which is another host factor for tbsv, play non-overlapping functions to enhance (+)-strand synthesis. altogether, the two host factors enhance tbsv replication synergistically by interacting with the viral (−)rna and the replication proteins. in addition, we have developed an in vitro assay for flock house virus (fhv), a small rna virus of insects, that also demonstrated positive effect on fhv replicase activity by the added ded p helicase. thus, two small rna viruses, which do not code for their own helicases, seems to recruit a host rna helicase to aid their replication in infected cells. all eukaryotic plus-stranded (+)rna viruses have similar replication cycles in infected cells. after translation of their mrna-sense genomic rna(s), the viral rna and the viral replication proteins are recruited to the site of viral replication in membranous compartments. after the assembly of the membranebound viral replicase complexes (vrc), the viral replicase uses the viral rna as a template to produce complementary ( ) rna. this is then followed by (+)-strand synthesis in an asymmetric manner, producing excess amounts of (+)-strand progeny, which is released from replication for other viral processes. for efficient replication, (+)rna viruses recruit numerous host proteins [ ] [ ] [ ] [ ] [ ] . among the identified host proteins are rna-binding proteins, such as translation factors, ribosomal proteins and rnamodifying enzymes [ ] . the co-opted host proteins likely affect several steps in viral rna replication, including the assembly of the replicase complex and/or viral rna synthesis. however, the functions of host factors in (+)rna virus replication are known only for a small number of host factors [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . tomato bushy stunt virus (tbsv) is a model plant rna virus that is used intensively for identification and characterization of host factors [ , [ ] [ ] [ ] . the single genomic rna codes for two replication proteins, p and p pol , which are sufficient to support tbsv replicon (rep)rna replication in yeast (saccharomyces cerevisiae) model host [ , ] . p and p pol are components of the membrane-bound vrc that also requires the tombusviral (+)reprna as a platform during its assembly and activation [ , [ ] [ ] [ ] . recent genome-wide screens and global proteomics approaches with tbsv based on yeast revealed a large number of host factors interacting with viral components or affecting tbsv replication. a large fraction of the identified host proteins are rna binding proteins, which might affect viral rna synthesis [ , [ ] [ ] [ ] . the highly purified tombusvirus vrc is known to contain at least six permanent resident host proteins, including the heat shock protein chaperones (hsp , ssa / p in yeast) [ ] [ ] [ ] [ ] , glyceraldehyde- -phosphate dehydrogenase (gapdh, encoded by tdh and tdh in yeast) [ ] , pyruvate decarboxylase (pdc p) [ ] , cdc p e ubiquitin conjugating enzyme [ ] [ ] [ ] , eukaryotic translation elongation factor a (eef a) [ , ] , eukaryotic translation elongation factor bgamma (eef bc) [ ] and two temporary resident proteins, pex p shuttle protein [ ] and the vps p adaptor escrt protein [ , , ] . although the functions of several of these proteins have been studied in some details, additional host proteins might be also present in tombusvirus vrc [ , , [ ] [ ] [ ] ] . one of the intriguing questions is if tombusviruses use host-coded helicases for their replication. this is because, unlike larger rna viruses, the tombusvirus genome does not code for a protein with helicase function. thus, can tombusviruses and other small rna viruses replicate without the use of a helicase or they subvert a cellular helicase(s) to assist their replication? dead-box proteins constitute the largest family of rna helicases that are involved in all aspects of cellular metabolism and perform rna duplex unwinding and remodeling of rna-protein complexes in cells [ ] [ ] [ ] . ded p, which is essential for yeast growth, is among the best-characterized dead-box helicases [ ] . it is involved in initiation of translation of every yeast mrna [ ] [ ] [ ] and down-regulation of ded p level also reduced p and p levels in yeast [ ] . therefore, not surprisingly, tbsv replication also decreased significantly in yeast with reduced level of ded p. due to its essential role in translation, it is critical to separate the possible role of ded p in viral replication from its effect on viral translation. in this paper, a yeast-based cell-free tbsv replication assay and recombinant ded p was used to test the role of ded p in tbsv replication. we define that ded p plays a role in enhancing plusstrand (+)rna synthesis by the viral replicase. we also find that ded p is a component of the tombusvirus vrc and binds to the -end of the viral minus-stranded ( )rna. the obtained data support the model that ded p unwinds the -end of the tbsv ( )rna, rendering the rna compatible for initiation of (+)strand synthesis. interestingly, we find that ded p and gapdh play non-overlapping functions to enhance (+)-strand synthesis. altogether, the two host factors enhance tbsv replication synergistically by interacting with the viral ( )rna and the replication proteins. reduced level of tbsv rna replication in yeast cell-free extract depleted for ded p dead-box rna helicase since ded p is an essential translation factor for all yeast mrnas [ , ] , it is difficult to separate its direct versus indirect effect on tbsv replication in yeast model host. to circumvent this problem, we used whole cell extracts (cfe) prepared from yeast containing reduced level of ded p ( figure s ) to support cell-free tbsv replication. as we have shown previously, tbsv (+)rna can perform one complete cycle of replication in the cfe-based replication assay when purified recombinant p and p pol replication proteins are provided [ , ] . the cfe-based replication assay showed that both (+) and ( )rna synthesis decreased by over -fold when ded p was down-regulated as compared with the control cfe prepared from yeast with high level of ded p expression ( figure s , lanes - versus - ). thus, these data confirm that ded p is important for tbsv replication. however, the observed decrease in the cfe-based tbsv replication could be due to either reduced replication when ded p is limiting (direct effect) or lesser amounts of other critical host factors needed for tbsv replication in the cfe with reduced level of ded p (indirect effect due to ded p's role in host translation). to address these points, we first studied if ded p is present within the tombusvirus replicase and then, what is the mechanistic role of ded p during tbsv rna synthesis. to examine if ded p is present within the tombusvirus replicase complex, we flag affinity-purified the tombusvirus replicase from yeast cells actively replicating tbsv reprna [ , ] . this yeast also expressed ha-tagged ded p from yeast chromosome based on the native promoter. we found that the tombusvirus replicase preparation, which is highly active on added templates in vitro (not shown), contained ded p ( figure , lane ), while ded p was undetectable in the control yeast sample obtained using the same affinity purification ( figure , lane ). the control yeast also expressed the tombusvirus replication proteins (including the his-tagged p , but lacking flag-tag) and the ha-tagged ded p from yeast chromosome based on the native promoter and supported tbsv reprna replication (not shown). we also flag-affinity-purified his/flag-tagged ded p from the membrane-fraction of yeast co-expressing ha-p and ha-p and found that the purified ded p preparation contained ha-tagged p ( figure s a , lane ). the affinity-purified ded p preparation also showed tbsv replicase activity in vitro on added tbsv template ( figure s b , lane ), suggesting that the membrane-bound ded p is associated with the active tombusvirus replicase. altogether, these data support the model that ded p is part of the tombusvirus replicase. figure . co-purification of ded p with the p replication protein from yeast. the flag/ his-tagged p hf or his-tagged p h was purified from yeast extracts using a flag-affinity column (lanes - ). top panel: western blot analysis of ded p tagged with ha, expressed from the natural promoter in the chromosome, with anti-ha antibody in the purified p preparations. bottom panel: western blot analysis of hf-tagged p or his-tagged p with anti-his antibody. note that ''total'' represents the total protein extract from yeast expressing the shown proteins. each experiment was repeated three times. doi: . /journal.ppat. .g subverted host factors play a role in plus-strand rna virus replication. small rna viruses do not code for their own helicases and they might recruit host rna helicases to aid their replication in infected cells. in this paper, the authors show that the ded p dead-box helicase, which is an essential translation factor in yeast, is recruited by tomato bushy stunt virus (tbsv) into its replicase complex. they also show that ded p binds to the viral ( )rna and promotes (+)-strand tbsv synthesis when added to a yeast-based cell-free extract depleted for ded p. an atpase defective ded p mutant failed to promote tbsv replication in vitro, suggesting that the helicase activity of ded p is essential for its function during tbsv replication. in addition, the authors also show that another host protein, which also binds to the ( )rna, namely glyceraldehyde- -phosphate dehydrogenase (gapdh), further enhances tbsv (+)rna when added together with ded p to yeast-based cell-free extract. in summary, the authors show that the major functions of ded p and gapdh host proteins are to promote tbsv replication via selectively enhancing (+)-strand synthesis. recombinant ded p dead-box helicase enhances tbsv rna replication in yeast cell-free extract to address if ded p could play a direct role in tbsv replication, we added purified recombinant ded p to yeast cfe with reduced level of ded p (figure a ). we observed a , -fold increased level of tbsv rna replication (based on total singlestranded reprna level) in the presence of recombinant ded p when compared with the mbp control ( figure b , lane versus ). interestingly, the amount of double-stranded rna, which correlates with ( )-strand levels [ ] , did not increase in the presence of ded p in the cfe assay ( figure b, lanes versus ) . this finding suggests that ( )-strand rna did not contribute to the -fold increase in total tbsv rna levels when recombinant ded p was added to the cfe-based replication assay. therefore, we conclude that the added recombinant ded p selectively increased tbsv (+)-strand rna synthesis in vitro. to demonstrate if the atpase/helicase activity of ded p is important for the enhancement of tbsv rna replication in the cfe-based replication assay, we tested an atpase defective mutant of ded p (d mutant) [ ] . this mutant could not enhance tbsv replication in vitro ( figure c , lanes - versus - ), suggesting that the atpase activity of ded p is important for tbsv replication. to further test what step(s) ded p promotes during tbsv replication, we performed a two-step replication assay based on yeast cfe. in this assay, the first step includes the assembly of the replicase complex on the endogenous membranes present in cfe in the presence of the viral (+)reprna, the p /p replication proteins and atp/gtp [ ] ( figure a , step ). under these conditions, the viral replication proteins recruit the reprna to the membrane and the replicase becomes partially rnase and protease insensitive, but it cannot initiate minus-strand synthesis yet, due to the absence of ctp/utp [ ] . then, centrifugation and washing the membranes will remove all the proteins and molecules not bound to the membrane. this is followed by addition of atp/ctp/gtp/utp to initiate rna synthesis during the second step. addition of purified ded p to the cfe during the first step did not increase tbsv replication ( figure b , lane versus ). this suggests that ded p did not facilitate the assembly of the replicase complex, unlike other host factors, such as hsp and eef a [ , ] . moreover, ded p was likely lost during the centrifugation/washing step in this assay due to its low association with the membrane. however, addition of ded p exclusively during the second step of the cfe assay resulted in , -fold increase in tbsv rna replication ( figure b , lane versus ), similar to the stimulatory effect of ded p during standard cfe replication assay ( figure ). interestingly, a ded p mutant (d ) deficient in atpase activity could not stimulate tbsv rna synthesis in this assay ( figure b , lane ), while a mutant (d , figure s c ) with increased atpase activity [ ] promoted tbsv rna synthesis by , -fold ( figure b , lane ). altogether, these findings indicate that ded p has a direct stimulatory function during tbsv rna synthesis, while ded p is unlikely to affect viral rna recruitment for replication or vrc assembly. to further test the direct effect of ded p on rna synthesis, we utilized detergent-solubilized and affinity-purified tombusvirus -stranded rna products produced in the cell-free tbsv replication assay. the dsrna products are also shown on the right subpanel with high contrast for better visualization. odd numbered lanes represent replicase products, which were not heat treated (thus both ssrna and dsrna products are present), while the even numbered lanes show the heat-treated replicase products (only ssrna is present). the % of dsrna and ssrna in the samples are shown. note that, in the nondenatured samples, the dsrna product represents the annealed ( )rna and the (+)rna, while the ssrna products represents the newly made (+)rna products. right replicase from ded p-depleted yeast ( figure a ). this purified replicase can only synthesize complementary rna products on added tbsv templates, but, unlike the above membrane-bound replicase in the cfe-based assay, it cannot perform a complete cycle of rna synthesis [ , ] . we found that addition of purified recombinant ded p to the purified tombusvirus replicase programmed with the ( )reprna stimulated (+)-strand synthesis by , . - -fold ( figure b , lanes - versus - ; and figure s d , lane versus ). interestingly, ded p stimulated the production of the full-length (+)-strand rna product, while the amount of -terminal extension product ( tex; due to self-priming by the end of the template [ ] [ ] [ ] , figure s b ) decreased in the presence of added recombinant ded p. therefore, we suggest that ded p facilitates the de novo initiation on the ( )rna template by the tombusvirus replicase. the atpase inactive mutant d ( figure b , lanes - and s d, lane ), d and d ( figure s d , lanes and ) could not promote (+)-strand synthesis, suggesting that the atpase activity of ded p is required for the above stimulatory effect on tbsv rna synthesis. also, two active atpase mutants, d and d ( figure s c ), did facilitate (+)-strand synthesis ( figure s d , lanes and ), confirming that the atpase activity of ded p is required during tbsv (+)-strand rna synthesis. interestingly, ded p mutant d , albeit has increased atpase activity, has wt-like strand displacement activity in vitro [ ] . we found that d behaved similarly in tbsv replicase assay ( figure s d ) to wt ded p, suggesting that the unwinding activity of ded p is important during tbsv replication. to test if ded p can also stimulate the activity of the tombusvirus replicase on (+)-stranded rna templates, we used di- (+) rna in the purified tombusvirus replicase assay ( figure s a ). the tbsv (+)rna is known to carry a replication silencer element (rse) at the end that inhibits ( )rna synthesis in vitro [ ] . addition of ded p to the tombusvirus replicase assay did not enhance ( )rna synthesis ( figure s b , lane versus - ). also, d mutant deficient in atpase activity had not much effect on ( )rna synthesis ( figure s b , lane versus - ). based on these data, we suggest that ded p does not affect ( )rna synthesis by the tombusvirus replicase in vitro. to identify the region(s) of the tbsv rna bound by the recombinant ded p, we performed electrophoresis mobility shift assay (emsa) with purified components. comparison of plabeled tbsv (+) and ( )rnas in binding to purified recombinant ded p revealed that ( )rna bound more readily to ded p in vitro than (+)rna ( figure b , lanes - versus - ). additional emsa experiments using the four regions in di- ( )reprna ( figure a ) as unlabeled competitors revealed that ri( ) was the most efficient in outcompeting the p-labeled tbsv ( )reprna in binding to ded p ( figure c , lanes [ ] [ ] . this is important since ri( ) is the end of ( )reprna and contains important cisacting elements, such as the promoter and a short enhancer sequence for (+)rna synthesis [ , ] . further testing of ded p binding to (+)reprna regions revealed that riv(+), representing the noncoding region in tbsv rna, was bound more efficiently by ded p ( figure s , step-wise cell-free tbsv replication assay does not support a role for ded p helicase in the assembly of the tbsv vrc. (a) scheme of the cfe-based tbsv replicase assembly and replication assays. purified recombinant p and p pol replication proteins of tbsv and in vitro transcribed tbsv di- (+)reprna were added to the whole cell extract prepared from ded p-depleted yeast strain in step . the assay either contained or lacked the purified recombinant ded p ( . mg) or mbp during step . note that the assay was performed in the presence of atp/gtp to facilitate tbsv vrc assembly, but prevent rna synthesis in step . after step , centrifugation was used to collect the membrane fraction of the cfe, and after washing the membranes, step was performed in the presence of atp/ctp/gtp and p-utp to allow tbsv rna replication. in the samples presented in right panel, ded p or mbp were added at the beginning of step . (b) denaturing page analysis of the p-labeled tbsv reprna products obtained in the cfe assays in the presence of wt and various ded p mutants ( . mg each) ( figure s c ) or mbp. see further details in figure . note that the various preps of d mutant of ded p showed high variation in in vitro activity (for reasons currently unknown)-see figure b . each experiment was repeated at least three times and the data were used to calculate standard deviation. to test if ded p can interact with the tbsv p replication protein, we used the membrane-based split-ubiquitin assay [ ] . we found that ded p interacted with p protein in yeast ( figure s a ). the pull-down experiments with mbp-p and mbp-p also showed that ded p bound to the tombusvirus replication proteins in vitro ( figure s b ). this was further supported by the reverse pull-down experiments with gst-tagged ded p, which resulted in the co-purification of p and p ( figure s c ). to test if a dead-box helicase from plants might have similar stimulatory function on the activity of tombusvirus replicase, we have cloned and purified rh cytosolic dead-box helicase from arabidosis thaliana, which shows high degree of similarity to yeast ded p ( figure s ). addition of the recombinant atrh to the cfe prepared from ded p-depleted yeast strain increased tbsv reprna replication by , -fold ( figure a, lane ) . also, adding the recombinant rh to the replicase assay led to almost yeast with depleted ded p co-expressing p and p pol replication proteins and di- (+)reprna were used to affinitypurify the rna-free tombusvirus replicase. the in vitro assays were programmed with di- ( )reprna, and they also contained purified recombinant ded p, mutants or mbp in addition to atp/ctp/gtp and p-utp. (b) representative denaturing gel of p-labeled rna products synthesized by the purified tombusvirus replicase in vitro in the presence of . mg or . mg of purified recombinant ded p or its mutants is shown. the level of complementary rna synthesis producing ''reprna'' (marked as ''fl'', the full-length product, made via de novo initiation from the -terminal promoter) in each sample was compared to that of the replicase activity obtained in the absence of added recombinant protein (lane ). note that this replicase preparation also synthesizes de novo internal initiation products (''ii'') and -terminal extension products ('' tex''). each experiment was repeated three times. (c) representative denaturing gel of p-labeled rna products synthesized by the purified fhv replicase in vitro in the presence of . mg of purified recombinant ded p or its mutants is shown. note that the fhv replicase can use tbsv ( )reprna as a template in vitro. each experiment was repeated three times. (d) representative denaturing gel of p-labeled rna products synthesized by the purified fhv replicase in vitro in the presence of . mg of purified recombinant ded p or its mutants is shown. note that the fhv di- ( )reprna was used as a template in the fhv replicase assay. doi: . /journal.ppat. .g -fold increase of the activity of the purified tombusvirus replicase on tbsv ( )rna template ( figure b , lanes - versus ). to test if atrh can interact with the tbsv p replication protein, we used the membrane-based split-ubiquitin assay [ ] . we found that atrh , similar to ded p, interacted with p protein in yeast ( figure s a ). altogether, these data strongly suggest that plants also have dead-box helicases that could play similar role to the yeast ded p dead-box helicase in tombusvirus (+)rna synthesis. to test if flock house virus (fhv) replicase is affected by ded p, first we have developed an in vitro fhv replication assay based on affinity-purified fhv replicase preparation that can be programmed with exogenously added rnas ( figure c , lanes versus ). the in vitro fhv replicase assay revealed that the purified recombinant ded p increased (+)-strand rna synthesis on the ( )-stranded fhv template by , -fold ( figure d figure s d , lane versus ). it is likely that the observed stimulation of fhv replicase activity by ded p is direct, since we found that ded p bound to the fhv reprna ( figure s a ) and protein a rdrp protein in vitro ( figure s b ). altogether, these data show that, similar to the tombusvirus replicase, the activity of fhv replicase is stimulated by ded p only on the ( )rna templates, but not when using the (+)rna templates. ded p facilitates initiation by the tombusvirus replicase on rna/dna duplex to gain further insights into the mechanism of ded p-driven stimulation of tbsv and fhv rna synthesis, we exploited template structures, such as an rna/dna duplex, that are known to hinder rdrp-driven rna synthesis [ , ] . since ded p is an rna helicase [ , ] and the atpase/helicase function of ded p is needed for stimulation of (+)rna synthesis by the tombusvirus and fhv replicases, we wanted to examine if ded p might facilitate rna synthesis on a partial dna/rna duplex. we chose partial duplex for this assay, since ded p and other dead-box helicases are not processive enzymes and can only unwind short duplexes [ ] . also, we have shown previously that short dna oligos hybridized to the promoter region of ( )rna can inhibit (+)rna synthesis by the tombusvirus replicase in vitro [ , , ] . interestingly, addition of purified ded p to the tombusvirus replicase assay containing the short rna/dna duplex ( figure a non-overlapping functions of ded p and gapdh in promoting initiation by the tombusvirus replicase gapdh (tdh p in yeast) rna binding protein is also a host factor stimulating (+)rna synthesis by the tombusvirus replicase [ , ] . to test if ded p and gapdh could play a complementary role during (+)rna synthesis, we added the purified recombinant ded p and tdh p to the in vitro tombusvirus replicase assay based on the purified preparation ( figure a ). while ded p mostly stimulated de novo (+)rna synthesis initiated from the end of the ( )reprna up to , -fold ( figure b , lane ), tdh p enhanced both de novo (+)rna synthesis and tex by , -and , -fold, respectively ( figure b, lane ) . interestingly, the two host proteins together had the largest ( . -fold) effect on (+)rna synthesis, while their effect on tex was only , -fold. based on these data, we suggest that ded p and tdh p have a we also performed similar experiments with a short partial rna/dna duplex as a template. both ded p and tdh p alone could enhance (+)rna synthesis on the short rna/dna duplex ( figure c , lanes - versus ) but the largest stimulatory effect (i.e., close to -fold increase) was seen when both host factors were included in the assay ( figure c, lane ) . altogether, these data further support that ded p and gapdh play synergistic roles in (+)rna synthesis by the tombusvirus replicase. [ ] . since the viral rna plays multiple roles during infection, it is likely that remodeling of the viral rnas and rnp complexes during the switch from one step to another requires rna helicases or rna chaperones. accordingly, the larger rna viruses all code for rna helicases [ , ] . however, rna viruses with shorter than kb genomes usually do not code for rna helicases. they could still use rna helicases during infections if they can subvert selected host helicases for viral purposes. indeed, we show here that ded p helicase is recruited for tbsv replication to aid (+)strand rna synthesis. it is not yet known if ded p is the only host helicase needed for tbsv replication, since genome-wide screens and global proteomics approaches with tbsv have identified additional host helicases as well [ , , ] . however, ded p helicase seems to be an ideal host factor to be recruited for viral replication because it is involved in mrna and viral rna translation, thus it is colocalized with the viral rna prior to replication. also, by recruiting ded p, viruses could affect rna degradation (p-body formation; [ ] ) and initiation of translation of new rnas, likely affecting subsequent host translation (including virus-induced mrnas coding for anti-viral proteins or required for anti-viral signaling). the essential nature of ded p for host mrna translation, however, makes characterization of ded p function as a host factor difficult. therefore, the combined use of in vivo and in vitro approaches might be necessary to dissect the function of ded p in rna virus replication as shown in this paper. by using a ded pdepleted yeast cfe in combination with recombinant ded p allowed us to define that the atpase (helicase) activity of ded p is required for efficient tbsv (+)-strand synthesis (see below). ded p also increased the rdrp activity of the fhv replicase, suggesting that recruitment of dead-box helicases might also be useful for additional small rna viruses (see below). co-purification experiments revealed that ded p is present in the tombusviral vrc (figure ). in addition, ded p affected (+)strand synthesis, but not the assembly of the vrc in vitro (figures - ) , indicating that ded p is likely present in the vrc. surprisingly, ded p, unlike other previously tested host factors hsp , gapdh, or eef a [ ] [ ] [ ] , ] , was able to affect tbsv reprna replication even after the replicase assembly step took place in vitro (figure ). this suggests that ded p can enter the membrane-bound vrc, possibly due to its interaction with p ( figure s ) and the helicase activity of ded p might lead to some remodeling of vrc. it is not yet known if additional members of the large helicase family could perform similar function to ded p during tbsv replication. our in vitro data with atrh plant helicase protein, which is very similar to ded p ( figure s ), suggests that this protein can likely perform similar function in plant infections. for example, atrh has been shown to boost tbsv (+)rna synthesis in vitro ( figure ) and bind to p replication protein ( figure s a ), which can facilitate its recruitment into the vrc. since more than rna helicases are present in plants, further experiments will be needed to define if additional host helicases might also be involved in tbsv replication. the in vitro data, based on the cfe assay containing the membrane-bound vrc as well as the solubilized/purified tombusvirus replicase, showed the ded p can mainly stimulate tbsv (+)-strand synthesis, while its effect on ( )rna synthesis is less pronounced. the atpase activity of ded p is required for this stimulatory effect, suggesting that the helicase function of ded p is likely important for unwinding the secondary structure of ( )rna template with the purified tombusvirus replicase or destabilizing the replication intermediate with the membrane-bound vrc, which might contain dsrna structure. this function of ded p can explain why yeast with down-regulated ded p level produced small amount of (+)-stranded rna progeny, albeit it has also shown that depleted ded p reduced the level of p /p in yeast [ ] . however, the decreased p /p levels are expected to reduce both (+) and ( )rna levels [ ] ( figure s ). since the recombinant ded p enhanced (+)-strand synthesis by the purified recombinant tombusvirus replicase, we propose that ded p directly affect tbsv rna synthesis via affecting the structure of the rna templates. however, we cannot exclude that ded p could also affect the activity of the vrc due to its interaction with p , albeit the assembly of the vrc was not affected by ded p in the cfe-based assay ( figure ) . overall, the recruitment of a host dead-box helicase for replication of a small rna virus is remarkable, since rna viruses with less than kb genomes are usually do not code for their own helicases [ ] . these viruses are thought to replicate without needing a helicase by using rna chaperones or possibly recruiting host helicases. the emerging picture with tbsv is that this virus utilizes both the viral-coded p rna chaperone [ ] and the host ded p helicase for replication by promoting (+)-strand synthesis. both p and ded p have been shown to open up short dna/rna duplexes, although the activity of ded p was more robust than that of p in vitro [ ] . why would tbsv utilize both an rna chaperone and an rna helicase? it is possible that both proteins are needed for robust (+)rna synthesis to make excess amount of progeny rna. also, ded p helicase could be involved in remodeling the viral rna bound by the viral rdrp or host proteins prior or during rna synthesis. since ded p can work in both -to- and -to- directions [ ] , it could be used for multiple purposes during rna synthesis. based on the available data, it seems that tbsv (+)rna synthesis is not only affected by the p /p replication proteins, but by gapdh and ded p host proteins as well. since the viral replication proteins were shown to bind to tbsv ( )rna nonspecifically [ ] , we propose that the above host proteins, which bind strongly to the tbsv ( )reprna, are involved in facilitating the proper and efficient recruitment of the p rdrp protein to the ( )rna template (or alternatively to the dsrna intermediate) within the vrc as shown in figure . for example, ded p might unwind either local secondary structure in ( )reprna or dsrna region and that, in turn, could favor binding of gapdh or p to the ( )rna template. this is followed by binding of gapdh to an au-rich internal site and proper positioning of the p rdrp (bound to gapdh, [ ] ) over the (+)-strand initiation promoter, leading to (+)rna synthesis. the synergistic effect of these host proteins could promote efficient recycling of the viral rdrp resulting in multiple rounds of (+)rna synthesis ( figure ). thus, the roles of these host proteins are to serve as ''matchmakers'' between the viral rna template and the viral rdrp. it is intriguing that two host proteins, eef a and eef bc, are proposed to serve somewhat similar functions during ( )rna initiation for tbsv [ , ] . therefore, the emerging picture is that tbsv utilizes different host proteins for promoting ( ) versus (+)rna synthesis. this strategy could be beneficial for the virus by allowing asymmetric rna synthesis, thus leading to excess amount of progeny (+)rna. overall, host dead-box or related rna helicases have been shown to affect many different aspects of virus infections, including translation of viral proteins [ ] [ ] [ ] ; viral rna replication [ ] [ ] [ ] [ ] ; reverse transcription [ ] ; the activity of anti-viral proteins [ , ] , and virus-mediated regulation of host gene transcription [ ] . interestingly, ded p is known to affect minus-strand synthesis during replication of the l-a dsrna virus of yeast [ ] , suggesting that the use of dead-box helicases is wide-spread among rna viruses. saccharomyces cerevisiae strain by (mata his d leu d met d ura d ) and tet::ded yeast strain (ythc library, mata his d leu d met d ura ::cmv-tta) was obtained from open biosystems (huntsville, al, usa). the plasmid pesc-his-gal-his /gal-di- expressing cucumber necrosis virus (cnv) his-tagged p and the tbsv di- reprna was described earlier [ ] . the cnv p protein has very high sequence identity with the closely related tbsv p , but the cnv p is expressed better and more active in yeast than tbsv p . recombinant yeast tdh p protein was produced in e. coli as gst fusion using plasmid pgex-tdh , described earlier [ ] . recombinant ded p helicase proteins were produced in e. coli as maltose binding protein (mbp) fusions [ ] . the expression plasmid pmal-ded was prepared by pcr using primers # (ccagctg-cagtcaccaccaagaagagttg)/ # (ccaggaatt-tggctgaactgagcgaacaag) and the yeast genomic dna as a template. the plasmids pmal-dx, expressing different ded p mutants, were prepared by pcr using # / # primers and plasmids containing mutated ded sequences [ , ] . the pcr products obtained from ded wt and mutant sequences were digested with ecori and psti and inserted between ecori and psti sites in pmalc- (new england biolab). to express recombinant fhv protein a in yeast, we generated plasmid pgad/cup/fhv/protein a/c-term/ha/flag. the following sequence was fused downstream to the full length fhv protein a coding region by repeated pcr and cloning steps: the pcr product was amplified with oligos # (ccgat-catgactctaaaagttattcttggag) and # (gg-agctcgagttacttatcgtcatcgtc) followed by digestion with pagi and xhoi. it was cloned into ncoi and xhoi digested pcuphis [ ] . expression and purification of the recombinant mbp-tagged host proteins and the mbp-tagged tbsv p and p replication proteins and ded p helicase from e. coli were carried out as described earlier with modifications [ ] . briefly, the expression plasmids were transformed separately into e. coli strain bl (de ) codonplus. protein expression was induced using isopropyl b-dthiogalactopyranoside (iptg) for h at uc in the case of host proteins and at uc in the case of p and p , then the cells were collected by centrifugation ( , rpm for min). the cells were suspended and sonicated in mbp column buffer containing mm hepes-koh ph . , mm nacl, mm edta, mm b-mercaptoethanol. the extract was then centrifuged at , g for min, followed by incubation with amylose resin (neb) for min at uc. after washing the resin times with the column buffer, the proteins were eluted with column buffer containing . % (v/w) maltose. purification of gst-tagged tdh (pgex-tdh ) [ ] was carried out using glutathione resin and eluted with mm glutathione, mm ß-mercaptoethanol in the column buffer following the same protocol as mbpproteins. eluted proteins were aliquoted for storage at uc. protein fractions used for the replication assays were at least % pure, as determined by sds-page (not shown). we have previously shown that our ded p preparation has helicase activity on short rna/dna duplexes in vitro [ ] . the p-labeled or unlabeled full-length di- (+) and ( )rnas and the four separate regions (ri-iv), were generated as described [ ] . transcripts for replicase or cfe replication assays were purified as described earlier [ ] . the amounts of transcripts were quantified by uv spectrophotometer (beckman). to obtain fulllength fhv-derived di- rna [ ] , we used primers # (gtaatacgactcactatagtaaacaattccaagttcc-aaaatgg) and # (accttagtctgttgacttaaa-ctgg) or # (gtaaacaattccaagttcc) and # (gtaatacgactcactatagggaaccttagtctgttg-acttaaac) for (+) or ( )di- rnas, respectively, and pdi [ ] as a template in pcr reactions. the rna transcripts were synthesized on the pcr templates using t based transcription [ ] . in vitro tbsv replication assay in cell-free yeast extract cfes from by or tet::ded strains capable of supporting tbsv replication in vitro were prepared as described earlier [ , ] . briefly, the in vitro tbsv replication assays were performed in -ml total volume containing ml of cfe, . [ , ] . host proteins were added in different amounts as indicated in the figure legends. the reaction was performed as described [ , ] . the newly synthesized p-labeled rna products were separated by electrophoresis in a % polyacrylamide gel (page) containing . tris-borate-edta (tbe) buffer with m urea. to detect the double-stranded rna (dsrna) in the cell-free replication assay, the p-labeled rna samples were divided into two aliquotes: one half was loaded onto the gel without heat treatment in the presence of % formamide, while the other half was heat denatured at uc for min in the presence of % formamide [ ] . fractionation of the whole cell extract was done according to [ , ] . the total extract was centrifuged at , g at uc for min to separate the ''soluble'' (supernatant) and ''membrane'' (pellet) fraction. the pellet was re-suspended and washed with buffer a ( mm hepes-koh ph . , mm potassium acetate, and mm magnesium acetate) followed by centrifugation at , g at uc for min and re-suspension of the pellet in buffer a. in vitro tbsv replication in the fractions was performed as described [ , ] . yeast strains (by and tet::ded ) were transformed with plasmids pgbk-hisflagp (or pgbkhisp as a control), pgad-hisflagp (or pgad-hisp as a control) and pyc-di . the his-flag double-tagged hf-p and p were expressed from the adh promoter and di- reprna was under the gal promoter. transformed yeast were pre-grown on sc-ulh media containing % glucose at uc. after centrifugation at , rpm for min and washing pellet with selective media containing % galactose and mg/ml doxycycline, yeast were grown for hours in sc-ulh media containing % galactose at uc. the replicase purification was done according to a previously described procedure [ ] with the following modification. briefly, mg of yeast cells were resuspended and homogenized in tg buffer [ mm tris-hcl [ph . ], % glycerol, mm mgcl , mm kcl, . m nacl, , and % [v/v] yeast protease inhibitor cocktail (ypic)] by glass beads using fastprep homogenizer (mp biomedicals). the membrane fraction containing the viral replicase complex was solubilized with ml tg buffer containing % triton x- , % [v/v] ypic as described [ , ] . after affinity purification of hf-p on anti-flag m -agarose affinity resin (sigma), the resinbound replicase complex was eluted in ml elution buffer , % glycerol, mm mgcl , mm kcl, mm nacl, % triton x- , and . mg/ml flag peptide (sigma)]. in vitro rdrp activity assay was performed by using di- ( ) or (+) rna template transcribed in vitro by t transcription [ ] . to measure the effect of host proteins in the replicase assay with di ( ) rna containing short double-stranded region at the end, a heat denatured rna transcript ( uc for min) was annealed with a -nt oligodeoxynucleotide (# ) (in : molar ratio) complementary to the end of di- ( )rna in ste buffer ( mm tris, ph . , mm edta, and mm nacl) and then slowly (in min) cooled them down to uc . rnase one digestion to remove single-stranded p-labeled rna was performed at uc for min in a rnase one buffer containing . ml of rnase one (promega). to obtain fhv replicase preparation, by yeast strain was transformed with plasmid pgad/cup/fhv/proteina/c-term/ ha/flag and pesc-his-gal ::fhvrna framshift. after selection of transformed yeast on sc-lh plates, yeast were pre-grown overnight in selective media containing % glucose at uc. after centrifugation at , rpm for min and washing pellet with selective media containing % galactose, yeast were grown hours at uc in sc-lh media containing % galactose and mm cuso to induce fhv rna replication. affinity-purification was done similarly as for tombusvirus, except for using different buffers: homogenization buffer consisted of the split-ubiquitin assay was based on the dualmembrane kit (dualsystems). the bait construct, pgad-bt -n-his , expressing the cnv p replication protein has been described earlier [ ] . the prey constructs were made by pcr amplification of individual genes using gene specific primers: # (ccagctg-cagtcaccaccaagaagagttg) / # (ccagccat-ggccaccaagaagagttg) followed by digestion with ecor and nco for ded and # (ccagggatccatgac-ttacggtggtagag) / # (ccagccatggatagt-ttgaacgacctc), # (ccagggatccatgagtcgc-tacgatagccg) / # (ccagccatgggctccaccc-tcttctgctc) followed by digestion with bamh and nco in the case of rh gene, respectively. digested pcr products were fused to nubg at either the -or -termini (nubg-x and x-nubg) by cloning into pprn-n-re or pprn-c-re vectors [ ] , respectively, using the same enzymes. yeast strain nmy was co-transformed with pgad-bt -n-his and ppr-n-re or one of the prey constructs carrying the cdna for a given helicase and plated onto trp /leu synthetic minimal medium plates. transformed colonies were picked with a loop, re-suspended in water, and streaked onto tlha (trp /leu /his /ade ) plates to test for p -helicase protein interactions as described [ ] . protein co-purification with the viral replicase s. cerevisiae strain ded :: ha-hphnt was generated by homologous recombination using strain by . pcr was performed using plasmid pym- (euroscarf) [ ] as template and primers # (gcagaaaacgaagaatcct-caccctagtttgtctgaaatcaatcgatgaattcgag-ctcg) / # (ggctggggtaacagcggtggttcaaa-caactcttcttggtggcgtacgctgcaggtcgac). the pcr products were transformed to by and recombinant yeast colonies were selected in ypd plates supplemented with hygromycin. recombinant yeast strains were transformed with plasmids pgbk-hisflagp , pgad-hisflagp and pyc-di [ ] . his/flag-tagged hf-p and hf-p were expressed from adh promoter and di- transcript was under gal promoter. after selection of transformed yeast on sc-ulh plates, yeast were pre-grown overnight in selective media containing % glucose at uc. after centrifugation at , rpm for min and washing the pellet with selective media containing % galactose, yeast were grown for hours in sc-ulh media containing % galactose at uc. ml of pelleted yeast were used to affinity-purify hf-p and hf-p with anti-flag m agarose as described previously (see also text s ) [ , ] . hf-p and hf-p were detected with anti-his antibody ( / , dilution) and ap-conjugated anti-mouse antibody ( / , ). ded - ha protein was detected with anti-ha antibody from rabbit (bethyl; / , dilution) and ap-conjugated anti-rabbit ( / , ) followed by nbt-bcip detection. figure s reduced tbsv replication in cfe prepared from ded -depleted yeast. cfes were prepared from tet::ded yeast strain cultured in the absence of doxycycline (i.e., ded p is expressed) or in its presence ( mg/ml) (i.e., ded p is depleted). the cfes were programmed with recombinant p /p and di- (+) reprna as described in ref . note that the dsrna product represents the annealed ( )rna and the (+)rna, while the ssrna products represents the newly made (+)rna products. the cfes contained comparable amounts of host proteins (not shown). figure s ded p mutants with atpase activity promote plusstrand synthesis by the affinity-purified tombusvirus replicase. (a) scheme of the tombusvirus replicase assay. yeast with depleted ded p co-expressing p and p pol replication proteins and di- (+)reprna were used to affinity-purify the rna-free tombusvirus replicase. the in vitro assays were programmed with di- ( )reprna, and they also contained purified recombinant ded p. (b) representative denaturing gel of p-labeled rna products synthesized by the purified tombusvirus replicase in vitro in the presence of . mg of purified recombinant ded p. the level of complementary rna synthesis producing ''reprna'' (marked as ''fl'', the full-length product, made via initiation from the -terminal promoter) in each sample was compared to that of the replicase activity obtained in the absence of added recombinant protein. note that this replicase preparation also synthesizes internal initiation products (''ii'') and -terminal extension products ('' tex''). rnase one digestion was used to confirm the replicase products: the de novo products are insensitive, while the tex product changes migration after rnase treatment yeast as a model host to explore plant virus-host interactions organelle-like membrane compartmentalization of positive-strand rna virus replication factories cytoplasmic viral replication complexes emerging picture of host chaperone and cyclophilin roles in rna virus replication the dependence of viral rna replication on coopted host factors yeast as a model host to dissect functions of viral and host factors in tombusvirus replication host factors involved in west nile virus replication viral and cellular proteins involved in coronavirus replication diverse roles of host rna binding proteins in rna virus replication global genomics and proteomics approaches to identify host factors as targets to induce resistance against tomato bushy stunt virus multiple roles of viral replication proteins in plant rna virus replication the roles of host factors in tombusvirus rna recombination yeast as a model host to study replication and recombination of defective interfering rna of tomato bushy stunt virus purification of the cucumber necrosis virus replicase from yeast cells: role of coexpressed viral rna in stimulation of replicase activity role of an internal and two -terminal rna elements in assembly of tombusvirus replicase specific binding of tombusvirus replication protein p to an internal replication element in the viral rna is essential for replication defining the roles of cis-acting rna elements in tombusvirus replicase assembly in vitro systematic, genome-wide identification of host genes affecting replication of a positive-strand rna virus yeast genome-wide screen reveals dissimilar sets of host genes affecting replication of rna viruses identification of essential host factors affecting tombusvirus rna replication based on the yeast tet promoters hughes collection a temperature sensitive mutant of heat shock protein reveals an essential role during the early steps of tombusvirus replication a key role for heat shock protein in the localization and insertion of tombusvirus replication proteins to intracellular membranes in vitro assembly of the tomato bushy stunt virus replicase requires the host heat shock protein proteomics analysis of the tombusvirus replicase: hsp molecular chaperone is associated with the replicase and enhances viral rna replication tomato bushy stunt virus co-opts the rna-binding function of a host metabolic enzyme for viral genomic rna synthesis ubiquitin-conjugating enzyme is a component of the tombusvirus replicase complex and ubiquitinates p replication protein translation elongation factor a facilitates the assembly of the tombusvirus replicase and stimulates minus-strand synthesis translation elongation factor a is a component of the tombusvirus replicase complex and affects the stability of the p replication co-factor synergistic roles of eukaryotic translation elongation factors bgamma and a in stimulation of tombusvirus minus-strand synthesis the host pex p plays a role in peroxisomal localization of tombusvirus replication proteins ubiquitination of tombusvirus p replication protein plays a role in virus replication and binding to the host vps p escrt protein a unique role for the host escrt proteins in replication of tomato bushy stunt virus mrna export: rnp remodeling by dead-box proteins bent out of shape: rna unwinding by the dead-box helicase vasa the dead-box protein family of rna helicases dead-box proteins can completely separate an rna duplex using a single atp ded p, a dead-box protein required for translation initiation in saccharomyces cerevisiae, is an rna helicase yeast rna helicases of the dead-box family involved in translation initiation authentic replication and recombination of tomato bushy stunt virus rna in a cell-free extract from yeast motif iii in superfamily ''helicases'' helps convert the binding energy of atp into a highaffinity rna binding site in the yeast dead-box protein ded internal initiation by the cucumber necrosis virus rna-dependent rna polymerase is facilitated by promoter-like sequences mechanism of di rna formation in tombusviruses: dissecting the requirement for primer extension by the tombusvirus rna dependent rna polymerase in vitro partial purification and characterization of cucumber necrosis virus and tomato bushy stunt virus rna-dependent rna polymerases: similarities and differences in template usage between tombusvirus and carmovirus rna-dependent rna polymerases a replication silencer element in a plus-strand rna virus enhancement of rna synthesis by promoter duplication in tombusviruses analysis of minimal promoter sequences for plus-strand synthesis by the cucumber necrosis virus rna-dependent rna polymerase rna chaperone activity of the tombusviral p replication protein facilitates initiation of rna synthesis by the viral rdrp in vitro use of double-stranded rna templates by the tombusvirus replicase in vitro: implications for the mechanism of plus-strand initiation direct inhibition of tombusvirus plus-strand rna synthesis by a dominant-negative mutant of a host metabolic enzyme, gapdh, in yeast and plants evolution and taxonomy of positive-strand rna viruses: implications of comparative analysis of amino acid sequences role of rna chaperones in virus replication the dead-box rna helicase ded p affects and accumulates in saccharomyces cerevisiae p-bodies direct inhibition of tombusvirus plus-strand rna synthesis by a dominant negative mutant of a host metabolic enzyme, glyceraldehyde- -phosphate dehydrogenase, in yeast and plants host transcription factor rpb p affects tombusvirus replication and recombination via regulating the accumulation of viral replication proteins unwinding by local strand separation is critical for the function of dead-box proteins as rna chaperones characterization of the rna-binding domains in the replicase proteins of tomato bushy stunt virus a mutant allele of essential, general translation initiation factor ded selectively inhibits translation of a viral mrna rna helicase a modulates translation of hiv- and infectivity of progeny virions autogenous translational regulation of the borna disease virus negative control factor x from polycistronic mrna using host rna helicases cyclosporin a associated helicase-like protein facilitates the association of hepatitis c virus rna polymerase with its cellular cyclophilin b a host rna helicase-like protein, atrh , interacts with the potyviral genome-linked protein, vpg, associates with the virus accumulation complex, and is essential for infection identification of rna helicase a as a new host factor in the replication cycle of foot-and-mouth disease virus cellular rna helicase p relocalization and interaction with the hepatitis c virus protein and the potential role of p in hcv rna replication ddx dead-box rna helicase inhibits hepatitis b virus reverse transcription by incorporation into nucleocapsids dexh-box protein dhx is required for optimal function of the zinc-finger antiviral protein regulating intracellular antiviral defense and permissiveness to hepatitis c virus rna replication through a cellular rna helicase, rig-i kaposi's sarcoma-associated herpesvirus viral protein kinase interacts with rna helicase a and regulates host gene expression ded p, a conserved dexd/ h-box translation factor, can promote yeast l-a virus negative-strand rna synthesis in vitro screening of the yeast ythc collection identifies essential host factors affecting tombusvirus rna recombination comparison of turnip crinkle virus rna-dependent rna polymerase preparations expressed in escherichia coli or derived from infected plants flock house virus replicates and expresses green fluorescent protein in mosquitoes a versatile toolbox for pcr-based tagging of yeast genes: new fluorescent proteins, more markers and promoter substitution cassettes the authors thank dr. daniel barajas for valuable comments. ded mutants are the generous gift of dr. n.k. tanner (centre medical u., geneva, switzerland). dr. shou-wei ding (uc riverside) is thanked for the fhv clones. the escherichia coli expression plasmids gst-tdh was provided by dr. tyng-shyan huang (nagy lab). key: cord- -ybtk cp authors: carey, brian d.; akhrymuk, ivan; dahal, bibha; pinkham, chelsea l.; bracci, nicole; finstuen-magro, sarah; lin, shih-chao; lehman, caitlin w.; sokoloski, kevin j.; kehn-hall, kylene title: protein kinase c subtype δ interacts with venezuelan equine encephalitis virus capsid protein and regulates viral rna binding through modulation of capsid phosphorylation date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: ybtk cp protein phosphorylation plays an important role during the life cycle of many viruses. venezuelan equine encephalitis virus (veev) capsid protein has recently been shown to be phosphorylated at four residues. here those studies are extended to determine the kinase responsible for phosphorylation and the importance of capsid phosphorylation during the viral life cycle. phosphorylation site prediction software suggests that protein kinase c (pkc) is responsible for phosphorylation of veev capsid. veev capsid co-immunoprecipitated with pkcδ, but not other pkc isoforms and sirna knockdown of pkcδ caused a decrease in viral replication. furthermore, knockdown of pkcδ by sirna decreased capsid phosphorylation. a virus with capsid phosphorylation sites mutated to alanine (veev cpd) displayed a lower genomic copy to pfu ratio than the parental virus; suggesting more efficient viral assembly and more infectious particles being released. rna:capsid binding was significantly increased in the mutant virus, confirming these results. finally, veev cpd is attenuated in a mouse model of infection, with mice showing increased survival and decreased clinical signs as compared to mice infected with the parental virus. collectively our data support a model in which pkcδ mediated capsid phosphorylation regulates viral rna binding and assembly, significantly impacting viral pathogenesis. introduction to be key for functionality, demonstrated by phosphorylation of sindbis virus (sinv) e glycoprotein which is necessary for viral maturation [ ] and the hypervariable domain of nsp which is important for minus strand synthesis [ ] [ ] [ ] . we have recently shown that veev capsid protein is phosphorylated on t , t , s , and t residues and that phosphorylation is in part regulated by the phosphatase protein phosphatase α (pp α) [ ] . here, we extended our study to determine the kinase responsible for capsid phosphorylation and to further elucidate the importance of phosphorylation for viral replication and pathogenesis. phospho-prediction site server, netphos . is an online tool that predicts phosphorylation sites in eukaryotic proteins using ensembles of neural networks. analysis of the primary amino acid sequence of capsid by netphos . [ ] resulted in a list of potential phosphorylation sites on capsid based on known kinase consensus sequences (table ) . any potential sites of tyrosine phosphorylation were removed as previous results indicated capsid was phosphorylated on serine and threonine residues. next, any residues with a score below . were removed from the analysis because scores below . indicate less than % confidence that the site is actually phosphorylated. the software also listed potential kinases for each residue. our previous data showed that capsid is phosphorylated on t , t , s , and t [ ]. netphos . predicted an unknown kinase and protein kinase c (pkc) to be the kinases responsible for phosphorylation of t , t , and s . t was not predicted to be phosphorylated by the netphos . software with any statistical significance. in order to determine if pkc associates with capsid, co-immunoprecipitation experiments were performed. vero cells were infected with veev tc- (a live attenuated vaccine strain of veev) or mock infected. following infection, cells were lysed and samples were immunoprecipitated with an antibody against veev capsid. western blot analysis was performed on immunoprecipitated samples with antibodies against pkc isoforms α, δ, μ, and z. analysis demonstrated an association between pkcδ and capsid but no other pkc isoforms tested ( fig a) . pkcδ was also found to interact with stat , a known pkcδ interacting partner [ ] (s a fig). a reverse co-immunoprecipitaion confirmed the interaction of pkcδ with capsid ( fig b) , but pkcδ did not interact with the viral k/tf protein (s a fig). to determine at what point of the viral life cycle this interaction was most prevalent, cells were infected with veev tc- and collected at , , , and hours post infection (hpi). samples were immunoprecipitated with an antibody against pkcδ and western blot analysis for veev capsid was performed. results indicated the association between capsid and pkcδ is highest at and hpi ( fig b) with a slight interaction observed at hpi. the detection at later time points could, however, be due to increased levels of capsid at later time points making the interaction easier to detect. furthermore, confocal microscopy was performed to visualize co-localization between veev capsid and pkcδ. cells were either mock infected or infected with veev tc- and incubated for hours. cells were fixed, permeabilized, and stained with antibodies against pkcδ and either veev capsid (fig a) or veev e (fig b) . consistent with immunoprecipitation results, a clear co-localization between veev capsid and pkcδ was detected. co-localization analysis of z-stack images was performed and a scatter plot was created comparing the intensities of the red pixels (x-axis) with the green pixels (y-axis) (fig c) . the scatter plot shows the intensities of every pixel within the image, the region within the yellow gate and indicated by the arrow corresponds to the regions of interest of which the pearson's correlation was calculated (arrows in fig a) . a pearson's correlation at the regions of interest was . indicating a relatively strong correlation. co-localization analysis of e and pkcδ ( fig d) using the same gating as the capsid:pkcδ analysis did not show a strong correlation of colocalization with a pearson's correlation of . . interestingly, localization of pkcδ in uninfected cells was fairly compact and perinuclear, suggesting localization in the er/golgi. however, after veev infection pkcδ localization displayed a more diffuse phenotype. collectively, these results suggest that veev capsid interacts with pkcδ. pkcδ is a serine/threonine kinase and our previous work indicated veev capsid is phosphorylated on specific serine/threonine residues [ ] . we set out to determine if pkcδ is responsible for the phosphorylation of veev capsid. cells were treated with sirna targeting either pkcδ or a scrambled control, and then transfected with a plasmid expressing the structural polyprotein of veev. following hours of incubation, cells were collected, lysed, and samples were immunoprecipitated with an antibody against veev capsid. anti-phospho-serine and anti-phospho-threonine antibodies were used to detect phosphorylation via western blot analysis. pkcδ sirna had no effect on cell viability ( fig a) and pkcδ protein expression was reduced by~ % (fig b) . capsid threonine phosphorylation was decreased by % and serine phosphorylation by % following pkcδ sirna treatment (fig c & d) . loss of pkcδ also resulted in decreased phosphorylation of stat serine , which is a known pkcδ in order to determine if the decrease in phosphorylation is important for veev replication, viral production was measured after sirna knockdown of pkcδ. transfection of sirna against pkcδ caused a significant time-dependent decrease in veev tc- viral titers ( fig fig . a) with more than a one log decrease at hpi. similar results were observed with virulent bsl- veev trd, with~ log reduction of viral replication at hpi. however, loss of pkc had no impact on eastern equine encephalitis virus (eeev) or western equine encephalitis virus (weev) (fig b) . an alignment of the primary amino acid sequences for the capsid proteins of veev, eeev, and weev showed that while both serine and threonine are conserved amongst all three viruses only serine is predicted to be phosphorylated in all three viruses. interestingly, threonine was also not predicted to be phosphorylated in veev capsid, but was detected by lc-ms/ms analysis ( ). threonine and were not conserved in eeev or weev. these data suggest that pkcδ is important for veev replication, but not related alphaviruses. to further investigate the importance of the phosphorylation of veev capsid, we designed a capsid phosphorylation-deficient (cpd) mutant virus using the veev tc- backbone. we have previously shown that capsid is phosphorylated on four residues in the rna-binding domain [ ] . veev cpd was produced by mutating the previously identified phosphorylation sites to alanine (t a, t a, s a, and t a) ( fig a) . cells infected with veev cpd to examine the link between veev cpd and pkcδ, cells were treated with sirna targeting pkcδ or a scrambled control and then infected with either veev tc- or veev cpd. veev cpd replication was not affected by pkcδ knockdown while veev tc- exhibited a similar decrease in titer as with earlier sirna experiments ( fig f) . these results indicate that replication of veev cpd is not sensitive to the loss of pkcδ suggesting that pkcδ plays a role in the phosphorylation of capsid. to determine the impact of capsid phosphorylation on viral replication, viral growth kinetics of veev tc and veev cpd were measured at , , , , and hpi in vero cells. growth kinetics were also observed in human primary astrocytes, given that astrocytes are a proven an analysis of extracellular genomic copies to particle forming units (pfu) ratio was performed and the genomic copies to pfu ratio was determined to be lower in veev cpd vs. veev tc- ( fig c) . these data suggest that cells infected with veev cpd output more functional viral particles than veev tc- , potentially due to more efficient viral packaging and this phenotype is due to loss of capsid phosphorylation. to determine the stability of the veev cpd mutant sites, we passaged veev cpd in vero cells times and purified six different plaques for viral sequencing. no sequence reversion at the mutated residues was observed in any of the six plaques tested. sequencing of the entire capsid region revealed only one amino acid mutation in one plaque: gly to tyr in position . this residue is found within the sd domain of capsid which is involved in rna binding and nucleocapsid assembly [ ] . these results suggest that there is limited selective pressure for the phosphorylation sites to revert in vitro and are consistent with the unaltered replication kinetics of veev cpd. we have previously shown that inhibiting the dephosphorylation of capsid results in less viral rna (vrna) binding to capsid [ ] . given these results and that our capsid phosphorylation deficient virus displays a lower genomic copies to pfu ratio, we hypothesized that capsid phosphorylation mediates vrna binding. in order to test this hypothesis, we investigated whether veev cpd had an effect on vrna binding to capsid. cells were infected with either veev tc- or veev cpd, fixed in paraformaldehyde, lysed, and immunoprecipitated for veev capsid. after immunoprecipitation, rna was isolated and rt-qpcr was performed with primers targeting the canonical veev rna packaging signal [ ] . results indicated that almost times more viral rna bound to capsid in the veev cpd vs. parental virus at all times post infection tested (fig a) . loss of pkcδ through sirna transfection also resulted in increased capsid viral rna binding, further solidifying the link between veev cpd and pkcδ (fig b) . we next wanted to understand how capsid phosphorylation globally affects viral rna binding. previous work with sindbis virus (sinv) indicated that sinv capsid bound to multiple regions of viral rna in the cytoplasm [ ] . to this end, clip-seq studies were performed to determine if veev capsid interacts with viral rna at sites other than the packaging signal. briefly, hek cells were infected with a gfp-reporter expressing strain of veev tc- , and at hpi the cells were washed and cross-linked via ultraviolet irradiation. after the generation of whole cell lysates, anti-capsid or nonspecific antibodies were used to immunoprecipitate rna:protein complexes. the rna:protein complexes were fragmented via rnase treatment on the bead, and after extensive washing the immunoprecipitated rna fragments were eluted via proteinase k digestion and used as the starting material for cdna library generation. the clip-seq libraries were then aligned to the reference genome to reveal sites of veev capsid:rna interaction. clip-seq analysis of the cytoplasmic veev capsid:rna interactions revealed distinct sites of significant enrichment, including an enriched cluster within the coding region of the gfp fluorophore (as shown in s fig) . the precise implications of this interaction, or its biological impact on viral infection are unknown; however, as the enrichment of this site could inadvertently mask the statistical significance of the interaction identification process by reducing the significance of all other viral rna regions, and all downstream analyses utilized parental tc- , we elected to censor the region from our downstream analyses. thus, similarly to what was observed for sinv, distinct viral interaction sites were observed for the veev capsid protein and the vrna (fig c) . interestingly, no significant enrichment was observed at the region of the genome corresponding to the veev packaging signal (ps). protein kinase c modulates veev capsid phosphorylation however, it should be noted that a similar phenomenon was observed for sinv ( ). curiously, unlike sinv, veev capsid:rna interactions were observed in both the nonstructural and structural coding regions. comparative analysis of the molecular composition and . cells were collected at the indicated time points, cross-linked, and cell lysates were immunoprecipitated with either α-ha or α-veev capsid antibodies. rt-qpcr against the veev packaging signal was performed on rna isolated from immunocomplexes and normalized relative to a veev rna standard curve generated during the same reaction. genomic copies per reaction from α-ha immunoprecipitated samples were subtracted from the associated genomic copies from α-capsid immunoprecipitated samples to remove background and then the % rna bound to capsid was determined by dividing the genomic copies from the input rna by the normalized immunoprecipitated rna. rna:capsid binding of veev cpd was normalized relative to veev tc- samples. b) u mg cells were transfected with nm scramble control or pkcδ sirnas. seventy-two hours posttransfection, cells were infected with veev tc- (moi . ). samples were collected at hpi and processed in the same manner as panel a. c) a graphical representation of the veev capsid clip-seq data. on the y-axes are the mean depths of coverage for the veev anti-capsid data sets (top) and relative statistical significance (bottom) plotted with respect to nucleotide position (x-axis). shown above the graphs is a schematic representation of the veev tc- reference genome (drawn to scale with the x-axis). d) vero cells were infected with veev tc- or veev cpd (moi . ). cells were collected at hpi, cross-linked, and cell lysates were immunoprecipitated with either α-ha or α-veev capsid antibodies. rt-qpcr against the veev canonical packaging signal and the sites identified in panel c was performed on rna isolated from immunocomplexes and normalized relative to a veev rna standard curve generated during the same reaction. data analysis was performed as described in panel a. data is average of biological replicates. � = p< . , �� = p< . , ��� = p< . , ���� = p< . . https://doi.org/ . /journal.ppat. .g structure for the sinv and veev capsid:rna interaction sites (including those of the gfp fluorophore) fails to identify any particular motif or structural element. nonetheless; the lack of an identifiable canonical interaction motif may be entirely due to the limited number of interaction sites observed for each virus, rather than a differential set of binding proclivities. to determine the impact of veev capsid phosphorylation on vrna binding, we utilized quantitative rna-ip experiments to probe the interaction between the capsid protein and the vrna at sites identified as highly enriched, intermediately enriched, and non-enriched by our clip-seq studies. in addition to the capsid:rna interaction sites, we also assessed binding at the veev ps ( ). as shown in fig d, the veev cpd mutant had enhanced binding relative to parental tc- at all sites tested, including those with no significant enrichment as determined via clip-seq. thus, these data suggest that phosphorylation of capsid is globally inhibitory to viral rna binding. viral genomic copies to pfu ratios have been shown to be a determinant of viral attenuation. ebola virus becomes attenuated in macaques as the genomic copies to pfu ratio decreases [ ] , whereas increased genomic copies to pfu ratio shows attenuation in bunyamwera virus [ ] . to determine if the mutations in veev capsid phosphorylation sites cause attenuation of veev tc- in vivo, six-week-old female c h/hen mice were challenged intranasally with a lethal dose of veev tc- or veev cpd. mice were monitored for survival over days and were observed daily for clinical symptoms of disease, weight loss, and body temperature. sixty percent of mice infected with veev tc- succumbed to infection by days post-infection while % of mice infected with veev cpd survived the infection (fig a) . furthermore, survivors infected with veev cpd showed less severe clinical signs of infection compared to tc- infected mice (fig b) . mice infected with tc- displayed mild signs of clinical illness (decreased activity, weight loss) by day post-infection and increased in severity (scruffy, hunched, lethargic, significant weight loss) by day / post-infection. surviving tc- mice recovered by day (fig a) . meanwhile, mice infected with cpd mutant virus displayed mild signs of clinical illness (decreased activity, weight loss) with only of the mice increasing to more severe signs and one mouse succumbing to infection (fig b and s fig) . these data suggest that veev cpd is attenuated in mice, warranting further study into its potential for a vaccine strategy. however, future studies identifying viral load and distribution of mice infected with veev cpd are required. our data demonstrated that pkcδ interacts with veev capsid, as indicated by its co-immunoprecipitation with capsid and co-localization by confocal microscopy during veev infection. pkc is a family of serine/threonine protein kinases that have been implicated in numerous cellular processes including transcriptional regulation, membrane structure modulation, immune mediation and cell growth regulation [ ] . pkc has been implicated in several different viral life cycles. inhibition of pkcα causes an increase in dengue virus replication, potentially by acting as a restricting mechanism that regulates dengue virus replication [ ] . pkc is also involved in the lytic cycle switch of epstein-barr virus by modulating microtubule depolymerization [ ] . additionally, hepatitis b virus inhibits pkcδ causing decreased stat activation during infection [ ] . pkcδ, specifically, has been shown to be involved in hiv- , sinv, and porcine reproductive and respiratory syndrome virus replication [ ] [ ] [ ] . pkcδ is a novel isoform of the pkc family of kinases and plays conflicting roles in cell survival and cell death signaling. it is pro-survival and anti-apoptotic during cytokine receptor-initiated cell death, but it protein kinase c modulates veev capsid phosphorylation also can be a pro-apoptotic protein during dna damage signaling [ , ] . in our study, we find that pkcδ regulates veev capsid binding to viral rna. since our data indicated pkcδ is associated with veev capsid, we tested the importance of pkcδ during infection. a significant decrease of viral titers was observed starting at hpi and continuing for at least hpi after pkcδ knockdown by sirna. to more specifically assess the importance of veev capsid phosphorylation, a veev capsid phosphodeficient mutant on a tc- backbone was constructed. interestingly, the phospho-deficient mutant virus, veev cpd, displayed no significant difference in titer when compared to the parental tc- . these data are contrary to the sirna data which show a decrease in viral titer after knockdown of pkcδ. these data could indicate that pkcδ is important in more than one aspect of the viral life cycle and that knockdown of pkcδ prevents another unknown mechanism of viral replication from functioning properly. it is also possible the phosphorylation of individual residues plays independent roles and pkcδ is only impacting one or some of the phosphorylated residues. future studies with viruses containing single mutations and/or double and triple mutant combinations will be necessary to determine the importance of individual phosphorylation sites. our results show that knockdown of pkcδ and mutation of capsid phosphorylated residues decreases the level of phosphorylation on veev capsid to almost undetectable levels. we identified the phosphatase responsible for capsid dephosphorylation, pp α, in our previous study [ ] , but no kinase had been identified up to that point. the knockdown of pkcδ coupled with the drop in capsid phosphorylation strongly suggests that pkcδ is the kinase responsible for phosphorylating capsid. our previous data suggest that higher levels of capsid phosphorylation results in less viral rna binding and it has been shown in the related togavirus, rubella virus, that phosphorylation of capsid protein regulates its rna binding and viral replication [ ] . we compared the amount of rna bound to capsid in either veev tc- or veev cpd and our results indicated that there is more viral rna bound to capsid in the mutant strain. coupling these data with our previous study that shows that higher levels of phosphorylation causes less viral rna bound to capsid, we propose that capsid phosphorylation/dephosphorylation is a cycle that regulates rna binding (fig ) . interestingly, veev cpd also displayed a lower genomic copy to pfu ratio and decreased pathogenicity. as veev cpd replicated to similar levels as parental veev tc in vitro, these data suggest that capsid phosphorylation is not necessary for viral replication, but rather regulates viral pathogenesis. the altered pathogenesis may be linked to veev cpd producing less non-infectious particles. multiple viruses have been shown to produce non-infectious particles, including influenza virus [ , ] , hepatitis b virus [ ] , herpes simplex virus [ ] , and yellow fever virus [ ] . the non-infectious particles can be immature viruses, defective interfering particles, or semi-infectious particles with varying roles in pathogenesis ranging from increasing disease, stimulating the innate immune response, serving as a decoy for antibodies, or facilitating recombination [ ] [ ] [ ] . our data support a model whereby capsid phosphorylation facilitates the release of non-infectious particles which aid in disease development. future studies will be aimed at determining how capsid phosphorylation regulates pathogenesis. pkcδ has previously been shown to phosphorylate the capsid protein of rubella virus which regulates binding of viral rna to capsid [ ] however, our study is the first to put forth a mechanism for any alphavirus capsid phosphorylation cycle. while pkcδ is at least one kinase that regulates veev capsid phosphorylation, there could potentially be other kinases that are important during the life cycle of veev such as dnapk and pka, as predicted in fig . working model of the capsid phosphorylation and its impact on viral pathogenesis. our working model is that capsid phosphorylation is a mechanism important for regulating capsid:viral rna binding. capsid is dephosphorylated by pp α increasing its ability to bind to viral rna and conversely phosphorylation by pkcδ decreasing viral rna binding. capsid phosphorylation regulates veev's genomic copy to pfu ratio and viral pathogenesis. https://doi.org/ . /journal.ppat. .g protein kinase c modulates veev capsid phosphorylation silico. pkc was also predicted to phosphorylate capsid proteins of eeev and weev, but loss of pkc through sirna transfection had no impact on viral replication. however, there were multiple residues of eeev and weev capsid proteins that are predicted to be phosphorylated and by alternative candidate kinases such as pka. this is an area of importance for future study as phosphorylation of alphavirus capsid proteins may be used as a conserved mechanism to regulate capsid functionality. cellular and viral protein phosphorylation is critical during the life cycle of several viruses [ - ]. furthermore, elucidating the mechanism of phosphorylation during infection and the purpose it serves can help lead to attenuation of viruses for potential vaccine candidates. several viruses can be attenuated through mutation of their phosphorylation sites including t on the ns protein of influenza a virus [ ] as well as s and s on the measles virus phosphoprotein [ ] . our data add to the list of viruses attenuated by ablating an important phosphorylation event. it shows that mutating the phosphorylation sites on veev capsid attenuates the virus in vivo. additionally, surviving mice infected with veev cpd display only mild clinical symptoms of infection suggesting that veev cpd does not induce the pathogenesis observed with veev tc- . further studies are being performed in our lab to determine viral kinetics in vivo and if veev cpd at a lower dose can potentially eliminate clinical symptoms observed from this study and also protect against lethal challenge with aerosolized veev trd. vero, hek t (atcc, crl- ) and u mg (atcc, htb- ) cells were maintained at ˚c, % co in dulbecco's modified eagle medium (dmem) supplemented with % fetal bovine serum (fbs), % glutamine, and % penicillin/streptomycin. bhk- (atcc, ccl- ) cells were maintained at ˚c, % co in modified eagle medium (mem) supplemented with % fetal bovine serum (fbs), % glutamine, and % penicillin/streptomycin. human primary astrocytes (lonza cc- ) were maintained at ˚c, % co in astrocyte growth medium bullet kit (lonza cc- ). protein lysates were obtained using lysis buffer consisting of mm tris-hcl ph . , mm nacl, mm edta, . % np- , mm naf, . mm na vo , complete protease inhibitor cocktail (sigma-aldrich, ). following cell lysis, mg of total protein per sample was immunoprecipitated with μg of anti-pkcδ (cell signaling, ) rabbit primary antibody, anti-veev-capsid (bei resources, nr- ) goat primary antibody, or anti-ha (cell signaling, ) rabbit primary antibody at ˚c overnight. following immunoprecipitation, antibody:antigen complexes were bound to protein g dynabeads (thermo fisher, d) . beads containing the protein complexes were washed one time in tne with . % np- , twice in tne with . % np- , and once in pbs. western blot loading buffer, consisting of novex tris-glycine sample loading buffer sds (thermo fisher, lc ), t-per tissue protein extraction reagent (thermo fisher, ), edta, complete protease inhibitor cocktail, mm naf, . mm na vo , and mm dtt, was added to antibody:dynabead complexes and samples were run on nupage - % bis-tris protein gels (thermo fisher, np ). following transfer to a pvdf membrane, membranes were blocked in either % bsa-tbs-tween or % milk-pbs-tween and incubated in the indicated primary antibody [anti-pkcδ (cell signaling, ) : dilution), anti-pkcμ (cell signaling, , : dilution), anti-pkcα (cell signaling, , : dilution), anti-pkcz (cell signaling, , : dilution), anti-veev-capsid (bei resources, : dilution), anti-phosphoserine (millipore, ab , : dilution), anti-phosphothreonine (millipore, ab , : dilution), anti-stat (cell signaling d z g, : ratio) or anti-v (biorad mca , : dilution)] overnight at ˚c. following primary antibody incubation, membranes were washed and incubated for hour in the appropriate secondary antibody, either anti-rabbit hrp-conjugated (cell signaling, ), anti-mouse hrp-conjugated (cell signaling, ) or anti-goat hrp-conjugated. membranes were imaged on a chemidoc xrs molecular imager (biorad) using the supersignal west femto maximum sensitivity substrate kit (thermo fisher, ). non-immunoprecipitated samples were processed in the same way without performing the immunoprecipitation steps. the identification of cytoplasmic veev capsid:rna interactions was conducted as described in [ ]), with minor differences. specifically, x hek cells were infected with veev tc- .dsgfp at an moi of pfu/cell, and after a hour incubation period the cells were washed and irradiated with x μjoules per square centimeter. after crosslinking whole cell lysates were generated via the addition of ripa buffer ( mm tris [ph . ], mm nacl, . % np- , . % sodium deoxycholate, . % sds). the whole cell lysate was solubilized via repeated passage through a -gauge needle, and insoluble materials were removed from the lysate via centrifugation at , xg for minutes at ˚c. the clarified lysates (~ μl per reaction) were then incubated with μl packed volume of protein g sepharose beads (pierce) for minutes at ˚c. the resin was removed by gentle centrifugation at , xg for minutes, and the pre-blocked lysates were transferred to fresh microfuge tubes containing polyclonal anti-capsid or nonspecific igg. after hours of incubation at ˚c on a rotating mixer, μl of protein g sepharose was added to each tube and the incubation proceeded as before for another hours. after the formation of the immunoprecipitation complex, rnase a was added to each tube and the immunoprecipitations were incubated for minutes at room temperature to fragment the bound rna:protein complexes. then the nuclease treated resin was collected via centrifugation at , xg for minutes and washed three with excess volumes of ripa buffer. after the removal of the contaminating rnase and rna protein kinase c modulates veev capsid phosphorylation fragments, the bound resin was washed twice more with xpbs, and the rna fragments were eluted from the resin by way of proteinase k digestion. the purified fragments were extracted using trizol and precipitated prior to the generation of cdna libraries using the nebnext small rna sequencing (neb) kit, as per the manufacturer's instructions. the resulting libraries were sequenced on a hiseq platform (illumina). the resulting cdna library data was trimmed to remove indices and adaptors and trimmed for quality (> over a nt scanning window) and length (> nt). the resulting cdna data was aligned to the veev tc- .dsgfp reference genome using bowtie with the default parameters. only the reads which aligned to the positive-sense rna genome were mapped, and sequence coverage was clustered at a nucleotide level of resolution. the analysis of the clustering data was performed identically to that described in [ ] . the sequencing data and their analyses as relevant to the studies described herein are available in the supplemental files accompanying this manuscript as s data. in addition, the entire rna sequencing data set has been deposited in the national center for biotechnology information (ncbi) gene expression omnibus, and can be accessed by the following url: https://urldefense.proofpoint.com/ v /url?u=https- a__www.ncbi.nlm.nih.gov_geo_query_acc.cgi- facc- dgse &d=dw ibag&c=oag lqnacbdgugvbenj swhr tmtjs-x o_kuappgy&r=habxqcvdssepsf q - oxf _xjrptra mamiryitxktm&m=mn_eswz wg_rigqhybbazs w pxzfckymqnj povc tk&s=zwhexjsjw_vfkc s i li bn v ki iuxiiayn s &e= to enhance the clarity of data presentation and enable congruence between the nucleotide positions and the native tc- sequence, we have censored the region corresponding to the gfp reporter during the presentation of the data. nonetheless, the data deposited to the ncbi consists of the data set in its entirety, and the presentation of the uncensored alignment data may be found in s fig. infected cells were collected, washed in pbs, and cross-linked with % paraformaldehyde. cross-linked cells were lysed in ripa lysis buffer ( mm tris-hcl, ph . , % np- , . % sodium deoxycholate, . % sodium dodecyl sulfate, mm edta, complete protease inhibitor cocktail tablet, and mm nacl) and ultrasonicated. lysates were clarified by centrifugation and immunoprecipitated as described above with α-ha and α-veev capsid antibodies. following immunoprecipitation, antibody:antigen complexes were bound to protein g dynabeads as described above. beads bound to antibody complexes were washed in high stringency ripa buffer ( mm tris-hcl, ph . , % np- , % sodium deoxycholate, . % sodium dodecyl sulfate, mm edta, m nacl, m urea, and complete protease inhibitor cocktail tablet) and resuspended in teds buffer ( mm tris-hcl, ph . ), % sodium dodecyl sulfate, mm edta, and mm dtt). samples were then processed for rna extraction. supernatants were collected to analyze extracellular viral rna, and infected cells were lysed and collected in trizol (thermo fisher, ) to analyze intracellular rna. extracellular viral rna was isolated using ambion's magmax viral rna isolation kit (thermo fisher, am ) while intracellular rna and immunoprecipitated rna (and input rna) was isolated using the direct-zol rna miniprep kit (zymo research, r ) by following the manufacturer's instructions. rt-qpcr was performed as described previously [ ] for viral rna using the integrated dna technologies primer pairs (table ) and taq-man probe ( -carboxyfluorescein-tcgtccgcactgacatctgttgc-carboxytetramethylrhodamine) against the viral rna packaging signal (nt - ) ( ) or by sybr green for non-probe based assays. the absolute quantification was determined using stepone software v . based on the threshold cycle relative to the standard curve. the standard curve was determined using serial dilutions of veev-tc- rna at known concentrations. relative rna:capsid binding was determined by subtracting the background rna bound to antibody and dynabeads (α-ha ip samples) from the experimental samples and compared to input rna levels. results were normalized to tc- for comparison. for cell viability assays, cells were cultured as described above and transfected with sirna at a final concentration of nm. cells were incubated for hours and atp production was measured as an indication of cell viability using promega's cell titer-glo (promega, g ). assays were performed in white-walled, -well plates (corning, ) seeded with , cells per well following the manufacturer's protocol. luminescence was detected on a beckman coulter dtx plate reader after ms integration per well. for sirna transfections, u mg cells were transfected with dharmafect transfection reagent (ge lifesciences, t- ) for hours prior to veev infection. for sirna either on-target plus smartpool for prkcd (ge lifesciences, l- - ) or all stars sirna (qiagen, si ) negative control were used. for experiments with plasmids, we previously described a veev structural polyprotein construct [ ] that was transfected using polyethylenimine (pei) at a ratio of μg pei: μg dna. vero cells were grown on poly-l-lysine coated cover-slips in a -well plate, infected with veev tc- or mock-infected, and processed for immunofluorescence analysis as previously described [ ] . anti-veev-capsid goat primary antibody ( : dilution) and alexa fluor donkey anti-goat secondary antibody (thermo fisher, a , : dilution) were used to probe for capsid. anti-pkcδ rabbit primary antibody ( : dilution) and alexa fluor protein kinase c modulates veev capsid phosphorylation donkey anti-rabbit secondary antibody (thermo fisher, a , : dilution) were used to probe for pkcδ. anti veev-e mouse primary antibody ( : dilution) and alexa fluor anti-mouse secondary antibody (thermo fisher, a , : dilution) were used to probe for e . slides were imaged using an oil-immersion x objective lens on a nikon eclipse te -u confocal microscope, with all samples subjected to four line averaging. at least four images were taken of each sample, with one representative image shown. each image was processed using nikon nis-elements ar analysis . software. z-stack analysis was performed on nikon nis-elements ar analysis . software using the co-localization function. briefly, the red (x-axis) and green (y-axis) channels were selected for comparison and the resulting scatter plot shows the intensity of each pixel within the image. the gate function was used to highlight the pixels that were the most intense for both channels. pixels within the gates were analyzed for pearson's correlation. six-week-old female c h/hen mice were obtained from charles river laboratories. groups of mice were individually identified via tattoo and had temperature transponders (biomedic data systems) implanted subcutaneously days prior to the start of the study. mice were infected intranasally with a dose of x pfu/mouse of veev tc- or veev cpd. animals were observed for survival over the course of days. mice were observed daily for signs of clinical illness as determined by our clinical sign scoring sheet developed for tc- animal studies. mice were scored individually on the following parameters: appearance, mobility, attitude, and body condition. appearance was scored as follows: -smooth coat, bright eyes; slightly scruffy and/or hunched at rest; -scruffy and/or hunched at rest; -very scruffy and/ or hunched at rest, mild eye crust; and -very scruffy and/or hunched at rest, closed inset eyes. mobility was scored as follows: -active, exploring cage; -less active, walking; -slow movement; -no movement; and -unresponsive. attitude was scored as follows: -alert, -mildly lethargic, -lethargic, and -unaware. body condition was scored as follows: -normal or overweight; -underconditioned; and -emaciated. scores for each parameter were summed for a total score and mice scoring - were observed once daily, mice scoring - were observed twice daily, and mice scoring an or greater were humanely euthanized. measurements of well-being, weights and body temperatures were recorded each day. personnel performing clinical observations, weights and body temperatures were blinded to the animal groups. experiments were performed in animal biosafety level (absl- ) laboratories in accordance with the national research council's guide for the care and use of laboratory animals ( ) and under george mason university iacuc protocol number . statistical analysis was performed using graphpad prism software (http://www.graphpad. com). all experiments were done with at least biological replicates and statistical significance was evaluated using student's t-test. p-values are indicated within the figure by an asterisk where � = p< . , �� = p< . , ��� = p< . , and ���� = p< . . all the causative agent of infectious equine encephalomyelitis in venezuela venezuelan equine encephalitis the systemic pathology of venezuelan equine encephalitis virus infection in humans eastern and venezuelan equine encephalitis viruses differ in their ability to infect dendritic cells and macrophages: impact of altered cell tropism on pathogenesis epidemiological aspects of venezuelan equine encephalitis virus infections vaccines for venezuelan equine encephalitis nucleotide sequences of the s mrnas of the viruses defining the venezuelan equine encephalitis antigenic complex venezuelan equine encephalitis virus capsid-the clever caper deletion analysis of the capsid protein of sindbis virus: identification of the rna binding region the old world and new world alphaviruses use different virus-specific proteins for induction of transcriptional shutoff venezuelan equine encephalitis virus capsid protein forms a tetrameric complex with crm and importin alpha/beta that obstructs nuclear pore complex function structure of semliki forest virus core protein the alphaviruses: gene expression, replication, and evolution proteome-wide post-translational modification statistics: frequency analysis and curation of the swiss-prot database the structure and mechanism of protein phosphatases: insights into catalysis and regulation the two-component system. regulation of diverse signaling pathways in prokaryotes and eukaryotes phosphorylation events during viral infections provide potential therapeutic targets the severe acute respiratory syndrome coronavirus nucleocapsid protein is phosphorylated and localizes in the cytoplasm by - - -mediated translocation phosphorylation of a herpes simplex virus dut-pase by a viral protein kinase, us , dictates viral pathogenicity in the central nervous system but not at the periphery phosphorylation and dephosphorylation events play critical roles in sindbis virus maturation functional analysis of nsp phosphoprotein mutants of sindbis virus deletion and duplication mutations in the c-terminal nonconserved region of sindbis virus nsp : effects on phosphorylation and on virus replication in vertebrate and invertebrate cells localization and phosphorylation of semliki forest virus non-structural protein nsp expressed in cos cells from a cloned cdna particle-to-pfu ratio of ebola virus influences disease course and survival in cynomolgus macaques attenuation of bunyavirus replication by rearrangement of viral coding and noncoding sequences the extended protein kinase c superfamily inhibition of protein kinase c promotes dengue virus replication microtubule depolymerization activates the epstein-barr virus lytic cycle through protein kinase c pathways in nasopharyngeal carcinoma cells hepatitis b virus polymerase impairs interferon-alpha-induced sta t activation through inhibition of importin-alpha and protein kinase c-delta protein kinase c-delta regulates hiv- replication at an early post-entry step in macrophages pkcdelta is required for porcine reproductive and respiratory syndrome virus replication infection of glioma cells with sindbis virus induces selective activation and tyrosine phosphorylation of protein kinase c delta. implications for sindbis virus-induced apoptosis protein kinase c as a tumor suppressor posttranslational modification of the au-rich element binding protein hur by protein kinase cdelta elicits angiotensin ii-induced stabilization and nuclear export of cyclooxygenase mrna phosphorylation of rubella virus capsid regulates its rna binding activity and virus replication biological activities of 'noninfectious' influenza a virus particles influenza virus reassortment is enhanced by semi-infectious particles but can be suppressed by defective interfering particles enhancement of hepatitis b virus infection by noninfectious subviral particles the following reagents were obtained through the nih biodefense and emerging infections research resources repository, niaid, nih: venezuelan equine encephalitis virus, tc- , nr- , venezuelan equine encephalitis virus, trinidad donkey, nr- , and polyclonal anti-venezuelan equine encephalitis virus, tc- (subtype ia/b) capsid protein (antiserum, goat), nr- . we thank farhang alem for assistance with the confocal microscopy data analysis. key: cord- -b aa lri authors: seiradake, elena; henaff, daniel; wodrich, harald; billet, olivier; perreau, matthieu; hippert, claire; mennechet, franck; schoehn, guy; lortat-jacob, hugues; dreja, hanna; ibanes, sandy; kalatzis, vasiliki; wang, jennifer p.; finberg, robert w.; cusack, stephen; kremer, eric j. title: the cell adhesion molecule “car” and sialic acid on human erythrocytes influence adenovirus in vivo biodistribution date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: b aa lri although it has been known for years that adenoviruses (ads) interact with erythrocytes ex vivo, the molecular and structural basis for this interaction, which has been serendipitously exploited for diagnostic tests, is unknown. in this study, we characterized the interaction between erythrocytes and unrelated ad serotypes, human (had ) and (had ), and canine (cav- ). while these serotypes agglutinate human erythrocytes, they use different receptors, have different tropisms and/or infect different species. using molecular, biochemical, structural and transgenic animal-based analyses, we found that the primary erythrocyte interaction domain for had is its sialic acid binding site, while cav- binding depends on at least three factors: electrostatic interactions, sialic acid binding and, unexpectedly, binding to the coxsackievirus and adenovirus receptor (car) on human erythrocytes. we show that the presence of car on erythrocytes leads to prolonged in vivo blood half-life and significantly reduced liver infection when a car-tropic ad is injected intravenously. this study provides i) a molecular and structural rationale for ad–erythrocyte interactions, ii) a basis to improve vector-mediated gene transfer and iii) a mechanism that may explain the biodistribution and pathogenic inconsistencies found between human and animal models. adenoviruses (ads) are nonenveloped double-stranded dna pathogens that infect all vertebrate classes. to date, all ads display a characteristic, icosahedral symmetry in which subunits of the trimeric hexon protein form the facets and copies of the penton, comprising the pentameric penton base protein and the externally projecting trimeric fiber, form the vertices [ ] . while the stoichiometry of the penton base and hexon is apparently conserved, the fiber can exist as a single or double copy at each vertex [ ] . at least in vitro and for most cell types, the fiber mediates the initial attachment to primary receptors, such as the d domain of the coxsackievirus and adenovirus receptor (car), sialic acids, cd , and others (for review see zhang & bergelson [ ] ). interaction with auxiliary receptor(s), in particular some of the dimeric integrins via the arg-gly-asp sequence (an integrininteracting motif) on the penton base, may induce internalization of some serotypes. however, other auxiliary receptors or mechanism of internalization may exist for human serotypes and (had / ), and canine serotype (cav- ), which have no identifiable integrin-interacting motif in the penton base [ , ] . greater than ad serotypes have been isolated. approximately of these are currently classed as human pathogens that, in most cases, generate subclinical ocular, respiratory and gastrointestinal tract infections. in the immunocompromised host however, lethal had infections can spread, via unknown mechanisms, to the kidney, liver and brain [ , ] . the human ads (hads) are divided into subgroups (or species or subgenera) a -f. the triage of the human serotypes into subgroups is based in part on serotype-specific ex vivo erythrocyte cross-linking (or hemagglutination) [ ] . the clinical hemagglutination assays use lysates from virus-infected cells to crosslink erythrocytes from a handful of species. this highly heterogeneous lysate contains whole virus particles, empty capsids, penton monomers, penton dodecahedrons, fiber monomers, hexon etc. by using fractionated infected cell lysate, a handful of laboratories found that the multivalent complexes containing fiber were responsible for hemagglutination [ ] [ ] [ ] [ ] . erythrocyte membranes contain highly sialated glycoproteins and glycolipids. one of the most abundant glycoproteins on erythrocytes is glycophorin a (, copies/cell). with its high sialic acid content, glycophorin a is the main contributor to the net negative cell-surface charge and is critical for minimizing cellcell interactions and preventing erythrocyte aggregation [ ] . sialic acid is a collective term for a family of -carbon monosaccharides, which are often found as terminal sugar residues on glycans of glycoproteins and glycolipids (usually a - , - or - linked). in addition to some hads, a number of viruses, including orthomyxoviruses, paramyxoviruses, picornaviruses, papovaviruses, coronaviruses, reoviruses and parvoviruses bind to sialic acids [ ] [ ] [ ] [ ] [ ] . possibly because erythrocytes from different species vary in their sialic acid content, the hemagglutination properties of sialic acid-binding viruses may also diverge [ ] . had subgroup d (serotypes , , p and ) and b: (serotypes a, p and a) erythrocyte binding depends on the fiber head and several attempts have been made to define the region(s) responsible [ ] [ ] [ ] [ ] . among the hads, serotype is unusual: its fiber head can bind car, cd and sialic acid, but the virus appears to use only the latter two as functional receptors [ ] [ ] [ ] . burmeister et al. found that the sialic acid moiety bound to a basic patch close to the center of the trimeric fiber heads of had and had p [ ] . interestingly, the putative hemagglutination domain in subgroup d heads partially aligns with the sialyl-lactose binding site, which on the basis of sequence alignments is likely to be conserved in other members of this subgroup. these observations raise the question as to whether ad-erythrocyte interaction is a consequence of fiber head-sialic acid interaction. in this study we initially characterize the erythrocyte binding of unrelated serotypes, had , had and cav- . although both had and cav- agglutinate human erythrocytes at low particle-to-cell ratios, they use different receptors (sialic acid and cd vs. car) [ , , ] , have different clinical tropisms (ocular vs. respiratory tract) and infect different species. we found that had -erythrocyte interaction is primarily due to sialic acid binding. we show by structural analysis that the cav- fiber head also contains a sialic acid binding site, but in contrast to had this site is modestly involved in hemagglutination. unexpectedly, our biochemical and competition analyses suggested that cav- erythrocyte interactions also depend on binding to car on human and rat erythrocytes. using a transgenic mouse that expresses car on erythrocytes [ ] we demonstrate that car-binding ads can be sequestered by car-expressing erythrocytes, and prevent liver infection. our study provides a molecular and structural rationale for the -year-old enigma of ex vivo aderythrocyte interactions. in addition to the relevance for ad pathogenesis and vector biology, the expression of car by human erythrocytes may shed light on the role of cell adhesion molecules during erythropoiesis. consistent with previous reports, we found that at a modest physical particle (pp)-to-erythrocyte ratio (, ) had efficiently agglutinates human erythrocytes ( figure a) . when compared to had , cav- hemagglutinates at , -fold lower ratio (i.e. more efficiently, figure b ). in our hands, and consistent with others [ ] , had also agglutinated human erythrocytes, but at a higher ratio (. pp/erythrocyte) ( figure c ). to assay the roles of had and cav- capsid proteins, we incubated erythrocytes with chimeric had vectors harboring the fiber from had [ ] or the fiber head from cav- [ ] . we found that the had -had f and had -cav- h hybrid capsids induced agglutination at lower ratios than had but not quite to those of had and cav- ( figure d and e). pre-incubating erythrocytes with neuraminidase, which removes sialic acid from their membranes, eliminated agglutination by had and a had -had f hybrid capsid ( figure , right hand column). in contrast, removing sialic acid from erythrocyte membranes only modestly reduced cav- and the hybrid had -cav- h agglutination (data summarized in table ). together, these data suggest that the cav- fiber head, like in subgroup d and b: hads, is responsible for hemagglutination and that sialic acid binding plays a more significant role in had binding of human erythrocytes than it does for cav- . to mimic the hemagglutination caused by had fiber head in a virion-like context, we generated a multivalent protein complex similar to the ''complete hemagglutinin'' described for had by norrby et al. [ ] . for this purpose had penton dodecahedra were incubated with chimeric ''mini fibers'' consisting of had fiber tail and shaft motifs + and had fiber head. in addition, we generated had fiber heads containing mutations in the sialic acid binding site [ ] (see table for a list of fiber head mutants). the had penton dodecahedra without a fiber did not cause hemagglutination (data not shown). the dodecahedra containing a wild type had fiber head (had h wt ) ( figure s ) agglutinated erythrocytes at low protein concentrations (about -fold less mass than had virions) ( table ) . similar to had , this ''complete hemagglutinin'' poorly agglutinates neuraminidasetreated erythrocytes ( table ). the dodecahedra containing a had fiber with a lys to glu mutation at amino acid in the sialic acid binding site (had h sa ), had at least a -fold reduced hemagglutination activity compared to the dodecahedra containing had h wt . the chimeric wild type cav- fiber head (cav- h wt ) did not bind to had penton dodecahedra, which precluded equivalent experiments. these data suggest that the had fiber shaft, hexon and pix do not play key roles during binding and that the sialic acid binding site is involved in agglutination of human erythrocytes. to assay the binding of recombinant fiber heads, we incubated had h wt and mutant fiber heads with erythrocytes and then added anti-fiber head antibodies or antiserum. we then quantified attachment by flow cytometry. had h wt (figure a ) and in most cases, adenoviruses are thought to initially enter the host via contact with epithelial cells and spread within the host via an unknown mechanism. most adenovirus serotypes use a cell adhesion molecule dubbed ''car'' to attach to cells. to assess, predict and understand adenovirus biology and vectorology, many in vivo studies use mice and monkeys. these animal models have been considered reliable models in the realm of viral pathogenesis and gene transfer. one of the implications of our study suggests that the rat may be a more appropriate model during intravenous adenovirus delivery because like humans, and unlike mice and monkeys, they also express car on their erythrocytes. the identification of car on human erythrocytes explains a -year-old enigma of adenovirus hemagglutination, helps us better understand adenovirus in vivo biology and may open new avenues to understand the role of cell adhesion molecules during erythropoiesis. mutants carrying point mutations in the car binding site (had h car and had h car , mutation in glu and ser , data not shown) [ ] bound to erythrocytes in a dosedependent manner. however, had h sa and had h sa (a tyr to ala mutation at amino acid ), which harbor mutations in the sialic acid binding site, poorly bound erythrocytes ( figure a ). in addition, all of the had fiber heads poorly bound neuraminidase-treated erythrocytes (figure a , bottom row and data not shown). together, these data suggest that the had fiber head sialic acid binding site is a key determinant in the attachment to human erythrocytes. using the above approach, we detected minimal binding of cav- h wt to erythrocytes (data not shown), possibly because the affinity of single cav- fiber heads is low, the high affinity polyclonal antibody binding out-competed the weaker erythrocyte binding, and/or the number of cav- fiber head receptors on erythrocytes is low. to address the potential competition, we incubated fluorescently labeled cav- fiber heads with erythrocytes to circumvent the use of antibodies. here we detected low, but reproducible, binding of cav- h wt to mock-, as well as neuraminidase-treated, erythrocytes ( figure b ). together, these data suggest that cav- erythrocyte binding is notably less dependent on sialic acid than had , and the lower binding may be due to reduced affinity or fewer receptors. to address sialic acid-fiber head interaction using a cell-free system, glycophorin or asialoglycophorin (asialogp) were immobilized on biacore sensor chips and increasing concentrations of had fiber heads were assayed. all binding curves had a square shape, suggesting high on and off rates ( figure s ). had h wt bound to glycophorin, but less efficiently to asialogp. compared to had h wt , had h sa bound equally to asialogp, but less to glycophorin. interestingly, had h sa (lys glu) bound less efficiently to glycophorin and asialogp compared to had h wt and had h sa , which suggests that had agglutination may be charge-dependent. consistent with the erythrocyte binding data, we found very little binding between cav- h wt and glycophorin or asialogp (data not shown). overall, the spr results reflect the results had -had f, a hybrid capsid containing the had fiber on the had capsid; and e. had -cav- h, a hybrid capsid containing the cav- fiber head on the had capsid. agglutination is visualized by the lack of erythrocyte sedimentation (no red spot) at the bottom of the conical well (see figure s for graphic description). the pink shaded well is the first well that does not significantly agglutinate. all assays were performed at least three times. doi: . /journal.ppat. .g structural analysis of the cav- fiber head suggests a sialic acid binding site our results suggested that had hemagglutination was due to sialic acid binding via the sialic acid binding site in the fiber head. although we have no evidence suggesting that cav- can use sialic acid as a functional receptor, it is possible that the efficient cav- agglutination of human erythrocytes is due to multiple and coordinated binding of sialic acid. for example, each of the homotrimeric fiber heads on the end of the flexible cav- shaft [ ] could bind three sialic acid moieties (theoretically up to /capsid). because the predicted subgroup d fiber head hemagglutination domains (in the cd and gh loops) appear to be well conserved, we tried to identify the corresponding domain in the cav- fiber head using a mutagenesis strategy based on sequence homology ( figure s ). this approach was unsuccessful (data not shown), suggesting that ad hemagglutination sites are not strictly conserved. to address possible sialic acid binding via an alternative approach, we soaked cav- fiber head crystals in solutions containing - sialyl-d-lactose. the crystal diffracted to . Å and the structure was solved by molecular replacement using our model of cav- fiber head [ ] (crystallographic details are summarized in table s ). the electron density maps showed clear density for six n-acetyl neuraminic acid (neu ac) moieties in the asymmetric unit, three per fiber head trimer. sialyl-lactose is composed of three sugar rings, the sialic acid moiety being linked to lactose (galactose-glucose). the density for the lactose moieties was weak, implying that these are flexible within the crystal. only one of the six galactose rings in the asymmetric unit could be modeled. this molecule is located in between the two fiber head trimers in the asymmetric unit and forms a distant hydrogen bond (distance . Å ) to lys via the galactose oxygen . it is unlikely that this interaction is enough to immobilize the galactose ring because the other five galactose moieties in the asymmetric unit are not equally well visible in the electron density. more likely, the stabilization is due to the spatial restrictions imposed by the proximity of the other fiber head trimer. we found that the sialic acid binding site on cav- head is distinct, both in sequence and location, from that found on the had fiber head. on the cav- fibers head, sialic acid binds further away from the three-fold symmetry axis at the periphery of the trimer ( figure a -d). the residues involved in binding (asn , ser , ser , gln and arg ) do not align with those involved in sialic acid-binding by had fiber head ( figure s ). the had sialic acid binding site consists of three residues forming hydrogen bonds (tyr , pro and lys ) and two residues contacting sialic acid in hydrophobic interactions (tyr and val ) ( figure e ). all seven interactions between cav- fiber head and sialic acid are hydrogen bonds or salt bridges ( figure f ), suggesting a relatively strong interaction compared to had . unlike had fiber head [ ] , no hydrophobic contacts contribute to sialic acid binding. finally, sialic acid binds within a basic patch in each head ( figure c and d). our results showing that the cav- fiber head contains a sialic acid binding site creates a paradox: both had and cav- fiber heads contain car and sialic acid binding sites -but had uses sialic acid and cd , while cav- uses car (cav- does not use cd to infect cells, unpublished data) to infect cells. to determine if sialic acid and car binding were mutually exclusive, we soaked cav- or had fiber head crystals in complex with car in solutions containing sialyl-d-lactose. crystals containing the complex with cav- fiber head diffracted to . Å and contained chains of fiber head bound to chains of car d and sialyl-d-lactose molecules in the asymmetric unit (space group i ). crystals containing the complex with had fiber head diffracted to . Å and contained one chain of fiber head in complex with one car d molecule and one sialyl-d-lactose molecule in the asymmetric unit (space group i ). for both structures the electron density maps showed clear density for sialic acid, but not lactose, at the expected positions on the fiber heads ( figure g and h). together, our data demonstrate that cav- and had sialic acid binding sites do not overlap with the car binding sites, and both fiber heads can bind car and sialyl-d-lactose simultaneously. arnberg and colleagues previously showed that had interaction with sialic acid was inhibited at high salt concentration [ ] . to determine if a cav- -erythrocyte interaction was at least partially charge dependent, we incubated a cav- vector expressing gfp (cavgfp) or a had vector expressing gfp (adgfp) with mock-or neuraminidase-treated erythrocytes in pbs containing increasing concentrations of ions. we then pelleted the erythrocytes by centrifugation, removed an aliquot of the supernatant, added it to cells, and assayed the cells for gfp expression by flow cytometry hr post-infection. using mocktreated erythrocytes (figure ), we found that at physiological salt concentrations , % of cavgfp was removed from the supernatant, while at higher salt concentration ( mm nacl) , % of cavgfp was removed. when using neuraminidasetreated erythrocytes, we again found that cavgfp was efficiently removed from the supernatant. all the test samples were figure . binding of wild type and mutant fiber heads to human erythrocytes. a. had fiber head binding to erythrocytes (mock-treated, top panels or neuraminidase-treated, bottom panels) as measured by flow cytometry. the shadowed profile = mock-treated erythrocytes without fiber heads. the line graphs correspond to four -fold dilutions ( , . , . and . mm) of three had fiber heads: had wt = wild type, had sa and had sa contain mutations in the had h sialic acid binding domain. the lowest dilution ( . mm = the dotted line in the top left panel. the darkest line (furthest to the right in the top left panel) = mm. except for the modest binding of mm had sa to neuraminidase-treated cells (bottom row middle panel), no other notable binding could be detected and therefore the line profiles overlap or are hidden by the shadowed mock-treated profile. b. fluorescently-labeled cav- fiber head (cav h wt ) binding to mock-treated (left) or neuraminidasetreated (right) erythrocytes as measured by flow cytometry. three -fold dilutions of cav h wt ( , . and . mm) covalently labeled with cy were assayed. the shadowed profile = mock-treated erythrocytes without fiber heads. as above the highest concentration of fiber head is in the darkest line profile. all assays were performed at least twice in triplicate. doi: . /journal.ppat. .g significantly (p, . ) different from the control, as well as from the mock-treated mm nacl (p, . ). consistent with other studies [ , ] adgfp was also efficiently ( %) removed from the supernatant after the incubation with mock-(or neuraminidase-) treated erythrocytes. these data suggest that, like had , a modest degree of cav- -erythrocyte interactions may depend on electrostatic interactions. in addition, both the car-tropic adenoviruses (had and cav- ) bind human erythrocytes at physiological salt concentrations. while our results show that had hemagglutination is due primarily to sialic acid binding, cav- hemagglutination appeared more complex. we therefore developed additional tests to address the molecular and structural basis. due its sensitive and semi-quantitative potential, we returned to hemagglutination assays to understand the erythrocyte interactions. freshly purified cav- h wt , which predominantly consists of individual trimeric fiber heads, did not cause hemagglutination, presumably because it is not sufficiently multivalent to crosslink erythrocytes. however, several his-tagged ad fiber heads, including those of had short fiber [ ] and had , form multimers that dissociate into single trimeric fiber heads upon removal of the histidine tag. similarly, his-tagged cav- fiber head forms multimers of a defined size that are stable on a sizeexclusion column and can be visualized with an electron microscope (not shown). removal of the his-tag yields single fiber heads. we found that his-tagged cav- h wt agglutinated erythrocytes, while his-tagged cav- h sa and cav- h sa (two cav- fiber heads with one or two mutations in the sialic acid binding site, see table ) showed reduced hemagglutination titers. however, we could not exclude the possibility that the reduced hemagglutination in these latter cav- h constructs was due to electrostatic interactions (the mutations modified the charge of the fiber heads). we next assayed cav- hemagglutination using a competition assay (see figure s for schema). in these competition assays, we pre-incubated erythrocytes with fiber heads, antibodies, salt and/ or neuraminidase. then the erythrocytes were incubated with cav- and compared to mock-treated erythrocytes. we found that hemagglutination could be . -fold reduced by preincubating cav- with anti-fiber head antibodies, or preincubating erythrocytes with cav- h wt (see figure a for specific example and b for cumulative data). pre-incubating the erythrocytes with the head from had or performing the assay in mm nacl led to modest to -fold reductions. unexpectedly, we found that like cav- h wt , cav- h sa notably reduced agglutination (, -fold), while a cav- fiber head with a mutation in the car binding site (cav- h car ) had a modest , -fold reduction. in most cases, pre-treating erythrocytes with neuraminidase had a fairly small additive effect of cav- hemagglutination. together, these data suggest that the car binding site, which is present in cav- h wt and cav- h sa but not cav- h car , is involved in cav- agglutination of human erythrocytes. that the cav- car binding site is involved in erythrocyte binding is consistent with the study by nicol et al. [ ] , which showed that a car-ablated had vector no longer agglutinated human and rat erythrocytes. the presence of car on erythrocytes would be inconsistent with other reports [ ] . among other functions, car acts as a homodimeric cell adhesion molecule at tight gap junctions [ ] . however, the expression of cell adhesion molecules during erythropoiesis is not unprecedented [ ] . erythrocytes from some species express cell adhesion molecules during the early stages of differentiation that are thought to be involved in interaction with macrophages. we therefore incubated erythrocytes with anti-car antibodies that recognize the extracellular domain of car and assayed expression using flow cytometry. we found low, but reproducible, car expression on the cell surface of human erythrocytes ( figure a ). to assay car expression using another approach, we used western blot analysis to screen erythrocytes from several species. using an anti-car ab that recognizes the cytoplasmic domain of car we found car expression on human and rat, but not on mouse, dog, rabbit and nonhuman primate erythrocytes ( figure b and data not shown). again, our results showed a relatively low level of car on human erythrocytes, which is consistent with the low level of cav- h wt binding to erythrocytes ( figure b) . these data suggest that car binding is a significant factor in cav- , and likely other car-tropic ads, interaction with human erythrocytes. this also is consistent with the fact that cav- agglutinates rat erythrocytes, but not erythrocytes from mice, dogs, rabbits or some nonhuman primates (as well as other species) ( figure s ). if car binding plays a role in cav- agglutination, then knocking down/blocking or artificially expressing car on erythrocytes should prevent/induce hemagglutination. eliminating car on mature enucleated erythrocytes is technically challenging. furthermore, to the best of our knowledge there are no known anti-car antibodies that completely block ad attachment. on the other hand, car has been expressed on erythrocytes using a transgenic mouse line (gata -car) with the car cdna downstream of the globin transcription factor promoter [ ] . using flow cytometry and western blot analysis ( figure a & b) , we compared the levels of human car expressed by gata -car and human erythrocytes. we then repeated the hemagglutination assays using erythrocytes from control (c bl/ ) and gata -car mice. we also found that cav- agglutinated gata -car erythrocytes at a low particle-to-cell ratio ( figure c ), while there was no agglutination of c bl/ erythrocytes. equally important, competition assays with recombinant cav- fiber heads gave profiles that were similar to when we used human erythrocytes ( figure d , and data not shown). together, our data demonstrate that car expression by erythrocytes can lead to agglutination by car-tropic adenoviruses. in spite of recent notable advances [ , ] , in vivo ad biodistribution, tropism and pathogenesis for the car-tropic hads are still poorly understood. group c had serotypes and are the prototype ads in terms of structure, tropism and pathogenesis. however, tropism has been primarily studied in vitro, ex vivo or in animal models. our results showing that human and rat erythrocytes harbor car on their external membranes while mice and nonhuman primates do not, suggests that these latter animals poorly mimic the in vivo environment that ads encounter. to better address in vivo biodistribution of car-tropic ads, we injected gata -car and control c bl/ mice with a had vector and quantified viral genome blood half-life and tissue distribution. we found that the viral load in the blood was fold higher (p, . ) in gata -car versus isogenic control mice (car-negative erythrocytes) during the first hr post-injection ( figure a ). these data are reminiscent of the studies where human blood cells are routinely positive by qpcr for wild type had sequences [ , ] . in addition, notably absent from the adgfp-injected gata -car mice was transgene expression in the liver. lack of transgene expression was also consistent with the significant (p, . ) , -fold difference in the mean viral load as quantified by qpcr ( figure b ). the lack of efficient liver infection is also consistent with the generally unexpected and poor infection of rat liver compared to mice and nonhuman primates following injection of car-tropic ad vectors. together, these data suggest that car expression by rat and human erythrocytes plays a significant role in had in vivo distribution and, in turn, determining which tissues are susceptible to infection. we initiated this study to examine a -year-old enigma of adenovirus biology. our results demonstrate that had interac-tion with human erythrocytes is primarily due to sialic acid binding via a conserved sialic acid binding site on this subgroup d had fiber head. cav- , a serotype that like the prototype had is ''cartropic'' and by most criteria is unrelated to had , interacts with human erythrocytes via a mechanism depending on several factors, including most notably binding to car. most subgroup d had sialic acid binding site residues are conserved [ ] , and structural analysis suggest that they bind sialic acid in an equivalent fashion. unexpectedly, we found that the cav- fiber head harbors a sialic acid binding site. but, in contrast to the well-conserved location of the car binding domains on some ad fiber heads, location of the sialic acid binding site is not conserved. following the structure-based identification of the amino acids in the cav- and had fiber heads that interact with sialic acid, figure . cav- hemagglutination assays using competition with fiber heads, increased salt concentrations and neuraminidase. a. examples of the results from competition assay (see figure s for the schema of the approach). the pink shaded well corresponds to the last well that shows agglutination. a shift to the left of the pink shade signifies decreased agglutination due to blocking of the virus binding site or removal of an attachment ligand. a -fold decrease in agglutination would correspond to a one-well shift to the left of the last well that showed agglutination. top control panel = agglutination of human erythrocytes by cav- (for example , pp/erythrocyte in the pink shaded well on top row). middle panel = erythrocytes preincubated with cav- h sa (the cav- fiber head that contains a mutation in the sialic acid binding site but can still bind car) prior to the agglutination assay (, pp/erythrocyte in the pink shaded well, top row), which equals an , to fold decrease from the control). bottom panel = the same as the middle panel except that the erythrocytes were pretreated with neuraminidase ( to -fold decrease). b. the graph shows the cumulative data from competition assays. cav- -agglutination of mock-treated erythrocytes was used as the starting point. the blocking agent was used with mock (black bars) or neuraminidase-treated (grey bars) erythrocytes. all assays were performed at least three times. doi: . /journal.ppat. .g we introduced mutations to assay the role of these sites in sialic acid binding and erythrocyte interaction. in a number of conditions and approaches we showed that hemagglutination by had depends primarily on sialic acid binding. first, removing sialic acids from erythrocytes with neuraminidase eliminated erythrocyte cross-linking by had and a chimeric capsid harboring the had fiber head. likewise, pre-incubation with neuraminidase significantly reduced hemagglutination caused by protein complexes containing multiple copies had fiber heads. second, mutating single residues in the sialic acid binding sites reduced the hemagglutination activity of these protein complexes. third, wild type had fiber heads bound more efficiently to erythrocytes or glycophorin than those carrying a point mutation in the sialic acid binding site. our binding assays using wild type and mutated had heads also suggested that the sialic acid affinity is partially charge-driven. in addition, although had head also binds car [ ] , in the context of the virus it does not appear to use car as a receptor, possibly due to its relatively short fiber. therefore, car on erythrocytes is likely to be less important for had biodistribution. in spite of our structural data showing a well-defined sialic acid binding site, we could not detect notable binding between cav- fiber head and glycophorin, a highly sialated protein. it was possible that the affinity of cav- fiber head to erythrocytes or glycophorin was lower than that of had fiber head, and that the efficient erythrocyte binding of cav- depended on the avidity of . car expression by erythrocytes leads to cav- induced agglutination. a. flow cytometry analysis of car expression by human, mouse and gata -car mouse erythrocytes using the anti-car e . , which recognizes the native form of car. b. western blot analysis of car expression using the polyclonal anti-car antibody car , which recognizes the denatured form of car: murine fibroblast (nih t cells, car ), human embryonic kidney cells ( cells, car + ) and mouse liver (car + ), and human, rat, mouse (c bl/ ) and gata -car mouse erythrocytes. an anti-actin staining was used to control the protein input. c. hemagglutination assay using c bl/ and gata -car mouse erythrocytes. no significant agglutination was found with c bl/ mice while gata -car erythrocytes agglutinated with , pp of cav- /erythrocyte (pink shaded wells). d. hemagglutination competition assay using gata-car erythrocytes and performed as described in figure using a higher concentration of the fiber head (as would be expected due to the higher levels of car on gata-car erythrocytes). doi: . /journal.ppat. .g multiple fibers. as the cav- fiber head is less positive than had fiber head (pi's of . and . respectively), this could have explained the lower affinity. the affinity between virus proteins and sialic acid is usually in the millimolar range; therefore it was conceivable that the affinity of single cav- fiber heads to erythrocytes or glycophorin was low. consistent with this assumption, we previously found that cav- is more neutrally charged than other ads [ ] . paradoxically though, cav- agglutinates at a lower particle-to-cell ratio than had and our crystallography data suggested that sialic acid binding should be at least as strong as had . however, one cannot reliably predict affinity from structural data. the identification of car expression by erythrocytes from species that are agglutinated by car-tropic ads is a crucial observation. using i) competition assays with recombinant fiber heads harboring point mutations in the sialic acid and car binding sites and ii) transgenic mice expressing car on erythrocytes, we characterized the unexpected and significant role of car in ad binding. as mentioned previously, ad serotypes differ in their ability to bind erythrocytes from various species. our study suggests that this may be due to i) an interaction with different chemical variants, linkages and ratios of sialic acid, ii) the presence and affinity to car, and/or iii) the pi's of the head [ ] . for example, some non-car-tropic ads that agglutinate human erythrocytes may preferentially bind neu ac, the most abundant sialic acid on human erythrocytes. with respect to charge, both had and had p bind sialic acid with equal affinity and the only two residues that differ between their heads are not close to the sialic acid-binding site [ ] . with the limited amount of data available, subgroup d hads tend to have heads with higher pi's and interact with sialic acid [ ] . interestingly, the residues that make up the cav- sialic acid binding site are conserved in the cav- fiber head (data not shown), suggesting a conserved sialic acid binding function. with respect to car affinity, the cav- fiber head binds human car with the highest affinity of any fiber head known [ ] , which may favor its efficient hemagglutination. in contrast to other ads, higher temperatures poorly inhibit cav- hemagglutination [ ] , again suggesting a role for the high-affinity attachment to car. this is also consistent with a rather large difference between had and cav- hemagglutination: to the best of our knowledge the had head does not harbor a sialic acid binding site and its pi ( . ) is less basic than had or cav- . finally, we cannot exclude a role of the inter-and intra-species differences in the quantity of car expressed by erythrocytes. phylogenetically, car expression by erythrocytes appears to be random; we tested several other species (dogs, mice, rabbits, lemurs and monkeys) and did not find car expression. however, car levels on rat erythrocytes were relatively high ( figure ) and consistent with our hypothesis that car-tropic ads agglutinates human and rat erythrocytes via car binding. it is possible, but unlikely, that our lack of detection of car on erythrocytes on some species was due to the limit of sensitivity of our bank of anti-car antibodies, which nonetheless detected car expression on other cell types from these species (not shown). in the context of the significant amount of had -mediated gene transfer data, our data highlights an important parallel. while a -gram mouse can be injected intravenously with up to pp of an had vector with minimal side effects, injection of the same dose in a -gram rat is normally lethal [ ] . whether our data have a bearing on the death following portal vein injection of a had vector during a phase i trial [ ] is unknown, but deserves consideration. similar to the car binding domain in had , we can only speculate about the importance of sialic acid binding in the biology of cav- . although cav- does not agglutinate dog erythrocytes ( figure s ), canis lupus familiaris may not be the original host of cav- : seroprevalence against cav- can be found in coyotes, bears, pandas, skunks, mongooses, raccoons and foxes [ ] [ ] [ ] . it is paradoxical that had binds cd , sialic acid and car -yet does not use car [ ] -while cav- binds both sialic acid and car and to the best of our knowledge uses only car as a receptor [ ] . we cannot exclude the possibility that in some cell types sialic acid binding provides a first low-affinity attachment to the cell surface, while car-binding is followed in a second step, providing a high-affinity binding. similar two-step mechanisms have been proposed for other viruses [ ] . it is tempting to speculate that sialic acid binding may play a role in the preferential transduction of neurons by cav- vectors [ ] [ ] [ ] [ ] . our crystal structure of cav- fiber head in complex with car and sialyl-dlactose demonstrates that the ternary complex of the three molecules is stable. there is no indication that this should not be the case also in vivo. our data and numerous reports describing the ex vivo interaction of car-binding had with human/rat erythrocytes [ , [ ] [ ] [ ] , , [ ] [ ] [ ] strongly suggest this interaction probably occurs after the intravascular injection of car-tropic vectors. notably, lyons et al. showed that that . % had vector dna was associated with blood cells following intratumoral injection during a clinical trial [ ] . it is likely that erythrocyte binding occurs during wild type infection of some hads (as well as coxsackie b viruses) that use car, which will certainly lead to altered biodistribution. had dna is routinely found in human blood cells by pcr. it is also a common misconception that ads are rapidly cleared following a classical immune response. numerous clinical cases strongly suggest that latent hads can readily resurface if the host is immunosuppressed. the fate of particles that stick to erythrocytes under natural or artificial (i.e. vector injections) conditions is probably complex. for example, had -induced liver disease in immunocompromised humans is relatively common. in contrast, immunocompromised nonhuman primates are rarely diagnosed with simian ad (sav)-induced liver disease [ ] . does car expression on erythrocytes lead to an advantage for host or virus? has car expression on erythrocytes put a selective pressure on ads (and coxsackie b viruses that also bind car) to avoid an erythrocyte virus trap [ ] ? or has car expression by erythrocytes allowed hads to thrive because it has allowed open access to so many more tissues and cell types? in summary, our results resolve a longstanding enigma of aderythrocyte interaction in vitro and in vivo. in addition, we provide new insights into virus-erythrocyte interactions that will allow us to better understand had pathogenesis and facilitate the engineering of safer, more efficient gene transfer vectors. although in most in vivo scenarios hemagglutination per se is unlikely to occur due to the turbulence that erythrocytes encounter in the circulation, sequestering of vector particles by erythrocytes must diminish gene transfer efficacy. interest in ad biology is continually growing due to the increasing incidence of had-induced morbidity and mortality during immunosuppression [ , ] , and because adderived vectors are the most commonly used vectors in gene therapy clinical trials. the identification of fiber head mutants that do not bind human erythrocytes may be of interest. equally important may be the need to screen fiber heads from other serotypes for sialic acid and car binding domains. combined with had interactions with vitamin k-dependent coagulation factors [ ] , our study adds another critical element in the interaction with blood components, biodistribution and pathogenesis. cav- (cavgfp), had (adgfp), had , and the hybrid had -cav- h (ad luc -ck) and had -had f (ad f) vectors were prepared as previously described [ , , , ] . briefly, cavgfp and adgfp are e -deleted vectors expressing gfp. the capsids contain no modifications. the hybrid had -cav- h vector contains the fiber head of cav- on the had capsid. had -had f vector contains the had fiber (shaft and head) on the had capsid. all vectors were purified by double banding on cscl gradients, cscl was removed using pd- columns (pharmacia). the vectors were stored in phosphate-buffered saline (pbs) containing % glycerol at uc. stock titers were . physical particles/ml with . infectious particle/ physical particles. anti-car antibodies tested in this study included e . the recombinant fiber heads and dodecahedral sample were loaded between the mica-carbon interface as described [ ] . the samples were stained using % sodium silico tungstate ph . and air-dried. images were taken under low-dose conditions in an ex -ii jeol electron microscope working at kv and with a nominal magnification of , . the images were scanned on a z/i imaging scanner (photoscan td) with a pixel size of mm ( . Å per pixel at the sample level). fiber head constructs were cloned into pproex htb (life technologies) and expressed with a cleavable his-tag as described previously [ ] . had fiber head constructs contain residues - and cav- fiber head constructs contain residues - . point mutations were introduced using the quikchange site-directed mutagenesis kit (stratagene) and polymerase chain reaction (pcr). protein purification was performed as described [ ] . briefly, cells were incubated in lysis buffer ( mm tris-hcl ph . , mm nacl, mm imidazole, (boehringer complete edtafree protease inhibitor cocktail), centrifuged, and fiber head bound to a ni-nta column (qiagen). eluted protein was either directly loaded onto a superdex column for hemagglutination experiments with tagged fiber head, or incubated overnight with / his-tagged tobacco etch virus (tev) protease at uc. proteins were then dialyzed against lysis buffer and uncleaved protein and tev protease bound to a ni-nta resin. untagged fiber head was loaded onto a superdex column using the same buffer as for tagged protein ( mm tris ph . and mm nacl). cav- h car was labeled using alexa microscale protein labeling kit (molecular probes). cav- h wt was dialyzed in pbs . m naco ph . and labeled using mono-reactive dyes (cy , cy or alex , amersham bioscience) for min at room temperature. the elution of labeled protein was performed with ml of pbs using nap column (ge healthcare) pre-equilibrated with ml pbs. the final dye/protein ratios (, . for each) were determined using nanodrop nd- spectrophotometer. untagged cav- fiber head was concentrated to mg/ml in crystallization buffer ( mm nacl, mm tris ph . , mm sialyl-d-lactose) and crystallized in hanging drops containing ml protein solution and ml well solution ( % peg , % isopropanol, . m hepes ph . ). single crystals were transferred and frozen in a cryoprotectant solution ( % well solution, % glycerol, approximately mm sialyl-d-lactose). the sialyl-d-lactose (sigma) corresponds to a - n-acetylneuraminosyl-d-lactose (according to the supplier). purification and crystallization of fiber head complexes containing car d and sialyl-d-lactose the cloning, expression and crystallization of had and cav- fiber head car d complexes (space groups i and i ) was performed as described previously [ ] . after crystal growth, sialyl-d-lactose (sigma) was added to the drop to a final concentration of to mm. crystals were frozen the next day without adding cryoprotectant solution to crystals with cav- fiber head complex, and with % glycerol in the crystallization condition for crystals with had fiber head complex. details of the data collection and refinement of the three structures (cav- +sialyl-lactose, cav- +car d +sialyl-lactose and had +car d +sialyl-lactose) are given in table s . all crystallographic data were collected at the european synchrotron radiation facility (esrf) and processed with xds [ ] . all structures were solved by molecular replacement using phaser [ ] and refined with refmac [ ] . coot [ ] was used for the visualization of all models and electron density maps, and for the superposition of different models. procheck [ ] and molprobity [ ] were used for the validation of the obtained models. representations of the protein models and the electron density were made with pymol [ ] and grasp [ ] . binding interfaces were visualized with dimplot [ ] . the structurebased sequence alignment was made with sarf, modified manually in seaview [ ] and visualized with espript [ ] . the cav- +sialyl-lactose structure contains two fiber head trimers in the asymmetric unit. the cav- +car d +sialyllactose contains four trimeric fiber head -car d complexes in the asymmetric unit. due to the modest resolution of this structure, ncs restraints and tls refinement was used. the had +car d +sialyl-lactose structure has one fiber head monomer with bound car d in the asymmetric unit. cloning and purification of had penton dodecahedra in complex with chimeric mini-fibers chimeric mini-fiber constructs were cloned by pcr in two steps. first, dna fragments were produced using as template had genomic dna (for fragment ) or pproex htb vectors coding for wild type or mutant had fiber head (for fragments and ). fragment coded for had tail and shaft motives + (primers were aat aat cca tgg cca agc gag ctc gg and gtt cca tgc tac caa gga tcc atc agt ag). fragments and coded for wild type and mutant (k e) had fiber head (primers used were cta ctg atg gat cct tgg tag cat gga ac and aat aat gaa ttc tca ttc ttg ggc aat ata gg). in a second pcr reaction, fragment was annealed to fragment or using primers aat aat gaa ttc tca ttc ttg ggc aat ata gg and aat aat cca tgg cca agc gag ctc gg. the resulting longer fragments coded for complete mini-fiber containing had tail+shaft and either wild type or mutant (k e) had fiber head. these were cloned into pproex htb, and expressed at uc in e. coli strain bl star (de ) (life technologies) together with an n-terminal cleavable his-tag. cells were re-suspended in lysis buffer and sonicated. the cell lysate was centrifuged for min at g and the supernatant loaded on a ni-nta column (qiagen). protein bound to the resin was washed with mm tris-hcl ph . , mm nacl, mm imidazole and eluted in mm tris-hcl ph . , mm nacl, mm imidazole. to remove the his-tag, mini-fibers were incubated overnight with / his-tagged tev protease at uc. imidazole was removed by dialysis against lysis buffer. uncleaved protein and tev protease were removed by binding to a ni-nta resin. had penton forms multimers (dodecahedra) consisting of twelve penton bases each [ , ] . p. fender (institut de biologie structurale, grenoble) generously supplied purified had dodecahedra. minifibers and dodecahedra were mixed at an approximate molecular ratio of : and incubated at uc overnight. the mixture was loaded onto a superdex column (amersham) to remove excess fiber. the buffer contained mm tris ph . and mm nacl. dodecahedra in complex with mini-fiber were eluted with the void volume and were visualized with an electron microscope. all minifiber constructs carrying had tail+shaft and a cav- fiber head were unstable, possibly because they were misfolded, or they did not bind to had dodecahedra. non-treated, mock-treated or neuraminidase-treated erythrocytes and non-tagged fiber heads were incubated for min at uc. cell samples serving as negative control were incubated with pbs instead of fiber head solution. the cells were pelleted at g, re-suspended in pbs containing : rabbit anti-had fiber head serum (gift from n. arnberg, university of umeå) or : purified rabbit antibodies against cav- fiber head, and incubated for min at uc. the cells were pelleted as before, re-suspended in pbs containing : anti-rabbit fitc labeled antibody (sigma) and incubated in the dark at uc for min. the cells were pelleted again, re-suspended in pbs, and injected into a facscalibur apparatus (bd biosciences). the results were analyzed using cellquest and flowjo software (bd biosciences). human, rat (wistar), dog (beagle), vervet (chlorocebus pygerythrus), cynomologous monkey, rhesus macaque, guinea pig, rabbit (new zealand white), mouse (c bl/ ) or gata -car [ ] mouse blood was collected in edta, heparin or alsever's solution. erythrocytes were purified using ficol gradients, washed twice in pbs edta ( min at rpm) and stored less than hrs in pbs containing mm edta. for the human and gata -car mouse erythrocytes two fractions of packed erythrocytes were re-suspended separately in neuraminidase buffer each. neuraminidase a( - ,- ,- and- ) from arthrobacter ureafaciens (sigma) or a( - ,- and - ) (ozyme) was activated as recommended, and added to one of the two erythrocytes fractions. both mock-(without neuraminidase) and neuraminidase-containing fractions were incubated for hr at uc. the cells were then washed times with pbs to remove neuraminidase and buffer, and resuspended in pbs containing mm edta. virus, dodecahedra chimeric mini-fibers and cav- fiber head multimers were diluted with pbs in -fold (in the first column) and then -fold (horizontal) steps and added to -well plates with cone-shaped bottoms. an equal number of purified erythrocytes (either nontreated, neuraminidase-treated, or mock-treated) was added to each well and incubated at room temperature or uc for at least hrs. the blocking protein (, mg/well) was incubated with erythrocytes for h at uc with slow rotation. all assays were repeated at least times. in a volume of ml, cavgfp or adgfp ( particle/cell) was incubated with . mock-treated or with neuraminidasetreated erythrocytes with pbs, or pbs supplemented with nacl to bring its final concentration to or mm. the samples were incubated at room temperature for min, and then centrifuged for min at rpm in a microfuge. aliquots of the supernatant were removed and incubated with cells in -well plates. the cells were trypsinized and assayed for gfp expression by flow cytometry hrs post-incubation. data were analyzed using cellquest. the assays were performed twice and in quadruplicate. control cells and tissues were lysed with ml sds buffer ( cells) and benzonase for h at uc. the liver, peripheral blood mononuclear cells, nih t cells (mouse fibroblasts) and cell (human embryonic kidney) extracts ( mg/ml) were resuspended in ml of sds-sample buffer. erythrocytes (, ml) were lysed in ddh o, the membranes were pellet for min at , rpm in a microfuge and resuspended in ml of sds-sample buffer. sds-page was performed using a % acrylamide/bis-acrylamide stacking gel and a % acrylamide/bis-acrylamide running gel. membranes were blocked with tbs-tween, % milk at room temperature. the rabbit anti-car antibody car was diluted -fold for h at rt in tbs-tween % milk. the secondary anti-rabbit antibody was used at a dilution of / for min at room temperature in tbs-tween % milk. four adult gata -car and four control c bl/ mice were injected with . pp of adgfp via the tail vein. blood (, ml) was taken by tail vein bleeds at . , , and hr. the mice were sacrificed at hr by lethal injection, and the organs were perfused with pbs via cardiac puncture. the liver, lung and spleen were recovered, divided into parts for qpcr or histology. the organs used for histology were fixed in % pfa for hr then placed in % sucrose for hr, and embedded in oct matrix (cellpath, powys, uk). sections ( -mm-thick) were stained with . mg/ml bisbenzimide hoechst (sigma-aldrich) and ng/ml phalloidin-tritc (sigma-aldrich) before being mounted. images were acquired using a zeiss microscope and processes using the metamorph (molecular devices, wokingham, uk). the experimental protocols involving animals were approved by the university of massachusetts medical school institutional animal care and use committee. total dna from blood and liver were extracted by using the high pure dna isolation kit (roche diagnostics). qpcr was performed with a light cycler (roche diagnostics) using the platinum taq dna polymerase (invitrogen) and sybr green qpcr master mix [ ] . the primer pairs used for gapdh were: gapdh forward, aca gtc cat gcc atc act gcc ; gapdh reverse, gcc tgc ttc acc acc ttc ttg ; and the egfp: forward, cag aag aac ggc atc aag gt ; egfp reverse, ctg ggt gct cag gta gtg g . data are expressed as a ratio of gapdh to egfp. data were analyzed using a one-way anova and post-hoc comparisons were made using an unpaired student's t-test. figure s electron micrographs of dodecahedra fiber. a) ad penton dodecahedra b) ad penton dodecahedra in complex with chimeric minifiber containing wild type had fiber head. to the right of the photos are computer generated models of the structures. below are enlarged images of the dodecahedra fiber. found at: doi: . /journal.ppat. .s ( . mb tif) figure s binding of wild type and mutant fiber heads to human erythrocytes. a) spr curves obtained with different concentrations of wild type had fiber head (had hwt) and immobilized glycophorin (gp). b) spr curves obtained with had hwt and mutant had fiber heads (had hsa- and had hsa- ) at mm and immobilized gp. c) spr curves obtained with had hwt and mutant had fiber heads at mm and immobilized asialogp. adenovirus complex structures the fibers of fowl adenoviruses adenovirus receptors adenovirus type lacks an rgd alpha(v)-integrin binding motif on the penton base and undergoes delayed uptake in a cells canine adenovirus type attachment and internalization: coxsackievirus-adenovirus receptor, alternative receptors, and an rgd-independent pathway clinical features and treatment of adenovirus infections adenoviruses in the immunocompromised host hemagglutination by adenoviruses separation and characterization of soluble adenovirus type components hemagglutination with adenovirus serotypes belonging to rosen's subgroups ii and fiber-associated incomplete and complete hemagglutinins of adenovirus type characterization of a canine adenovirus hemagglutinin red blood cell glycophorins reovirus receptors and pathogenesis glycoconjugate glycans as viral receptors update on bk virus entry and intracellular trafficking role of sialic acids in rotavirus infection sialic acids as receptor determinants for coronaviruses receptor determinants of human and animal influenza virus isolates: differences in receptor specificity of the h hemagglutinin based on species of origin epitopes and hemagglutination binding domain on subgenus b: adenovirus fibers recombinant fiber proteins of human adenoviruses ad , ad and ad : localization of the haemagglutination properties and the type-specific determinant molecular characterization of the type-specific gamma-determinant located on the adenovirus fiber molecular characterization of hemagglutination domains on the fibers of subgenus d adenoviruses structural and mutational analysis of human ad and canine adenovirus fiber heads in complex with the d domain of coxsackie and adenovirus receptor adenovirus type uses sialic acid as a cellular receptor membrane cofactor protein is a receptor for adenoviruses associated with epidemic keratoconjunctivitis crystal structure of species d adenovirus fiber knobs and their sialic acid binding sites the erythrocyte viral trap: transgenic expression of viral receptor on erythrocytes attenuates coxsackievirus b infection titer determination of ad in blood: a cautionary note structural analysis of a fiber-pseudotyped adenovirus with ocular tropism suggests differential modes of cell receptor interactions an adenovirus vector with a chimeric fiber derived from canine adenovirus type displays novel tropism threedimensional structure of canine adenovirus serotype capsid adenovirus type binds to cell surface sialic acid through a charge-dependent interaction adenovirus type interactions with human blood cells may compromise systemic delivery crystal structure of enteric adenovirus serotype short fiber head effect of adenovirus serotype fiber and penton modifications on in vivo tropism in rats the coxsackievirus and adenovirus receptor alpha beta integrin and erythropoietin mediate temporally distinct steps in erythropoiesis: integrins in red cell development adenovirus serotype hexon mediates liver gene transfer adenovirus fiber disrupts car-mediated intercellular adhesion allowing virus escape rodent nonclinical safety evaluation studies of sch , an adenoviral vector for the p gene fatal systemic inflammatory response syndrome in a ornithine transcarbamylase deficient patient following adenoviral gene transfer oral administration of an attenuated strain of canine adenovirus (type ) to raccoons, foxes, skunk, and mongoose serosurvey of ex situ giant pandas (ailuropoda melanoleuca) and red pandas (ailurus fulgens) in china with implications for species conservation serological survey of selected canine viral pathogens and zoonoses in grizzly bears (ursus arctos horribilis) and black bears (ursus americanus) from alaska virus receptors: binding, adhesion strengthening, and changes in viral structure car chasing: canine adenovirus vectors-all bite and no bark? gene transfer to the central nervous system: current state of the art of the viral vectors preferential transduction of neurons by canine adenovirus vectors and their efficient retrograde transport in vivo long-term in vivo transduction of neurons throughout the rat central nervous system using novel helper-dependent cav- vectors virus-erythrocyte interactions studies on adenovirus receptors on red cells adenoviral hepatitis in a sivinfected rhesus monkey (macaca mulatta) adenovirus-associated acute respiratory disease in healthy adolescents and adults: a literature review severe pneumonia due to adenovirus serotype : a new respiratory threat? canine adenovirus vectors: an alternative for adenovirus-mediated gene transfer fluorescently tagged canine adenovirus via modification with protein ix-enhanced green fluorescent protein an archaeal peptidase assembles into two different quaternary structures: a tetrahedron and a giant octahedron moving median filters for area-detector data likelihoodenhanced fast translation functions efficient anisotropic refinement of macromolecular structures using fft coot: model-building tools for molecular graphics aqua and procheck-nmr: programs for checking the quality of protein structures solved by nmr molprob-ity: structure validation and all-atom contact analysis for nucleic acids and their complexes high b-value diffusion imaging protein folding and association: insights from the interfacial and thermodynamic properties of hydrocarbons ligplot: a program to generate schematic diagrams of protein-ligand interactions seaview and phylo_win: two graphic tools for sequence alignment and molecular phylogeny espript: analysis of multiple sequence alignments in postscript adenovirus penton dodecahedron exhibits structural changes of the base on fiber binding performing quantitative reverse-transcribed polymerase chain reaction experiments we thank n. arnberg for had and antiserum, s. hemmi, j. zabner and s. carson for the anti-car antibodies, and p. boulanger and w. burmeister for advice. we thank embl and esrf staff for help on synchrotron beam lines id -eh and id . we thank c. cazevieille, cric montpellier for the help with the sem photo of the agglutinated cells. key: cord- -i cgkvq authors: zhou, bin; ma, jingjiao; liu, qinfang; bawa, bhupinder; wang, wei; shabman, reed s.; duff, michael; lee, jinhwa; lang, yuekun; cao, nan; nagy, abdou; lin, xudong; stockwell, timothy b.; richt, juergen a.; wentworth, david e.; ma, wenjun title: characterization of uncultivable bat influenza virus using a replicative synthetic virus date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: i cgkvq bats harbor many viruses, which are periodically transmitted to humans resulting in outbreaks of disease (e.g., ebola, sars-cov). recently, influenza virus-like sequences were identified in bats; however, the viruses could not be cultured. this discovery aroused great interest in understanding the evolutionary history and pandemic potential of bat-influenza. using synthetic genomics, we were unable to rescue the wild type bat virus, but could rescue a modified bat-influenza virus that had the ha and na coding regions replaced with those of a/pr/ / (h n ). this modified bat-influenza virus replicated efficiently in vitro and in mice, resulting in severe disease. additional studies using a bat-influenza virus that had the ha and na of a/swine/texas/ - / (h n ) showed that the pr ha and na contributed to the pathogenicity in mice. unlike other influenza viruses, engineering truncations hypothesized to reduce interferon antagonism into the ns protein didn't attenuate bat-influenza. in contrast, substitution of a putative virulence mutation from the bat-influenza pb significantly attenuated the virus in mice and introduction of a putative virulence mutation increased its pathogenicity. mini-genome replication studies and virus reassortment experiments demonstrated that bat-influenza has very limited genetic and protein compatibility with type a or type b influenza viruses, yet it readily reassorts with another divergent bat-influenza virus, suggesting that the bat-influenza lineage may represent a new genus/species within the orthomyxoviridae family. collectively, our data indicate that the bat-influenza viruses recently identified are authentic viruses that pose little, if any, pandemic threat to humans; however, they provide new insights into the evolution and basic biology of influenza viruses. bats are present throughout most of the world and account for more than a fifth of mammalian species. they are natural reservoirs of some of the most deadly zoonotic viruses, including rabies virus, ebola virus, henipaviruses, and sars coronavirus [ , ] . recently, nucleic acids obtained from bat samples indicated bats may be a reservoir of a new group of influenza viruses (batinfluenza) that are phylogenetically very distantly related to other influenza viruses [ , ] . type a, b, and c influenza viruses belong to the orthomyxoviridae family and their genomes are composed of - negative sense rna segments (vrnas). while influenza b (ibv) and c viruses mainly infect human hosts, influenza a virus (iav) has a broad host range; including humans, marine mammals, horses, pigs, waterfowl, and poultry. new subtypes of iav, which have novel hemagglutinin (ha) and/or neuraminidase (na) surface pandemic viruses are often reassortant viruses composed of vrnas that are derived from multiple iav lineages that previously circulated in swine and/or avian reservoirs (e.g., avian-human reassortant, avian-human reassortant, and avian-swinehuman reassortant). the discovery of putative bat-influenza viruses expands the known host species reservoirs that may serve as a source of novel viruses, which is a major concern for public and animal health [ , ] . infectious bat-influenza viruses couldn't be isolated [ , ] and although several structural and biochemical characterization studies have been conducted with the putative bat-influenza ha, na, and part of pa, none of the vrnas have been shown to be functional in a replicative virus [ , , [ ] [ ] [ ] [ ] [ ] . this has led to questions such as: ( ) are the putative bat-influenza vrna sequences identified derived from infectious viruses or are they merely nucleic acid relics harbored in bats [ ] , ( ) are the vrna segments sequenced from a single bat-influenza virus or are they from multiple potentially incompatible viruses, and ( ) were the sequences of the complete gene segments, which is a significant technical challenge, determined accurately. the inability to culture infectious viruses is the major hurdle to confirm the existence of these novel influenza viruses, and to answer important questions, such as pathogenicity in animal models, ability to reassort with other influenza viruses, and their potential risk to public health [ , ] . the goals of this study were to synthesize the complete viral genome, characterize the bat-influenza virus using non-infectious approaches, then generate a replicative virus, and use it as a model to better understand bat-influenza viruses. synthetic genomics generated bat-influenza virus-like particles but they were not infectious in many host cell substrates lack of infectious particles in the original bat specimens is a potential factor in the inability to isolate/culture bat-influenza using multiple host cell substrates [ ] . based on digital sequence information published by tong et al. [ ] , we synthesized the complete genome of a/little yellow-shouldered bat/guatemala/ / (h n ) (fig. s ) and cloned it into reverse genetics plasmids to rescue this putative bat-influenza virus (bat ). thousands of spherical influenza-like particles budded into the supernatants of human cells ( t) transfected with the bat reverse genetics plasmids (fig. a) . the supernatants were inoculated into embryonated chicken eggs and cell lines derived from many species (canine (mdck), mink (mv -lu), swine (st), african green monkey (vero), human (a , calu- ), and freetailed bat (tadarida brasiliensis, tb lu); however, none of the host cell substrates tested supported productive virus infection (determined by serial passage and subsequent real-time rt-pcr). previous biochemical and structural studies with purified proteins of bat hemagglutinin (ha) and neuraminidase (na) the identification of influenza virus-like sequences in two different bat species has generated great interest in understanding their biology, ability to mix with other influenza viruses, and their public health threat. unfortunately, bat-influenza viruses couldn't be cultured from the samples containing the influenza-like nucleic acids. we used synthetic genomics strategies to create wild type batinfluenza, or bat-influenza modified by substituting the surface glycoproteins with those of model influenza a viruses. although influenza virus-like particles were produced from both synthetic genomes, only the modified bat-influenza viruses could be cultured. the modified batinfluenza viruses replicated efficiently in vitro and an h n modified version caused severe disease in mice. collectively our data show: ( ) the two bat-flu genomes identified in other studies are replication competent, suggesting that host cell specificity is the major limitation for propagation of bat-influenza, ( ) bat-influenza ns antagonizes host interferon response more efficiently than that of a model influenza a virus, ( ) bat-influenza has both genetic and protein incompatibility with influenza a or b viruses, and ( ) that these bat-influenza lineages pose little pandemic threat. indicate that the ha doesn't bind to canonical sialic acid receptors of influenza viruses and the na doesn't have neuraminidase activity, which is characteristic of iav and ibv nas [ ] [ ] [ ] [ ] . to further examine if the ha and na proteins are the major blocks to the propagation of the bat virus, we attempted to rescue reassortant viruses that contained the internal protein coding vrnas (pb , pb , pa, np, m, and ns) from bat and the surface glycoprotein vrnas (ha and/or na) from a recombinant a/puerto rico/ / (pr ). pr is a lab adapted h n virus that has been used for many years in research and vaccine settings because it replicates efficiently in embryonated chicken eggs, cell lines (e.g., mdck) and in the mice, but has low risk to humans. however, the three pr -ha/na reassortant genotypes containing the bat internal protein vrnas couldn't be rescued following transfection (fig. b) . while the bat internal protein/vrnas are capable of generating proteins and producing influenza-like particles, they may have critical mutations that were inhibiting infectivity, or they can't cooperate efficiently with the pr -ha/na proteins/vrnas. to further address the inability to rescue bat or the bat :pr -ha/na reassortants, we created a modified ha vrna (mh ) that contained the protein coding region from pr -h flanked by putative cis-acting terminal packaging signals from bat that we hypothesized would be similar to the regions known to be central to packaging of a/wsn/ and pr [ , ] ( fig. c and fig. s ). the bat na gene segment was modified using a similar strategy to replace the na coding region with pr -n , while the putative bat na packaging signals were retained (mn ) (fig. c and fig. s ). co-expression of the mh and mn vrnas with the six bat internal protein vrnas efficiently rescued a reassortant bat :mh mn virus (fig. b) . the reassortant bat :mh mn formed particles similar to that of bat (fig. a) and replicated robustly in vitro and in ovo (fig. d, e ). next generation sequencing demonstrated that the consensus sequence of the virus stocks from passage in mdck cells or embryonated chicken eggs was identical to that of the reverse genetics plasmids. furthermore, even after passages in mdck cells, we still didn't identify any nucleotide polymorphisms accounting for . % of the genomic population that would suggest strong selective pressure on bat genes or the modified ha/na genes of pr . to investigate whether bat :mh mn is able to infect and replicate in mice, a mouse study was performed using the mouse adapted pr iav as a positive control. bat :mh mn replicated efficiently in mouse lungs ( fig. a) , and caused significant weight loss as early as at days post inoculation ( dpi) (fig. b) . the virulence of bat :mh mn ( % mortality) was close to that of the pr virus ( % mortality) (fig. c) . histopathological analysis showed that the bat :mh mn virus caused typical influenza-like lesions characterized by a varying degree of broncho-alveolar epithelial degeneration and necrosis, and interstitial pneumonia. the peribronchiolar and perivascular areas were infiltrated by moderate numbers of lymphocytes and plasma cells (fig. d) . the histopathology identified correlates with presence of virus antigen in the mouse lungs (fig. e) . next generation sequencing was used to determine if the bat vrnas were genetically stable in mice. although nucleotide polymorphisms (at the level of %- %) were detected at sporadic loci throughout the bat vrnas, each lung sample only had one such polymorphism on average, and none of the mutations were found in more than one mouse. nonetheless, serial passage of this virus in mice may identify mutations in the bat backbone critical to replication/pathogenesis in mice. we did identify a low level nucleotide polymorphism in the modified pr ha at residue at that emerged in multiple bat :mh mn inoculated mouse lung samples collected at and dpi (ha-k e, %- % of the genomic population). this unanticipated result may have also occurred in pr inoculated mice; however the lung specimens from these mice were not sequenced. the virulence of the bat :mh mn in mice could partly result from the h and n of the mouse adapted pr virus. to further investigate pathogenicity of bat -like viruses we rescued another modified bat virus that expresses h n surface glycoproteins from a/swine/texas/ - / (h n ) (tx ), which we have used in pigs previously [ ] . the ha/ na vrnas of bat :mh mn were modified using a similar strategy used to generate the mh /mn , whereby the coding regions of bat glycoproteins were replaced with tx h n , while the putative bat packaging signals were retained (mh / mn ) (fig. a) . the rescued bat :mh mn virus replicated to peak titers close to that of tx (fig. b ) and both viruses were inoculated into mice to compare the morbidity (weight loss), mortality and virus replication at various times post inoculation. all mice survived infection and both viruses (bat :mh mn and tx ) caused little effect on weight gain as compared to the mock inoculated animals (fig. c) , indicating little overall disease. titration of virus in the lung tissues showed that the bat :mh mn virus replicated as efficiently as the tx control in the mice at early time points, yet it appeared to be cleared more rapidly (fig. d) . this data suggests that some of the pathogenicity observed in the bat :mh mn infected mice likely results from the mouse adapted ha/na of pr . however, it is clear that the bat influenza internal protein vrnas do support replication of the modified viruses (bat :mh mn and bat :mh mn ) in vitro, in ovo, and in the mouse lungs. the slightly lower replication efficiency and pathogenicity of those two viruses compared to the corresponding pr and tx viruses could be ascribable to either the nature of the bat internal protein vrnas or the engineering of the modified has and nas. bat-influenza viruses appear to have diverged from iav a very long time ago and their internal protein vrnas have many unique features that are not seen in iavs [ , ] . therefore, the biological roles of the various vrna segments and their protein products are likely to have both similarities and intriguing differences. many deadly bat viruses (e.g., filoviruses) have evolved powerful molecular mechanisms that inhibit host (e.g., human) immune responses [ ] [ ] [ ] [ ] . therefore, to gain an understanding of how bat-influenza viruses may evade the host innate immune response we analyzed the bat ns protein using interferon induction experiments and carboxy-terminal truncation mutations known to attenuate iavs. the ns protein of iavs is critical for pathogenicity of many strains because of its ability to antagonize the host interferon response [ ] . to compare the direct effect of bat -ns and pr -ns on interferon-b production, we expressed the proteins ectopically in human hek- t and then infected them with sendai virus to stimulate the innate immune response. activation of interferon-b promoter was determined by a luciferase mediated reporter assay [ ] . bat -ns inhibited host interferon-b induction comparable to that of the pr -ns , and carboxy-terminal truncation of bat-ns protein (ns - and ns - , see fig. s c for diagram) decreased its ability to inhibit interferon-b production (fig. a) . these results are consistent with the attenuating effect that these ns truncations have on pr (fig. a ) and other iav ns proteins; thereby, providing a strategy to generate live attenuated influenza vaccines [ , [ ] [ ] [ ] . a vsv-luciferase virus mediated bioassay was also performed to compare the effect the ns truncations have on the bat viruses' ability to inhibit host innate immune response [ ] . the replication of the vsv-luciferase virus, which is sensitive to innate immune activation, is inversely correlated with type i interferon induced by influenza virus. truncation of the bat -ns modestly reduced vsv replication, whereas truncation of the pr -ns severely inhibited vsv replication (i.e., luciferase expression) (fig. b) . these results were confirmed by analysis of influenza virus replication kinetics in a human lung epithelial cell line (fig. c) . the bat -ns truncated viruses (bat :mh mn ss-ns - and bat :mh mn ss-ns - ) replicated to titers of - tcid /ml (near wild type ns ; bat :mh mn ss), whereas the pr -ns truncation mutants had - fold lower titers than pr (fig. c, fig. s for gene and virus diagrams). to analyze the impact of these bat ns truncation mutations in vivo we inoculated mice with the same panel of modified bat viruses, or the pr -ns - as a control. in contrast to the significant attenuation conferred by the truncated ns in pr (pr -ns - ), recombinant bat-influenza viruses with truncated ns genes (bat :mh mn ss-ns - and bat :mh m n ss-ns - ) replicated efficiently in the lungs (fig. a) , caused significant morbidity (fig. b) , and remained % lethal in mice (fig. c) . altogether the ns studies show that the bat ns protein inhibits host interferon-b production and carboxy-terminal we analyzed the bat pb gene because of its central role in the species specificity of iavs, and some of the critical residues involved are known to be virulence determinants in mice and ferrets [ ] [ ] [ ] [ ] [ ] [ ] . asparagine (n) in the pb protein is a mammalian-signature in iavs and when this residue was mutated to aspartic acid (d, an avian-signature) in the modified bat (bat- d), it decreased virus titers in lungs, morbidity (minor weight loss), and resulted in % survival (fig. ) . the batinfluenza pb also has a serine (s) residue at position , which is unlike either mammalian or avian iavs. replacing the serine with the mammalian-signature residue lysine (k) [ , ] in the context of d (bat- k/ d) increased virus replication in the lungs but only caused slightly more weight loss (compared to the bat- d virus) and it remained attenuated in mice (fig. ) . in contrast, introducing another virulence marker pb -e g [ ] into the pb -n d virus (bat- g/ d) dramatically increased the pathogenicity of the bat virus ( % mortality), which was higher than the bat virus with wild type pb (bat :mh mn , fig. ). in addition, introducing the pb -e g (bat- g) into the wild type pb resulted the most significant increase of virus replication, morbidity, and mortality ( fig. ) , indicating there is an additive effect between the two virulence determinants (pb - g and pb - n) in the bat pb . all viruses collected from mouse lungs were deep sequenced to confirm the stability of the engineered mutations and although sporadic nucleotide polymorphisms ( % - %) were detected in the viral genomes ( to such polymorphisms per mouse sample on average), none of them occurred at the engineered loci. the high genetic stability of the modified bat viruses in mice is consistent with the notion that the bat influenza viruses are mammalian viruses that have been evolving and adapting in the bats for a long period of time. to determine the molecular basis for the altered pathogenicity imparted by the various mutations in the pb we examined their effects on the viral polymerase activity in human t cells using a luciferase-mediated mini-genome replication assay (fig. ). at all temperatures tested, the pb -n d mutation decreased the polymerase activity and the pb -e g mutation enhanced the polymerase activity, consistent with the decreased and increased pathogenicity in mice, respectively (fig. ) . interestingly, the pb - s showed intermediate polymerase activity compared to the pb - k and pb - e (fig. ) . in addition, the polymerase activity of the pb - g and pb - e/k mutants decreased proportionally when they were combined with the pb - d mutation (fig. ) . this result is consistent with the observation that bat- g/ d appeared to be less pathogenic than the bat- g virus (fig. ) . collectively, the data collected on the bat pb show that amino acid residues known to be important in replication, species specificity, transmission, and/or pathogenesis of iav are important in the replication and pathogenesis of bat . internal protein-coding vrnas of bat-influenza don't efficiently reassort with iav or ibv reassortment of iavs is important in the evolution of iavs and generation of panzootic and pandemic strains. furthermore, efficient replication of bat-influenza internal protein vrnas in higher virus doses were used in this experiment based on the pr -ns - control virus, which caused significant weight loss but had low mortality at tcid so the attenuation of bat-ns truncated viruses can be appropriately compared to it. *, p, . , truncated bat :mh mn ss- and bat :mh mn ss- compared to pr -ns - . doi: . /journal.ppat. .g human cells and mice, as well as their pathogenicity, necessitated an assessment of reassortment potential between bat and other influenza viruses. replication of vrnas from different parental viruses is a factor critical in the generation of reassortant progeny. transcription/replication of mini-genome reporter constructs showed that the viral rna dependent rna polymerase (rdrp), which is a heterotrimer of pb , pb , and pa, from bat-influenza, iavs, and ibvs generally recognize and transcribe their cognate vrnas more efficiently than non-cognate vrnas (fig. s ) . intriguingly, the bat polymerase replicated the ibv reporter very efficiently (fig. s ) . additionally, most rdrp combinations (pb , pb , pa) between bat-influenza and iavs nearly abolished the polymerase activity in this very sensitive mini-genome reporter assay (fig. a-i) . interestingly, the np protein, which is a single-strand rna-binding nucleoprotein, is completely compatible between bat and iavs ( fig. a-i) , but it is incompatible between the bat-influenza and ibv (fig. j) . although some gene segment combinations showed limited polymerase activity in the mini-genome assays, we couldn't generate any reassortant viruses using reverse genetics between bat :mh mn and pr that contain partly compatible rdrp components (e.g., bat-pb /pr -pb /pr -pa), including the highly compatible np vrna/protein ( table and table ). instead, the pr -m segment could unidirectionally substitute for the bat -m segment ( table ) . this likely results from the highly conserved nature of the m vrna and proteins (m , m ). swapping the putative cis-acting packaging signals of the bat-np and known packaging signals of the pr -np, or between the bat-ns and pr -ns didn't enable rescue of viruses containing either the np or ns vrnas in a heterologous virus background ( table and see fig. s for diagrams). low efficiency of packaging at least some vrna segments from the heterologous virus is also a major restrictive factor for reassortment. for instance, a reassortant virus containing six internal protein vrnas from bat and the ha and na from pr couldn't be rescued, whereas the pr ha and na coding regions flanked by bat packaging regions (mh and mn ) can efficiently reassort with the bat internal genes ( fig. b and table ). nevertheless, pr ha and na can individually reassort ( : ) with the bat six internal protein vrnas when mn or mh were provided, respectively ( table ). the inability to rescue the : reassortant bat :pr -h n virus may result from compounding the low efficiency of packaging for each of the wild type pr -ha and pr -na vrnas into the bat-influenza backbone. the mn can also reassort with the other seven segments from pr , even when many silent substitutions (ss) were introduced into the n coding regions to disrupt the remaining pr packaging signals (table ) . actually, another modified na that contains the coding region from ibv na flanked by the putative packaging region of the bat -na (bat-n ps-flub-na) can also be rescued in the pr background, strongly suggesting that the bat-influenza na segment could be efficiently packaged into the pr virus, whereas the bat ha packaging signal didn't mediate efficient packaging of the mh into the pr backbone ( table ). while the generation of reassortants through plasmid-based reverse genetics is a powerful and sensitive way to rescue influenza viruses, it's difficult to generate every possible gene constellation and accompanying minor nucleotide variations that could give rise to progeny reassortants during co-infection. therefore, we attempted to generate reassortants between a modified bat virus and pr using a classical co-infection approach. however, when mdck cells were inoculated at a high multiplicity of infection (moi) with both pr and bat :mh mn viruses, reassortment between the two parental viruses was not detected. we plaque purified progeny viruses from the co-infection and of them were the parental pr virus and of them were the parental bat :mh mn virus. although more exhaustive classical reassortant studies are needed to completely evaluate the generation of natural reassortants between these viruses, the data indicate that pr and bat :mh mn don't efficiently reassort. recently, another bat-influenza virus a/flat-faced bat/peru/ / (h n ) (bat ) was identified in peru and phylogenetic analysis indicated this virus diverged from the batinfluenza viruses identified in guatemala (e.g., bat ) so long ago that genetic diversity between these two bat-influenza viruses is higher than that of iavs [ ] . reassortment of the pb , pb , pa, and np segments in mini-genome polymerase activity assay demonstrated that the bat and bat viruses were fully compatible (fig. k) . most importantly, successful reassortment between the two modified bat viruses (bat :mh mn ss and bat :mh mn ss) ( table and fig. s for diagrams of constructs) proved that these genetically divergent bat-influenza virus vrnas were highly interchangeable, in contrast to their very low compatibility with iav and ibv. interestingly, classical coinfection of the bat :mh mn and bat :mh mn viruses in mdck cells readily generated reassortant progeny viruses with various genotypes, and some were apparently preferentially selected (e.g., bat :bat -ns reassortant, table s ), demonstrating the merit of classic co-infection strategy in identification of gene constellations that may have certain advantages. collectively the mini-genome replication, reverse genetics reassortment, and co-infection reassortment experiments strongly suggest that two divergent bat-influenza viruses readily reassort with each other, whereas they won't reassort with canonical iavs in the natural setting. the generation of synthetic modified bat-influenza viruses (e.g., bat :mh mn ) that grow to high titers in commonly used influenza virus culture substrates and mice is an important step toward understanding these novel bat-influenza viruses. the rescue of bat :mh mn and bat :mh mn viruses demonstrates that the putative vrnas of bat function efficiently together and are probably derived from either one virus, or a group of compatible viruses, whose pb , pb , pa, np, m, and ns proteins efficiently replicate and package vrnas in host cells commonly used to culture influenza viruses (fig. ) . importantly, the data also shows that the bat-influenza ha and na were the sole determinants inhibiting bat virus rescue, and that the terminal regions of ha and na of bat-influenza viruses selected for our constructs contain cis-acting vrna packaging signals. although wild type bat-influenza virus (bat ) couldn't be propagated in the human, canine, mink, avian, porcine or bat cell lines we tested, consistent with tong et al. [ ] , it is likely that the bat-influenza virus can infect some other cell cultures from other species and/or tissues, especially cells derived from appropriate bat species. our bat :mh mn studies provide other unique insights, which can't be gleaned from non-infectious assays. for instance, non-infectious assays (interferon-b reporter assay, fig. a ) showed the bat ns carboxy-terminal truncationss (ns - and ns - ) were similar to the truncated pr ns (ns - and ns - ), which largely lost the ability to inhibit the host interferon response. however, mouse experiments with the replicative bat-influenza viruses revealed that the truncation of bat ns had minimal effects on the viral pathogenesis compared to the truncation of pr ns (fig. ) . differences in the attenuating impact observed in the pr -ns and the bat -ns truncated viruses suggests that bat has novel molecular mechanisms that have evolved in the amino terminal portion of ns and/or other internal protein vrnas to antagonize/evade the host innate immune response. the pb of iav plays important roles in replication, species specificity, transmission, and pathogenesis [ ] [ ] [ ] [ ] [ ] [ ] [ ] . our analysis of bat-influenza pb demonstrated that it is also a virulence determinant and as anticipated conversion of mammaliansignature residues at position to avian-signature (n d) attenuated the virus, and the e g substitution [ ] enhanced virulence. pb - is one of the most studied positions differentiating avian viruses (glutamic acid) and mammalian viruses (lysine) [ , ] . intriguingly, the bat-influenza pb has a serine at position , which is unlike mammalian or avian iavs. our data show that pb - s has intermediate polymerase activity compared to pb - e and pb - k in mammalian cells, suggesting an alternative evolutionary pathway that avian influenza viruses may be able to take for mammalian adaptation. reassortment of the segmented genomes of orthomyxoviruses is a powerful evolutionary mechanism that is central to the success of these pathogens. viruses within a genus readily reassort upon coinfection of a single host cell (e.g., avian and swine iav); whereas, viruses from a different genus (e.g., iav and ibv) don't reassort. the factors important for generation of reassortant progeny from two parental influenza viruses include: recognition and replication of vrnas by parental virus rdrp, protein-protein interaction/ compatibility (e.g, heterotrimeric rdrp), and vrna-protein interactions needed for virion morphogenesis. the rna transcription/replication promoter of each influenza vrna segment is formed by base pairing of highly conserved nucleotides at the and termini, which form a partially double-stranded structure. the iav genus has specific nucleotide variations within the termini that distinguish it from ibv. the termini of bat-influenza vrnas also show conserved and complementarity; however, they also have distinct nucleotide variation. therefore, we used mini-genome replication studies to analyze promoter recognition and rdrp activity of various combinations of the pb , pb , pa table . rescue efficiency of reassortants with np and ns containing modified packaging signals. table . for each combination, the rescue was repeated at least times. doi: . /journal.ppat. .t subunits in combination with various nps from iav, ibv, or batinfluenza. the data show that the wild type rdrp most efficiently replicate their cognate vrnas, and that both iav and ibv rdrp have - % reduction in activity with the bat-influenza minigenome. many pb , pb , pa combinations between bat-influenza and iav/ibv dramatically reduce activity, which demonstrates protein-protein incompatibility between the rdrp subunits. interestingly, the bat-influenza np and iav np were completely compatible in the mini-genome assay, however np reassortant viruses could not be generated ( table and table ) suggesting that the incompatibility of nps may also involve complicated protein-vrna interactions. iavs of various subtypes can infect and reassort in bat cell lines [ , ] , providing a permissive environment for them to reassort with bat-influenza viruses. however, our reassortant analysis indicates that while two divergent bat-influenzas readily reassort, bat-influenza and iavs don't easily reassort in co-infection experiments. reverse genetics reassortment studies showed the pb , pb , pa, np, and ns vrnas of bat-influenza don't efficiently reassort with the iav or ibv, and provide many additional tantalizing results. for example, reassortants were not rescued from relatively compatible rdrp combinations in the mini-genome assay (e.g. bat-pb /pr -pb /pr -pa, fig. a ) and demonstrate that divergent bat and bat can efficiently reassort with each other ( table ). the m segment is the most highly conserved gene among influenza a and b viruses. we found that the pr -m segment could substitute for the bat -m segment ( table ), indicating that the m vrnas/protein(s) of pr and bat have enough conservation in both cis-acting packaging signals and functional domains of the proteins (m /m ) to enable the replication of the modified bat virus. in contrast, putative packaging signal swapping of the np and ns segments didn't overcome reassortment defects suggesting that incompatibility at the protein-protein or protein-vrna level is likely to be a critical factor inhibiting reassortment between the bat-influenza and other influenza viruses. alternatively, one could argue that that since the vrna packaging signals of bat-influenza np and ns segments have not been delineated, the putative packaging regions incorporated in the batps-pr constructs may not be sufficient for packaging the modified vrnas. however, the well-defined pr packaging signals incorporated in our modified gene segments should be sufficient to package the corresponding bat-influenza np and ns vrnas (pr ps-bat-np and pr ps-bat-ns, fig. s d ) in the pr backbone. the failure to rescue the pr ps-bat np or ns viruses, as well as the pr :bat -m reassortant virus, strongly suggests protein-protein or protein-vrna level incompatibility and provides a unique opportunity to better understand the functional domains of these proteins through characterizing chimeric/mosaic proteins containing motifs/domains from both viruses. another caveat with our bat-influenza reassortment experiments is the focus on interactions with the laboratory adapted pr virus, which was chosen primarily due to biosafety concerns. reassortment between the bat :mh mn virus and other iavs, particularly avian viruses (e.g., h n , h n ) that appear to be more compatible in the mini-genome assay (fig. ) , are needed to fully assess reassortment potential of bat-influenza. however, based on our results from the np reassortment and the bat-pb / pr -pb /pr -pa reassortment experiments ( table and table ), the likelihood of rescuing a reassortant with rdrp components from both bat and iavs is very low. finally, since the has and nas of the bat influenza viruses can't be used to rescue viruses using contemporary influenza virus host substrates, we were not able to fully assess the ability of the ha or na to reassort with other influenza viruses (limited assessment provided in table ). however, the known bat influenza viruses (bat , bat ) could pose a pandemic threat if their ha and na acquire mutations that impart binding to canonical influenza virus receptors and rescuing the na for neuraminidase activity, or acquisition of binding and entry through alternative human cell surface receptors. collectively, our experiments suggest that the bat-influenza virus is unlikely to reassort with an iav or ibv and spread to other species even if they were to infect the same host cell. the restriction on reassortment appears to result from multiple levels of incompatibility (rna-rna, rna-protein, and/or protein-protein) that are either additive or synergistic. consequently, our data suggest that due to the extremely limited ability of genetic information exchange between bat-influenza and iav or ibv, the international committee on taxonomy of viruses could consider classifying these two bat-influenza virus lineages as a new genus or species within the orthomyxoviridae. this study also demonstrated the power of synthetic genomics in rapid characterization and risk assessment of an emerging virus, table . rescue efficiency of reassortants between bat :mh mn ss and bat :mh mh ss. h ps-h ss n ps-n ss h ps-h ss n ps-n ss h ps-h ss n ps-n ss h ps-h ss n ps-n ss h ps-h ss n ps-n ss h ps-h ss n ps-n ss h ps-h ss n ps-n ss h ps-h ss n ps-n ss h ps-h ss n ps-n ss h ps-h ss n ps-n ss h ps-h ss n ps-n ss h ps-h ss n ps-n ss h ps-h ss n ps-n ss h ps-h ss n ps-n ss h ps-h ss n ps-n ss h ps-h ss n ps-n ss h ps-h ss n ps-n ss h ps-h ss n ps-n ss h ps-h ss n ps-n ss h ps-h ss n ps-n ss +++ *rescue efficiency definition described in table . doi: . /journal.ppat. .t even when the virus itself is not readily cultured. the synthetic genomics/reverse genetics strategy employed provides an infinite supply of wild type bat-influenza particles that can be used to identify permissive cells or animals. the availability of our modified bat-influenza virus, opens many other avenues of investigation and discovery, including, for instance, to gain a better understanding of cis-acting signals in the vrnas that are important in bat-influenza transcription, replication, packaging/ particle morphogenesis, and to use forward genetics to elucidate viral protein-protein and/or viral protein-host protein interactions. finally, continued study of bat-influenza viruses and integration of data from other contemporary influenza viruses is important in the elucidation of the evolutionary history of influenza viruses. the study was reviewed and approved by the institutional biosafety committee at kansas state university (protocol # ), and by the institutional biosafety committee at the j. craig venter institute (protocol # ). we conducted the initial studies using pr gene fragments to generate the modified bat-influenza viruses and to test the reassortment potential because pr is a widely used lab/mouse adapted bsl virus that poses very low risk to humans or livestock. subsequently, tx h n genes were used in a few experiments because this is a bsl swine virus, which we have used previously and the viruses generated were considered low risk. the animal studies were performed in strict accordance with the recommendations in the guide for the care and use of laboratory animals of the national institutes of health. the animal protocol (protocol # ) was reviewed and approved by the institutional animal care and use committee at kansas state university. all animal studies were performed in a biosafety level facility located at the biosecurity research institute at kansas state university under the approved protocol # following the american veterinary medicine association guidelines on euthanasia. for virus inoculation, each mouse was anesthetized by inhaling % isoflurane. mice were euthanized if more than % of weight was lost after virus inoculation. euthanasia of mice was conducted by inhaling % isoflurane followed by cardiac puncture and cervical dislocation. no survival surgery was performed, and all efforts were made to minimize suffering. human embryonic kidney t (hek- t) cells, mouse rectum epithelial carcinoma (cmt- ) cells, and african green monkey kidney (vero) cells were maintained in dulbecco's modified eagle's medium (dmem) supplemented with % fetal bovine serum (fbs). madin-darby canine kidney (mdck) cells were maintained in minimum essential medium (mem) supplemented with % fbs. human lung epithelial (a ) cells, bat lung epithelial (tb lu) cells, mink lung epithelial (mv lu) cells and swine testis (st) cells were maintained in mem supplemented with % fbs. human lung epithelial (calu- ) cells were maintained in mem supplemented with % fbs, % nonessential amino acids, and mm sodium pyruvate. nucleotide sequences of the eight gene segments of a/little yellow-shouldered bat/guatemala/ / (h n ) (bat ) were retrieved from the genbank database. a total of oligonucleotides of - bases in length were designed for enzymatic assembly of the eight segments. the assembly and error correction processes were performed as recently described [ , ] , modified with increased time at all extension steps (from uc for min to uc for min) for efficient assembly of the polymerase segments. the synthesized segments (fig. s ) were cloned into the modified bidirectional influenza reverse genetics vectors pbz a [ ] using the recombination-based method [ ] and transformed into stella competent e. coli cells (clontech). colonies were selected and sequenced. the appropriate clones for each segment were propagated for plasmid preparation and verified by sequencing. the resulting plasmids are pbz a (pb ), pbz a (pb ), pbz a (pa), pbz a (ha), pbz a (np), pbz a (na), pbz a (m) and pbz a (ns). the whole process only took seven days to complete. the plasmids containing bat pb mutations were constructed by site-directed mutagenesis using the pbz a as template. the ns truncation constructs were generated by gibson assembly and details of the truncations are diagramed in fig. s c . the modified (m) bat ha and na (mh , mn , mh ss, and mn ss, see fig. s a , s b for diagrams, and fig. s for sequence alignment) were synthetized by gibson assembly from oligonucleotides. silent substitutions (ss) were introduced to disrupt the putative packaging signals in the pr ha and na terminal coding regions. the mh ss and mn ss are thus more appropriate than the mh and mn to assess the ha and na packaging signal compatibility between bat and pr . the batps-pr -np, pr ps-bat-np, batps-pr -ns, and pr ps-bat-np constructs were constructed similarly and diagramed in fig. s d . as a comparison of the speed of different synthesis strategies, the eight gene segments of a/flat-faced bat/peru/ / (h n ) (bat ) were synthesized by genewiz (nj, usa) in the vector plasmid of puc based on the genbank database and subcloned into phw vector. the resulting plasmids (phw-h -pb , phw-h -pb , phw-h -pa, phw-h -ha, phw-h -np, phw-h -na, phw-h -m and phw-h -ns) were confirmed by sequencing. the whole process took more than one month. the pb , pb , pa and np genes were also subcloned into the pdz vector for use in the mini-genome assay. diagrams of the mutant or modified genes of bat and bat are described in fig. s . the ppol -ns-luc reporters used in the mini-genome polymerase activity assay were described in fig. s e . sequences of all constructs used in this study were confirmed to ensure absence of unwanted mutations and the genbank accession numbers are km -km . briefly, . mg of plasmid for each gene segment was mixed and incubated with ml of mirus tranit-lt (mirus bio, madison, wi) at uc for min. the transfection mixture was transferred to % confluent t/mdck cell monolayers in a -mm tissue culture dish and incubated at uc with % co for h. the transfection supernatant was replaced with ml of opti-mem i medium (life technologies) supplemented with . % bovine serum albumin (bsa) fraction v (life technologies), mg/ml tosylsulfonyl phenylalanyl chloromethyl ketone (tpck)-trypsin (worthington, lakewood, nj), and % antibiotic-antimycotic (life technologies). three days post-transfection, culture supernatant (passage , p ) was collected and . ml of that was inoculated into mdck cells in -well plates at uc. supernatant (p ) was collected at days post-inoculation (dpi), or when severe cytopathic effect (cpe) was observed. the p supernatant was further passaged blindly for two passage before determined to be negative for rescue. titers of the viruses used in this study were determined by tcid assay in mdck cells. rescue efficiency definition. very easy (++++): p viral titer - tcid /ml, or severe cpe observed in p within dpi; moderate (+++): p titer - tcid /ml, or obvious cpe observed in p within dpi; difficult (++): p titer - tcid /ml, or weak cpe observed in p within dpi; very difficult (+): p titer lower than tcid /ml, or cpe not observed until p /p ; negative (neg): rescue failed, no cpe observed through passage . various transfection conditions including different transfection reagents, temperatures, and incubation time before supernatant collection were attempted to rescue the wild type bat virus and the reassortants between bat and pr . however, none of them generated any positive rescue results if they were negative under standard rescue condition described above. bat transfection supernatants were also transferred to various cells (mdck, mink lung mv -lu, swine testis, vero, a cells, calu- , bat lung epithelial tb lu) and embryonated chicken eggs and passaged at least three times. the real-time rt-pcr assays targeting bat and pr m genes were used to confirm negative results (primers and probes are possible upon request). to determine whether virus particles of bat and other viruses can be produced by reverse genetics system, a total of thirty-five ml of transfected t cell supernatants for each virus were collected at hours post transfection and centrifuged at rpm for minutes to remove the cell debris. then the clear supernatant was loaded on % (w/v) sucrose in centrifuge tubes and was concentrated at , rpm (optima le- k ultracentrifuge, beckman coulter) for hours. the virus pellets was dissolved in ml of water and the viral particles were fixed by incubating with . % paraformaldehyde at uc for hours. the fixed particles were dipped on a mesh copper grid and the grid was dried and stained with negative staining before observation under an electron microscope. mdck monolayers in -well plates were washed twice with pbs, and then ml of virus growth medium (vgm) was added to each well. the cells were inoculated at a multiplicity of infection (moi) of . tcid /cell with the bat :mh mn virus or pr virus (bat :mh mn virus or tx virus) and incubated at uc. supernatants were collected at , , and days post inoculation (dpi). inoculations of calu- cells were performed similarly, except that an moi of . tcid /cell was used for the following viruses: bat :mh mn ss, bat :mh mn ss-ns - , bat :mh mn ss-ns - , pr , pr -ns - , and pr -ns - . the vgm used for mdck cells was emem supplemented with . % bsa fraction v, mg/ml tpcktrypsin, and % antibiotic-antimycotic, and the vgm used for calu- cells was emem supplemented with . % bsa fraction v, mg/ml tpck-trypsin, and % antibiotic-antimycotic. all virus titers were determined by tcid assay using mdck cells. six of -day-old embryonated chicken eggs were inoculated with bat :mh mn or pr at tcid /egg. after days incubation at uc, allantoic fluid was collected from each egg and titrated individually. the eggs with the highest titers in each virus group was used to calculate the average titer and generate the graph in fig. e . a modified multi-segment rt-pcr [ , ] was used to amplify influenza-specific segments. the only modification to the procedure was the primers used for amplification were changed to match bat influenza termini. the oligonucleotide primers used were uni /inf- g ( -ggggggagcagaagcagg- ) and uni /inf- ( -cgggttattagtagaaacaagg- ). the m-rtpcr amplicons were used for illumina miseq library construction via nextera dna sample prep kit (illumina, inc.) and sequenced using the illumina miseq (illumina, inc.) according to manufacturer's instructions. snp variations were identified using custom software that applies statistical tests to minimize false positive snp calls that could be caused by the types of sequencespecific errors that may occur in illumina reads identified and described in nakamura, et al. [ ] . to overcome this problem, the protocol requires observing the same snp, at a statistically significant level, in both sequencing directions. once a minimum minor allele frequency threshold and significance level are established by the user, the number of minor allele observations and major allele observations in each direction and the minimum minor allele frequency threshold are used to calculate a p-value based on the binomial distribution cumulative probability, and if the p-values calculated in each of the two sequencing directions are both less than the bonferroni-corrected significance level, then the snp call is accepted. for our analyses, we used a significance level of . (bonferroni-corrected for tests in each direction to . ), and a minimum minor allele frequency threshold of % of the read population. to measure the ifn-antagonist function of ns , a luciferasebased, sendai virus-mediated ifn-b promoter activation assay was conducted as previously described [ ] . briefly, t cells in well plates were transfected with empty vector ( ng) or increasing amounts of wild type (wt) or carboxyl terminal truncated ns from bat and pr ( ng, ng, and ng of ns expression plasmids supplemented with ng, ng, and ng of empty vector, respectively). also co-transfected were ng of an ifn-b-promoter-luciferase reporter plasmid (pifnb-luc) and ng of a plasmid constitutively expressing renilla luciferase (prl-tk from promega). at hours post transfection, cells were infected with sendai virus to induce the ifn-b promoter. a dual-luciferase assay was performed at hour post virus inoculation, and firefly luciferase was normalized to renilla luciferase activity. the relative luciferase activity of the group with empty vector was set as %, and the other groups were presented relative to that. as previously described for the vsv-gfp virus mediated interferon bioassay [ ] , in the vsv-luciferase virus mediated bioassay, a cells were inoculated with one of the wild type or ns truncated viruses at an moi of tcid /cell, or were mock-inoculated; supernatants were then collected at hpi. supernatants were treated with uv irradiation to inactivate viruses and were then transferred to naïve a cells. following h of incubation at uc, supernatants were removed, and the cells were inoculated with vsv-luciferase virus [ ] , at an moi of tcid /cell. the firefly luciferase expression in the cells was measured using the luciferase assay system (promega) at hpi with vsv-luciferase. the luciferase-mediated mini-genome polymerase activity assay was performed as previously described, using a poli-driven reporter plasmid and pdz-based pb , pb , pa, and np bidirectional expression plasmids [ , ] . to determine the effects of pb mutations on polymerase activity (fig. ) t cells were co-transfected with . mg each of the pb (wt or mutant), pb , pa, np, and a ppol -flua-ns-luc (firefly luciferase flanked by a/ new york/ / [ ] ). as a control for transfection efficiency, . mg of the renilla luciferase plasmid prl-tk (promega) was also co-transfected. after hours of incubation at uc, uc, and uc, luciferase production was assayed using the dual-luciferase reporter assay system (promega) according to the manufacturer's instructions. firefly luciferase expression was normalized to renilla luciferase expression (relative activity). the relative activity of the pb -wt was set as fold, and the relative activities of the pb mutants were presented relative to that (fig. ) . to test the compatibility between rnps (pb , pb , pa, and np) and viral rna promoters from bat-influenza virus (bat ) (fig. s ) , iav (a/pr/ / ), and ibv (b/russia/ ), t cells were co-transfected with . mg each of the pb , pb , pa, np, and a ppol -ns-luc reporter plasmid, followed by incubation at uc for hours. three reporters were used in this study, including ppoli-bat-ns-luc (firefly luciferase flanked by bat ns non-coding regions), ppol -flua-ns-luc, and ppoli-flub-ns-luc (firefly luciferase flanked by b/russia/ ns non-coding regions) (fig. s d) . for each combination of rnp and ppoli-ns-luc reporter (from bat , a, or b type), three independent replicates were conducted. for each rnp, the luciferase activity with the reporter from the same virus (e.g., bat-rnp and ppol -bat-ns-luc) was set at %, and the activities with the other two reporters (e.g., ppol -flua-ns-luc and ppol -flub-ns-luc) were presented relative to that (fig. s ) . the pb , pb , pa, and np compatibility between bat and the following influenza viruses was examined in the study (fig. ) : (fig. k) , . mg each of the pb , pb , pa, np (from bat or bat ), and ppoli-bat-ns-luc plasmids were used (the ns non-coding regions of bat and bat have the same sequence). renilla luciferase was also co-transfected and dualluciferase reporter assay system was used. for each combination of pb , pb , pa, and np (from bat or another influenza virus), three independent replicates were conducted at uc, the luciferase activity of the all-bat -combination (bat -pb / bat -pb /bat -pa/bat -np) was set at %, and the activities of other combinations were presented relative to that. pathogenicity of pr , modified bat-influenza virus (bat :mh mn ) and pb mutants a total of female balb/c mice aged to weeks were randomly allocated to groups ( mice/group). six mice were intranasally inoculated with tcid of each virus (bat :mh mn , bat :mh mn -pb - d, bat :mh mn -pb - k d, bat :mh mn -pb - g d, bat :mh mn -pb - g, pr , or mem mock) in ml fresh mem medium while under light anesthesia by inhalation of % isoflurane. to determine the virus replication in mouse lungs, three mice from each group were euthanized on both and day postinoculation (dpi). another mice from each group were intranasally inoculated with tcid of viruses in ml mem medium; all eight mice were kept to monitor body weights and clinical signs. weights were recorded daily and general health status was observed twice daily. after the onset of disease, the general health status was observed three times daily. severely affected mice (i.e., more than % body weight loss) were euthanized immediately, and the remaining mice were euthanized on dpi. all control mice were intranasally inoculated with ml fresh mem (mock group), three control mice were necropsied at and dpi, the remaining mice were kept until the end of the animal study. during necropsy, the right part of the lung was frozen at uc for virus titration, and the left part of the lung was fixed in % formalin for histopathologic examination. for virus titration, the % lung homogenate was prepared in cold fresh mem medium by using a mini bead beater- (biospec products; bartlesville, ok). the homogenate was centrifuged at rpm for minutes, and the supernatant was titrated by infecting mdck cells in -well plates. for the histopathologic examination, lung tissues fixed in % phosphate-buffered formalin were processed routinely and stained with hematoxylin and eosin. the lungs were examined microscopically both for the percentage of the lung involved and for the histopathologic changes seen, including bronchiolar and alveolar epithelial necrosis, intraalveolar neutrophilic inflammation, peribronchiolar inflammation, and bronchiolar epithelial hyperplasia and atypia. for detection of virus np antigens in lung sections on day post infection, a rabbit anti-h n ( flu pandemic) np polyclonal antibody was used (genscript, usa). a pathologist examined each slide in a blinded fashion. pathogenicity of modified bat-influenza viruses (bat :mh mn ss) containing truncated ns genes a total of female balb/c mice aged to weeks were randomly allocated to groups ( mice/group). to determine virus replication, six mice were intranasally inoculated with tcid of each virus (bat :mh mn ss-ns -wt, bat :mh n ss-ns - , bat :mh mn ss-ns - , and pr -ns - ) in ml mem medium while under light anesthesia by inhalation of % isoflurane. three mice from each group were killed on both and day post-inoculation (dpi). another mice from each group were intranasally inoculated with tcid of each virus in ml mem medium for morbidity and mortality comparison. all the other procedures are same with described previously. a total of female balb/c mice aged to weeks were randomly allocated to groups ( mice/group). to investigate virus replication in mice, six mice from each group were intranasally inoculated with tcid of virus or mockinoculated with ml fresh mem medium while under light anesthesia by inhalation of % isoflurane. three of six inoculated mice from each group were euthanized at and day postinoculation (dpi). to evaluate viral pathogenicity in mice, the remaining eight mice from each group were intranasally inoculated with tcid of virus (bat :mh mn , and tx ) in ml fresh mem medium or mock-inoculated with ml fresh mem medium. the mice were monitored body weights and general health status daily. after the onset of disease, the general health status was observed twice per day. severely affected mice (i.e., more than % body weight loss) were humanly euthanized, and the remaining mice were euthanized and bloods were collected from each mouse to isolate serum for the hi assay at dpi. sample collection and analysis, and virus titration were performed as described above. to study the reassortment between bat :mh mn and pr or bat :mh mn , confluent monolayer of mdck cells in -well-plates were co-infected with both viruses (bat :mh mn and pr , or bat :mh mn and bat :mh mn ). both modified bat :mh mn and bat :mh mn viruses showed similar replication kinetics in mdck cells, whereas the pr replicated more efficiently than both modified viruses in mdck cells. therefore, for the co-infection study with pr and bat :mh mn viruses, the cells were infected with the pr at moi of and with the bat :mh mn at moi of (a ratio of both viruses is : ). for the co-infection study with bat :mh mn and bat :mh mn viruses, the cells were infected with each virus at moi of (a ratio of both viruses is : ). the co-infected mdck cells were incubated at uc with % co for hour. after hour of incubation, the supernatant was removed and the infected cells were washed with fresh mem for times. one ml of infection medium supplemented with mg/ml tpck-trypsin (worthington, lakewood, nj) was added on cells. the supernatant containing progeny viruses was collected at hours after inoculation. plaque assays were performed in mdck cells to select single virus from co-infected supernatants. the purified single virus (plaque) was amplified for further analysis. to identify the origin of each gene of the purified single virus, specific rt-pcr was used to differentiate internal genes from bat :mh mn , bat :mh mn and pr viruses (primers for specific rt-pcr are available upon request). the surface ha and na genes were differentiated by sequencing ha and na noncoding regions (packaging signals) since three parental viruses contain identical ha and na orf sequences and different sequences in non-coding region (it is difficult to differentiate them by rt-pcr). for the rt-pcr, rnas were extracted from each amplified single virus using a qiaamp viral rna mini kit (qiagen). cdna was synthesized by using the bat universal primer ( -agcagaagcagg- ) for the samples from the coinfection study with bat :mh mn and bat :mh mn viruses, and by using a mixture of an iav universal primer ( -agcraaagcagg- ) and the bat universal primer ( -agcagaagcagg- ) for the samples from the co-infection study with bat :mh mn and pr viruses. if the origin of internal genes determined by the specific rt-pcr was inconclusive, sequencing was performed to confirm the results from specific rt-pcr (all sequence primers are available upon request). luciferase activity, virus titers, and mouse weights were analyzed by using analysis of variance (anova) in graphpad prism version . (graphpad software inc, ca). one-way anova with dunnett's multiple comparison test was used to determine the significance of the differences (p, . ) among different groups. for simple comparisons, student's t test was used to examine the significance of differences observed. error bars represent standard deviation ( sd). figure s synthetic generation of the eight full-length genomic segments of a/little yellow-shouldered bat/ guatemala/ / (bat ). the products were assembled from oligonucleotides and error corrected. l: kb plus dna ladder from life technologies. (tif) figure s diagrams of select constructs and viruses used in this study. all constructs shown are in cdna sense complementary to viral rna. (a) modified ha (mh ) and modified na (mn ). to construct the mh , pr -ha coding region was flanked by the putative packaging regions from bat -ha and all atg in the bat -ha packaging region were mutated. to construct the mn , pr -na coding region was flanked by the putative packaging regions from bat -na and all atg in the bat -na packaging region were mutated. batps-b/na was constructed similarly with the packaging regions from bat -na and the coding region from b/russia/ -na. (b) mh ss was constructed by introducing of silent substitutions into the coding region of mh to disrupt the remaining packaging signals in the pr -ha coding region. mn ss was constructed by introducing of silent substitutions into the coding region of mn to disrupt the remaining packaging signals in the pr -na coding region. the mh ss was referred as h ps-h ss and the mn ss was referred as n ps-n ss in table bats: important reservoir hosts of emerging viruses isolation and characterization of a bat sars-like coronavirus that uses the ace receptor a distinct lineage of influenza a virus from bats new world bats harbor diverse influenza a viruses bat-derived influenza-like viruses h n and h n structural and functional characterization of neuraminidase-like molecule n derived from bat influenza a virus crystal structures of two subtype n neuraminidase-like proteins from bat influenza a viruses reveal a diverged putative active site hemagglutinin homologue from h n bat influenza virus exhibits divergent receptorbinding and ph-dependent fusion activities bat-derived influenza hemagglutinin h does not bind canonical avian or human receptors and most likely uses a unique entry mechanism the n-terminal domain of pa from bat-derived influenza-like virus h n has endonuclease activity the neuraminidase of bat influenza viruses is not a neuraminidase exploitation of nucleic acid packaging signals to generate a novel influenza virus-based vector stably expressing two foreign genes rewiring the rnas of influenza virus to prevent reassortment mutations in the ns protein of swine influenza virus impair anti-interferon activity and confer attenuation in pigs structural basis for marburg virus vp -mediated immune evasion mechanisms structural basis for dsrna recognition and interferon antagonism by ebola vp marburg virus evades interferon responses by a mechanism distinct from ebola virus ebola virus vp proteins inhibit the interaction of npi- subfamily karyopherin alpha proteins with activated stat the multifunctional ns protein of influenza a viruses influenza a and b viruses expressing altered ns proteins: a vaccine approach attenuation of equine influenza viruses through truncations of the ns protein ns-based live attenuated h n pandemic vaccines protect mice and ferrets pb residue is a pathogenic determinant of pandemic h n and h influenza a viruses in mice a single amino acid in the pb gene of influenza a virus is a determinant of host range identification of amino acids in ha and pb critical for the transmission of h n avian influenza viruses in a mammalian host molecular basis of replication of duck h n influenza viruses in a mammalian mouse model molecular basis for high virulence of hong kong h n influenza a viruses the viral polymerase mediates adaptation of an avian influenza virus to a mammalian host asparagine substitution at pb residue enhances the replication, pathogenicity, and transmission of the pandemic h n influenza a virus bat cells from pteropus alecto are susceptible to influenza a virus infection and reassortment differential sensitivity of bat cells to infection by enveloped rna viruses: coronaviruses, paramyxoviruses, filoviruses, and influenza viruses synthetic generation of influenza vaccine viruses for rapid response to pandemics analysis of recombinant h n wild-type and mutant viruses in pigs shows that the q l mutation in ha is important for transmission universal influenza b virus genomic amplification facilitates sequencing, diagnostics, and reverse genetics singlereaction genomic amplification accelerates sequencing and vaccine production for classical and swine origin human influenza a viruses influenza a virus molecular virology techniques sequence-specific error profile of illumina sequencers endosomal proteolysis of the ebola virus glycoprotein is necessary for infection we would like to thank hui he, haixia liu this manuscript was prepared while d. e. wentworth was employed at the j. craig venter institute. the opinions expressed in this article are the author's own and do not reflect the view of the centers for disease control and prevention, the department of health and human services, or the united states government. key: cord- -m y ozx authors: guo, fang; zhao, qiong; sheraz, muhammad; cheng, junjun; qi, yonghe; su, qing; cuconati, andrea; wei, lai; du, yanming; li, wenhui; chang, jinhong; guo, ju-tao title: hbv core protein allosteric modulators differentially alter cccdna biosynthesis from de novo infection and intracellular amplification pathways date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: m y ozx hepatitis b virus (hbv) core protein assembles viral pre-genomic (pg) rna and dna polymerase into nucleocapsids for reverse transcriptional dna replication to take place. several chemotypes of small molecules, including heteroaryldihydropyrimidines (haps) and sulfamoylbenzamides (sbas), have been discovered to allosterically modulate core protein structure and consequentially alter the kinetics and pathway of core protein assembly, resulting in formation of irregularly-shaped core protein aggregates or “empty” capsids devoid of pre-genomic rna and viral dna polymerase. interestingly, in addition to inhibiting nucleocapsid assembly and subsequent viral genome replication, we have now demonstrated that haps and sbas differentially modulate the biosynthesis of covalently closed circular (ccc) dna from de novo infection and intracellular amplification pathways by inducing disassembly of nucleocapsids derived from virions as well as double-stranded dna-containing progeny nucleocapsids in the cytoplasm. specifically, the mistimed cuing of nucleocapsid uncoating prevents cccdna formation during de novo infection of hepatocytes, while transiently accelerating cccdna synthesis from cytoplasmic progeny nucleocapsids. our studies indicate that elongation of positive-stranded dna induces structural changes of nucleocapsids, which confers ability of mature nucleocapsids to bind cpams and triggers its disassembly. understanding the molecular mechanism underlying the dual effects of the core protein allosteric modulators on nucleocapsid assembly and disassembly will facilitate the discovery of novel core protein-targeting antiviral agents that can more efficiently suppress cccdna synthesis and cure chronic hepatitis b. hepatitis b virus (hbv) is a small dna virus that chronically infects million people worldwide and causes approximately , deaths annually due to various severe liver diseases, including cirrhosis, hepatocellular carcinoma (hcc) and liver failure [ ] . currently approved direct-acting antiviral agents against hbv are six nucleos(t)ide analogues that inhibit viral dna polymerase with varying potency and barriers to drug resistance [ ] . although those viral dna polymerase inhibitors significantly reduce viral load and prevent liver disease progression, they rarely cure hbv infection due to their inability to eradicate cccdna [ ] . hbv core protein is a small polypeptide of amino acid residues. it exists in infected hepatocytes as several distinct quaternary structures and plays multiple roles in the viral replication cycle [ ] . the best characterized function of core protein is the assembly of pre-genomic (pg) rna and viral dna polymerase complex into nucleocapsids where hbv dna synthesis takes place [ ] . moreover, temporally and spatially regulated disassembly (or uncoating) of nucleocapsids is essential for delivery of viral relaxed circular (rc) genomic dna into the nuclei of infected hepatocytes [ , ] , where it is converted to covalently closed circular (ccc) dna [ , ] . furthermore, it has also been suggested that core proteins may associate with cccdna minichromosomes, in an as-yet undefined structural manner, to regulate its transcription [ ] . interestingly, it was also reported that core protein can be hijacked by host immune responses to recruit cytokine-induced dna cytosine deaminase apobec a to cccdna minichromosomes, which results in cytosine deamination and decay of cccdna [ , ] . due to their unique structures and essential roles in viral replication, disruption of, or interference with, nucleocapsid assembly and/or disassembly with small molecular core protein allosteric modulators (cpams) represents a new frontier in development of novel antiviral agents against hbv [ , ] . over the last two decades, at least five chemotypes of cpams have been reported [ ] . those compounds bind to a hydrophobic pocket, designated as the hap pocket, at the dimer-dimer interface near the c-termini of core protein subunits [ , ] . binding of these molecules in the hap pocket induces large scale allosteric conformational changes in core protein subunits and alters the capsid assembly kinetics and pathways [ , ] . while heteroaryldihydropyrimidines (haps), such as bay - and gls , misdirect capsid assembly to form non-capsid polymers of core proteins [ , ] , all other chemotypes of cpams, including sulfamoylbenzamides (sbas) and phenylpropenamides (ppas), represented by enan- and at- , respectively (s fig), induce the formation of morphologically "normal" empty capsids with distinct quaternary and/or tertiary structural changes and thus, preclude viral dna replication [ , ] . thus far, several haps and sbas have been shown to inhibit hbv replication in animal models and are currently under preclinical or clinical development [ , ] . inspired by the observation that a small molecule compound targeting the capsid protein of dengue virus has dual effects on both the assembly and disassembly (or uncoating) of the viral capsids [ ] , we hypothesized that hbv cpams may not only disrupt capsid assembly, but also alter the structure and function of assembled nucleocapsids and consequentially affect viral dna replication and/or cccdna synthesis. indeed, we have now obtained evidence showing that haps and sbas, but not ppas, induce disassembly of nucleocapsids from virions as well as double-stranded dna-containing cytoplasmic progeny nucleocapsids and consequentially interfere with cccdna biosynthesis from de novo infection and intracellular amplification pathways. discovery of human sodium taurocholate cotransporting polypeptide (hntcp) as the bona fide receptor for hbv infection of hepatocytes allows for establishment of convenient hepg derived hbv infection cell culture systems [ , ] . we have thus established a novel ntcpexpressing human cell line, designated as c a hntcp , and demonstrated its susceptibility to hbv infection. the parental cell line, c a, is a subclone of hepg cells that exhibits strong contact inhibition of growth and metabolic features that are more similar to normal hepatocytes [ ] . as shown in s fig, cccdna became detectable as early as day and reached maximum levels at day post infection by qpcr and conventional southern blot assays. hbv pgrna and core-associated viral dna replication intermediates as well as core protein also accumulated sequentially in infected cultures. hbsag was readily detectable by elisa as a product of hbv infection in the culture media. in order to investigate the effects of cpams on hbv infection, particularly cccdna synthesis from a de novo infection, c a hntcp cells were mock-treated or treated with representative hap (bay - , gls ) or sba (enan- ) and control compounds entecavir (etv) or myrcludex b (myrb), starting at h before hbv infection until harvesting at day and day post infection. as expected (fig ) , myrb, an acylated peptide derived from the hbv large envelope protein blocks virus entry [ ] , inhibited cccdna formation and consequential accumulation of pgrna and core dna. also as anticipated, etv, an hbv dna polymerase inhibitor, did not affect the synthesis of cccdna and accumulation of pgrna, but inhibited the synthesis of core dna [ ] . interestingly, gls , bay - and enan- significantly reduced the amounts of cccdna. viral pgrna and core dna were also proportionally reduced. a more detailed time course study spanning the first four days post infection revealed that the three cpams significantly inhibited cccdna formation, whereas etv and ifn-α did not (s fig) (fig d, lanes and ) . however, while etv did not reduce viral rna, ifn-α reduced the levels of viral pgrna and . / . kb mrna, presumably due to suppression of cccdna transcription [ , ] . to further characterize the inhibitory effect of cpams on cccdna biosynthesis, time-ofaddition and dose response experiments were performed. in agreement with its mode of action, myrb treatment starting at h before or at the time of infection efficiently blocked cccdna formation, whereas delayed treatment starting at h post infection completely failed to inhibit cccdna formation (fig a) . consistent with the kinetics of cccdna formation in this cell culture system (s fig), while cpam treatment starting at h before or at the time of infection reduced cccdna formation at similar efficiency, their inhibitory effects were significantly reduced when the treatment started at h post infection. moreover, all the three cpams inhibited cccdna formation in a concentration dependent manner (fig b) . those results are in agreement with a report published during the preparation of this manuscript that bay - and sba derivative jnj- inhibited hbv cccdna synthesis during hbv infection of human primary hepatocytes [ ] . to investigate the possibility that the observed inhibition of cpams on cccdna formation is due to inhibition of ntcp-mediated hbv entry, we examined their effects on hepatitis d virus (hdv) infection of c a hntcp cells. the results demonstrated that while myrb efficiently and cytoplasmic core dna (c) were quantified by real-time pcr assays. differences in viral cccdna, core dna or pgrna between mock-treated control and treated groups were statistically analyzed (t-test, * p < . , ** p < . , *** p < . ). (d) hybridization analyses of hbv replication intermediates in cells harvested at dpi. upper panel, hirt dna extracted from the cells harvested at dpi were denatured at ˚c for min to denature dp-rcdna to single-stranded dna and followed by restriction with ecori to convert cccdna into unit-length double stranded linear dna and detected by southern blot hybridization (labeled as ccc/ecori). unit-length hbv linear dna served as a molecular weight marker. lower panel, hbv rnas, pre-genomic rna (pgrna), . and . kb mrna specifying envelope proteins were determined by northern blot hybridization. s and s ribosomal rna (rrna) served as loading controls. https://doi.org/ . /journal.ppat. .g reduced genomic and antigenomic hdv rna by more than log, etv and ifn-α as well as bay- - and enan- did not apparently alter the amounts of hdv rna in the infected cultures (s fig). the results thus imply that inhibition of cccdna synthesis by the cpams is due to their interaction with hbv-specific components, most possibly nucleocapsids, but not the host cellular factor(s) in the entry pathway shared by hbv and hdv. cpams accelerate intracellular cccdna synthesis from cytoplasmic progeny nucleocapsid rcdna besides being synthesized from incoming virion dna in the de novo infection, cccdna in infected hepatocytes can also be amplified by delivery of rcdna from cytoplasmic progeny nucleocapsids into the nucleus and conversion to cccdna [ ] [ ] [ ] . this intracellular cccdna amplification pathway has been demonstrated to function in cultured cells and in vivo in duck hepatitis b virus (dhbv)-infected ducks, and is regulated by the large envelope protein and host cellular factors [ , ] . because cpams inhibit pgrna encapsidation and thus preclude viral dna replication and intracellular amplification of cccdna, their effects on intracellular progeny nucleocapsids and cccdna amplification cannot be investigated in cells directly treated with those compounds [ ] . to circumvent this problem, as depicted in fig a, we first arrested hbv dna replication by culturing hepad cells in medium without tetracycline, but containing the reversible viral dna polymerase inhibitor foscarnet (trisodium phosphonoformate, or pfa for short) for four days, which arrested hbv dna replication predominantly at the stages of incomplete and complete minus-strand dna (fig b) [ ] . the cells were then cultured in the presence of tetracycline to stop hbv pgrna transcription from integrated transgene in cellular chromosome, and also in the absence of pfa to allow viral dna replication and cccdna synthesis to resume. at the time of pfa withdrawal, mock treatment or treatment with etv, bay- - , enan- was initiated and cells were harvested at the indicated time points for analyses of hbv core dna and cccdna. synthesis and identity of cccdna in this cell culture system were confirmed by its resistance to heat denaturing and conversion into . kb unit-length dna after heat denaturing and ecori digestion (s fig). as shown in fig b, upon removal of pfa from culture medium, the incomplete and complete minus strand, partial double-strand and complete double strand hbv dna species (including rcdna) sequentially increased from to h (fig b) . deproteinized rcdna (dp-rcdna) and cccdna became readily detectable at and h, respectively ( fig c) . as expected, etv treatment inhibited the elongation of arrested hbv dna species and prevented the production of rcdna as well as dp-rcdna and cccdna. interestingly, although bay- - or enan- treatment did not inhibit elongation of minus-strand and partial double-strand dna in the cytoplasmic nucleocapsids, the rcdna was not accumulated in those cells ( fig b) . however, analysis of hirt dna indicated that the amount of dp-rcdna was not apparently reduced by cpam treatment and to our surprise, the cccdna was readily detected as early as h after the removal of pfa in cpam-treated cells. experiments with additional cpams and extended treatment duration further supported the notion that the selected hap and sba compounds reduced the accumulation of cytoplasmic rcdna-containing nucleocapsids (s fig cpams do not inhibit completion of positive strand dna synthesis, but confer dnase i sensitivity of mature forms of viral dna the apparent contradicting effects of cpams on cytoplasmic rcdna-containing nucleocapsid accumulation and kinetics of cccdna synthesis in hepad cells prompted us to investigate whether those compounds inhibited the completion of rcdna synthesis and/or destabilized the nucleocapsids containing mature forms of double-stranded dna. this latter possibility may explain the observed acceleration of cccdna formation from intracellular progeny nucleocapsids [ , ] . to this end, hbv capsids were purified from the cytoplasmic fraction of pfatreated cells and an endogenous dna polymerase assay were performed in vitro in the absence or presence of cpams. to probe the integrity of nucleocapsids, the accessibility of viral dna to dnase i digestion were tested at the completion of endogenous dna polymerase reaction, but before extraction of viral dna. southern blot analysis of viral dna species indicated that rcdna can be efficiently synthesized in the in vitro endogenous dna polymerase reaction when dntps were provided ( fig a) . furthermore, the presence of bay - , enan- or at- did not inhibit rcdna synthesis. however, the rcdna synthesized in the presence of bay - and enan- , but not at- , was susceptible to dnase i digestion (fig a and b ). hence, our results indicate that cpams do not inhibit hbv dna synthesis, but favor a hypothesis that the selected cpams specifically alter the structure of, and destabilize mature rcdna-containing nucleocapsids and facilitate rcdna nuclear delivery and synthesis of cccdna. because the mature rcdna-containing nucleocapsids only constitute a small fraction of total cytoplasmic capsids [ ] , it is not surprise that the cpam treatment did not alter the amounts and migration mobility of capsids, as revealed by a native agarose gel electrophoresis-based particle gel assay (fig a and b , lower panels) [ , ] . the differential effects of cpams on cccdna synthesis from the de novo infection and intracellular amplification pathways argues that the compounds may have a different effect on the nucleocapsids in virions and in the cytoplasm. to investigate this possibility, we purified hbv virion particles from the blood of a chronic hbv carrier and examined the effects of cpams on rcdna synthesis and integrity of nucleocapsids in an in vitro endogenous dna polymerase reaction as described above. similar to the results obtained with cytoplasmic nucleocapsids, the presence of bay - , enan- or gls did not inhibit rcdna synthesis from partially double-stranded virion dna, but conferred susceptibility of virion rcdna to dnase i digestion in a concentration-dependent manner ( fig a) . to further investigate whether cpams can directly induce structural change of the partially double-stranded dna-containing nucelocapsids, virion particles prepared from the patient serum were treated with the cpams in an endogenous dna polymerase reaction without dntp and followed by dnase i treatment prior to dna extraction. the results showed that each of the three tested cpams rendered all virion dna species susceptible to dnase i digestion in a concentration-dependent manner (fig b) . hence, the results indicate that irrespective to the maturation stage of viral dna, the nucleocapsids derived from virion particles are sensitive to cpam-induced structural changes that expose viral dna for dnase i digestion. elongation of positive-stranded dna gradually increases the vulnerability of cytoplasmic nucleocapsids to cpams encouraged by the observation that the cpam-induced virion dna susceptibility to dnase i did not depend on active dna polymerase reaction or ongoing dna chain elongation ( fig b) , we further examined the effects of cpams on capsids purified from the cytoplasm of hepad cells in an endogenous dna polymerase reaction buffer without dntps. as shown in fig a, while rcdna became susceptible to dnase i digestion at lower concentrations of [ , , ] . a kinetics study further revealed that while up to h incubation was required for enan- to induce significant exposure of rcdna, to h exposure was sufficient for gls and bay - to induce an extensive exposure of rcdna ( fig b) . to determine the kinetics of cpams induction of rcdna exposure in cells, hepad cells cultured in tet-free medium for days were treated with gls for the indicated periods of time. the cytoplasmic lysates were mock-treated or treated with dnase i before extraction of dna. as shown in s fig, treatment of gls for h induced extensive rcdna exposure for dnase i digestion. cpams promote uncoating of mature nucleocapsids although results presented above indicate that cpam treatment induces the exposure of rcdna in nucleocapsids to dnase i digestion, the extent of disruption on the structure of mature nucleocapsids by the different cpams remains to be determined. we therefore examined whether the mature viral dna species, rcdna and double-stranded linear (dsl) dna, were still associated with capsids after cpam treatment. the assumption is that if cpam treatment severely disrupts the mature nuclocapsids and results in releasing of viral dna, we anticipate that rcdna and dsldna species will not co-sediment with any form of capsids. however, if cpam treatment only mildly disrupts the mature nucleocapsids and causes the exposure of viral dna that is still associated with capsid structure, we anticipate to see the rcdna and/or dsldna species will co-sediment with capsids. to this end, hbv capsids prepared from hepad cells were mock treated or incubated with enan- , bay - or gls in endogenous dna polymerase reactions without dntps. the reactions were then fractionated by sucrose density gradient ultracentrifugation. the amounts of total capsids and capsid-associated dna in each of the fractions were analyzed by a . % native agarose gel electrophoresis-based particle gel assay [ ] . as shown in the upper panels of fig a to d , compared to mock-treated control, treatment with any of the three cpams did not alter the sedimentation profile of capsids and capsid associated hbv dna. these results are consistent with the observation that cpams only affect the mature nuclocapsids that constitute only a small portion of total capsids (fig and s fig) . however, analysis of viral dna by southern blot hybridization in each of the fractions revealed that rcdna and dsldna were lost or reduced from capsids sedimenting to % to % sucrose after bay - or gls treatment, but the majority of rcdna and dsldna still co-sediment with capsids after enan- treatment (fig a to d, lower panels) . due to the small amounts of rcdna and dsldna and low sensitivity of southern blot hybridization assay, we were not able to identify the location or distribution of the disassociated dna species in the gradients. nevertheless, those results indicate that while bay - or gls treatment most likely induced structural changes that were significant enough to cause loss of rcdna and dsldna from majority of the mature nucleocapsids, the less active compound enan- might only induce structural changes that render the dna content susceptible to dnase digestion. these two distinct degrees of conformational alteration may correspond to a subtle increase in "capsid breathing" for enan- , versus extensive rearrangement of the capsid subunit organization for bay- - and gls [ ] . the genomes of all viruses are wrapped with capsid protein(s) to form nucleocapsids. unlike viral enzymes that often have host cellular homologues, host cells do not encode proteins that are structurally and functionally similar to viral capsid proteins. therefore, viral capsid proteins are ideal and highly selective antiviral targets [ ] . in addition to serving as a vehicle for transmission of viral genomes between host cells, hbv nucleocapsids have other unique functions in the viral life cycle [ , , ] . as illustrated in fig , first, hbv dna replication occurs exclusively within the cytoplasmic nucleocapsids, by reverse transcription of viral pgrna first into negative-strand dna and then double-stranded rcdna. second, unlike all other viruses where the progeny nucleocapsids have only one destination, i.e., to be secreted as virions, the hbv progeny nucleocapsids can also deliver their rcdna into the nuclei to synthesize cccdna. due to superinfection exclusion [ , ] , it is reasonable to consider that the activity of the intracellular amplification pathway is the key determinant of cccdna pool size in infected hepatocytes [ ] . due to the large amounts of progeny nucleocapsids in the cytoplasm, this intracellular cccdna amplification pathway must be, and actually is, tightly controlled by viral and host factors [ , , , ] . finally, the exclusive replication in nucleocapsids and the spatially-controlled disassembly and delivery of rcdna into nuclei at nuclear pore complexes protect the viral dna from recognition by cytoplasmic dna sensors, and thus favor the persistent infection by hbv [ , , ] . hence, it has been speculated that targeting hbv core protein may disrupt multiple steps of hbv replication and activate innate immune response in virally infected cells. however, as mentioned, while several distinct chemotypes of hbv cpams have been shown to disrupt viral nucleocapsid assembly and consequentially inhibit viral genome replication, their effects on other aspects of nucleocapsid function have not been investigated. herein, we provide evidence suggesting that selected haps and sbas are able to interact with nucleocapsids from virions and mature forms of rcdna-containing nucleocapsids in the cytoplasm and subsequently interfere with their function of delivering viral rcdna to the nuclei for cccdna synthesis. although the structural basis for the cpams to target the highly selected sub-populations of nucleocapsids remains to be determined, our results are consistent with previous findings that mature hbv nucleocapsids are intrinsically unstable or fragile [ ] , probably due to the stiffness of rc and dsldna, which imposes bending energy and electrostatic repulsion to the capsid shell [ ] . moreover, viral dna synthesis in the nucleocapsids assembled from mutant dhbv core proteins with a serial n-terminal inertions destabilized mutant nucleocapsids, rendering mature viral dna selectively sensitive to nuclease digestion [ ] . therefore, it is possible that the intrinsic instability of mature nucleocapsids, due to the completion of double-stranded dna, confers the selective sensitivity for cpams to induce disassembly. considering the result that cpam treatment induces the accessibility of all forms of virion dna, either rcdna or partially double-stranded dna, to dnase i digestion, a possible explanation is that like many other viruses, structural shifts or maturation of nucleocapsids may occur during or after virion assembly and secretion, and confer susceptibility to cpams [ ] . moreover, a recent report showed that the carboxyl-terminal domain of core proteins is hypophosphorylated in dna-containing virions, but remains hyperphosphorylated in intracellular dna-containing nucleocapsids [ ] . although the c-terminal arginine-rich domain of hbv core protein is not directly involved in the binding of cpams [ ] , the dephosphorylation may alter the interaction between core protein and viral dna [ ] , and thus affect the nucleocapsid structure in a manner distinct from that of the hyperphosphorylated state [ ] . in addition, the sensitivity of partially double-stranded dna-containing cytoplasmic nucleocapsids to the increased concentrations of cpams strongly suggests that elongation of positive-stranded dna toward rcdna induces incremental, or gradual, structural changes favoring specific interaction with cpams. structural biology studies with purified double stranded dna-containing capsids may reveal those important structure differences. however, it is technically challenging to purify sufficient amounts rcdna-containing hbv capsids for cryo-em analyses. although our study revealed correlations between altered cccdna synthesis and induction of nucleocapsid destablization by cpams, the molecular mechanisms on how the cpaminduced nucleocapsid structural changes disrupt cccdna formation in de novo infection, but accelerate cccdna synthesis from progeny intracellular nucleocapsids remain to be determined. however, reasonable speculations can be made based on current knowledge. as illustrated in fig , on one hand, in de novo infection, cpams may interact with nucleocapsids in virions during endocytic entry or immediately after their release into the cytoplasm to induce viral dna exposure or release from nucleocapsids. the premature disassembly of nucleocapsids results in viral dna release and decay by cytoplasmic dnases before its arrival to the nuclear pore complex for nuclear import and subsequent cccdna synthesis. on the other hand, due to the shorter distance traveled as compared to the de novo infection, the intracellular progeny rcdna-containing nucleocapsids may reach nuclear pore complexes before a certain stage of their disassembly and cpam-induced uncoating actually accelerates the release of rcdna into the nuclei and thus cccdna formation. of course, a fraction of rcdna decay in the cytoplasm may also occur. in fact, this interpretation is in agreement with recent findings that enhanced destabilization of mature rcdna containing nucleocapsids, due to unidentified host cellular factors in mouse hepatocytes or a single amino acid substitution (i a) in core protein, significantly reduced the accumulation of rcdna, but increased the dp-rcdna and cccdna synthesis [ , ] . it had not escaped our attention that the released or exposed rc/dsldna in the cytoplasm may activate innate dna sensors [ ] [ ] [ ] [ ] . however, cytokine response was not detected in cpam-treated cells. whether this is due to the deficiency of cyclic gmp-amp synthase (cgas)-stimulator of interferon genes (sting) pathway in hepatocytes and hepatoma cells [ ] [ ] [ ] or efficient digestion of the released viral dna by cytoplasmic nucleases [ ] is currently under investigation. while inhibition of cccdna synthesis from de novo infection is obviously a beneficial therapeutic effect, the acceleration of intracellular cccdna amplification is detrimental. however, the potent inhibition of pgrna encapsidation by cpams will quickly block viral dna replication and production of mature progeny nucleocapsids and consequential cccdna synthesis. therefore, the only chance for cpams to accelerate cccdna synthesis during antiviral therapy is at the initial period of treatment to promote cccdna synthesis from pre-existing mature nucleocapsids in hbv infected cells. fortunately, treatment of hepad cells supporting steady-state hbv dna replication with etv and cpams to mimic the initial stage of antiviral treatment in vivo did not observe a significant increase of cccdna accumulation within first h of treatment. instead, similar to etv, cpams prevented amplification of cccdna pool during a prolonged treatment, as compared with mock-treated control (s fig). in fact, those results are consistent with the observation that cpam treatment of primary human hepatocytes after establishment of hbv infection did not alter the amount of cccdna [ ] . taking together, the results imply that cpams only significantly increase the amount of cccdna by acceleration of ongoing cccdna synthesis, such as under the condition of fast cccdna synthesis after release from pfa arrested hbv dna replication, but cannot accelerate the very low (or negligible) rate of cccdna synthesis to significantly increase the amount of cccdna in cells with established hbv infection. mechanistically, as stated above, it is possible that only when cpam-induced disassembly of mature nucleocapsids occurs at or near the nuclear pore complex may facilitate the import of rcdna into the nuclus for cccdna synthesis, but the mature nucleocapsids at such status may not exist in a significant amount in hepatocytes with established infection. nevertheless, careful monitoring of cccdna synthesis during the early phase of cpam treatment in vivo in animals and in clinical trials and pretreatment or combination with viral dna polymerase inhibitors might be considered. the hepad cell line (obtained from dr. christoph seeger at fox chase cancer center, philadelphia) supporting hbv pgrna transcription and subsequent viral dna replication in a tetracycline (tet)-inducible manner was maintained as previously described [ ] . c a cell line [a derivative of hepg (atcc hb- )] (atcc crl- ) was maintained in dmem/f- ( : ) medium supplemented with % fbs (gemini bio-products), u/ml penicillin, μg/ml streptomycin. entecavir (etv) is a gift from dr. william s. mason at fox chase cancer center, philadelphia [ ] . foscarnet was purchased from sigma. capsid assembly modulators enan- and bay - , gls and at- were described previously [ , ] . myrcludex b is a gift of dr. stephan urban at heidelberg university, germany [ ] . alpha-interferon (ifn-α) was purchased from pbl assay science. rabbit anti-hbc antibody was obtained from dako (b ). human sodium taurocholate cotransporting polypeptide (ntcp) gene coding sequence was amplified from a cdna clone purchased from origene (sc ). a carboxyl-terminal c tag was added by pcr amplification with the primers harboring c tag sequence and noti & bam hi restriction enzyme sites. the purified pcr fragments were digested with restriction enzymes not i and bam hi and subsequently cloned into a pqcxip vector (clontech). vsv g protein pseudotyped retroviruses were packaged in gp - cells as previously described [ ] . c a hntcp cell line stably expressing human ntcp was established by infection of c a cell line with the pseudotyped retroviruses and selected with medium containing μg/ml of puromycin. puromycin-resistant cells were expanded into cell line and designated as c a hntcp . proper expression of ntcp was confirmed by immunofluorescence and western blot assays. c a hntcp cells were seeded into collagen-coated -well plates at a density of × cells per well and cultured in complete dmem medium containing % dimethyl sulfoxide (dmso). one day later, the cells were infected with hbv prepared from hepad cell culture media at a moi of genome equivalents per cell in dmem containing % peg- . the inoculums were removed at h post infection and the cultures were maintained in complete dmem medium containing % dmso until harvesting. extraction and analyses of hbv dna and rna by hybridization and realtime pcr assays hbv core dna and rna extraction from infected c a hntcp or hepad cells, southern blot hybridization, and real-time pcr analyses were performed as described previously [ , ] . hbv cccdna from hbv-infected c a hntcp cells and hepad cells were extracted by a modified hirt dna extraction procedure [ ] . a fraction of hirt dna preparation was digested with unit of plasmid-safe adenosine triphosphate (atp)-dependent deoxyribonuclease (psad) (epicentre technologies) in a μl reaction for h at ˚c to remove rcdna. the dnase was inactivated by incubation of the reactions for min at ˚c. cccdna in the psad-treated samples were quantified by a real time pcr assay with primer sequences ggggcgcacctctcttta (forward) and ccacccaggtagctagagtcattag (reverse). the real-time pcr was performed using the sybr premix ex taq on a lightcycler ii (roche) as the following reaction procedure: ˚c for min then cycles of ˚c for s, ˚c for s, and ˚c for s. the amount of hbv cccdna in a dna preparation was determined by real-time pcr using a plasmid containing hbv genotype d genome as the standard. for southern blot hybridization, the hirt dna samples were denatured at ˚c for minutes and chilled in ice. this procedure completely denatures deproteinized rc-dna (dp-rcdna) into single stranded dna, whereas cccdna will remain as double stranded circular dna [ ] . the denatured hirt dna samples without or with further digestion with restriction enzyme eco ri to linearize cccdna into double-stranded linear dna were resolved in . % agarose gel and transferred onto hybond-xl membrane. the membrane was probed with α- p-utp labeled minus strand specific full-length hbv riboprobe. hdv production, infection and rna quantification. cell culture derived hdv particles were generated as described previously [ ] . briefly, huh cells (obtained from dr. christoph seeger at fox chase cancer center, philadelphia) were co-transfected with a plasmid containing three copies of hdv cdna sequence (psvld ) and a plasmid expressing hbv envelope proteins l, m and s (psv h) [ ] . hdv particles were harvested from the media between to and to days post transfection. after clarification by centrifugation, particles were precipitated in the presence of % peg- and the pellet was dissolved in tan buffer ( mm tris-hcl, ph . and mm nacl) at % volume of the original culture medium. for infection, c a hntcp cells were seeded into collagen-coated -well plates at a density of × cells per well and cultured in complete dmem medium containing % dimethyl sulfoxide (dmso). one day later, the cells were infected with hdv at a moi of genome equivalents per cell in dmem containing % peg- . the inoculums were removed at h post infection and the cultures were maintained in complete dmem medium containing % dmso until harvesting. intracellular hdv genomic and antigenomic rnas were quantified by qrt-pcr using strand-specific primers as described previously [ ] . purification of hbv virions and cytoplasmic capsids. hbv virions were prepared from a hbv carrier's serum (from bioreclamation ivt, the complete resources for all biologicals) by sucrose gradient centrifugation. briefly, ml of human sera was layered onto a -ml % (wt/vol) sucrose cushion in . m nacl, . m tris-hcl, ph . , and centrifuged for h at , rpm in sw rotor at ˚c. the supernatant fluid was removed and pellet was dissolved in tne buffer ( . m nacl; . m tris-hcl, ph . ; and . mm edta). to prepare intracellular hbv capsids, hepad cells were cultured in tetracycline-free medium with or without mm foscarnet (pfa) for days with daily medium change. the cells were then lysed with chilled lysis buffer containing mm tris-hcl (ph . ), mm edta, . % nonidet p- and % sucrose on ice for minutes, the lysate was centrifuged at , g for min to remove the nuclei and cell debris. the clarified supernatant was overlaid onto a - % (w/w) sucrose gradient and centrifuged at , rpm for h at ˚c using a beckman sw rotor. nineteen -ml fractions were collected from the bottom of the cushion. ten microliters of each fraction was dot-blotted onto the nitrocellulose membrane and hbv core protein was detected by incubation with a rabbit antibody against hbv core protein (dako, b ). the bound antibody was detected by irdye secondary antibodies and visualized by li-cor odyssey system. hbv core protein-positive fractions were pooled together and the sucrose concentration was adjusted to % by addition of tne buffer. the diluted sample was overlaid on a % sucrose cushion and centrifuged at , rpm for h at ˚c using beckman sw rotor. the pellet was resuspended in tne buffer. endogenous dna polymerase reaction. an endogenous dna polymerase reaction (epr) mixture was assembled with μl of hbv virion or capsid preparation, μl of x epr buffer which consisted of . m nacl, . m tris-hcl (ph . ), mm mgcl , mm dithiothreitol, . % (vol/vol) nonidet p- . dntp and capsid assembly modulators were added, as indicated, to . mm or a specified final concentration, respectively. water was added to bring the reaction volume to μl. after incubation at ˚c for an indicated period of time, the reaction was subjected for extraction of viral dna with or without prior dnase i digestion at ˚c for min. the viral dna were resolved in . % agarose gel and transferred onto hybond-xl membrane. the membrane was probed with α- p-utp labeled minus strand specific fulllength hbv riboprobe. particle gel assay. the cytoplasmic hbv capsids were analyzed by a native agarose gel electrophoresis-based assay. briefly, ten microliters of endogenous dna polymerase reaction or fraction of sucrose gradient centrifugation were resolved by electrophoresis through a . % agarose gel and transferred to a nitrocellulose membrane by blotting with tne buffer ( mm tris-hcl, ph . , mm nacl, and mm edta). hbv capsids on the membrane were detected with a procedure described previously [ ] . ultracentrifugation analysis of hbv capsid integrity. two hundred microliters of the purified hbv capsids were mixed with μl of reaction buffer containing . m nacl, . m tris-hcl (ph . ), mm mgcl , mm dithiothreitol, . % (vol/vol) nonidet p- . capsid assembly modulators were added as indicated and water was provided to bring the reaction volume to μl. after incubation at ˚c for the indicated period of time, the reaction was loaded onto a % to % linear sucrose gradient in tne buffer and spun at , rpm for hr (beckman, rotor sw ). . ml fractions were collected from the bottom of the centrifugation tube by blood collection set. sucrose concentration of each fraction was measured by refractor (mettler toledo). the rest of each fraction was mixed with ml tne buffer and pelleted by centrifugation at , rpm for hr. pellet was dissolved in μl tne buffer. μl of capsid solution were fractionated in a . % agarose gel electrophoresis and transferred to a nitrocellulose membrane to detect hbv capsid by probing with anti-hbcag antibody. viral dna was extracted from the remaining sample of reactions and detected by southern blot hybridization with α- p-utp-labeled minus strand-specific full-length hbv riboprobe. and cytoplasmic core dna (c) were quantified by real-time pcr assays. hbsag in culture medium was measured by using an elisa assay kit (autobio) (d). (e) the infected cells were fixed at and days post infection (dpi) and hbcag was detected by indirect immunofluorescent staining (red). the cell nuclei were stained with dapi (blue). the images were captured with a nikon x microscopy. (f) detection of hbv replication intermediates by hybridization assays. top panel, hirt dna extracted from the cells was denatured at ˚c for min to denature dp-rcdna into single stranded dna and followed by restriction with ecori to convert cccdna into unit-length dsldna (labeled as ccc/ecori) and detected by southern blot hybridization. unit-length hbv linear dna served as a molecular weight marker. middle panel, hbv pgrna and mrnas specifying envelope proteins (envrnas) were detected by northern blot hybridization. s and s ribosomal rna (rrna) served as loading controls. global epidemiology of hepatitis b virus infection: new estimates of age-specific hbsag seroprevalence and endemicity hepatitis b virus resistance to nucleos(t)ide analogues therapeutic strategies for a functional cure of chronic hepatitis b virus infection core protein: a pleiotropic keystone in the hbv lifecycle molecular biology of hepatitis b virus infection nuclear import of hepatitis b virus capsids and release of the viral genome alteration of mature nucleocapsid and enhancement of covalently closed circular dna formation by hepatitis b virus core mutants defective in complete-virion formation hbv cccdna: viral persistence reservoir and key obstacle for a cure of chronic hepatitis b metabolism and function of hepatitis b virus cccdna: implications for the development of cccdna-targeting antiviral therapeutics structural organization of the hepatitis b virus minichromosome specific and nonhepatotoxic degradation of nuclear hepatitis b virus cccdna interferon-gamma and tumor necrosis factor-alpha produced by t cells reduce the hbv persistence form, cccdna, without cytolysis the current status and future directions of hepatitis b antiviral drug discovery present and future therapies of hepatitis b: from discovery to cure high-resolution crystal structure of a hepatitis b virus replication inhibitor bound to the viral core protein small-molecule effectors of hepatitis b virus capsid assembly give insight into virus life cycle a heteroaryldihydropyrimidine activates and can misdirect hepatitis b virus capsid assembly hepatitis b virus capsids have diverse structural responses to small-molecule ligands bound to the heteroaryldihydropyrimidine pocket assembly-directed antivirals differentially bind quasiequivalent pockets to modify hepatitis b virus capsid tertiary and quaternary structure heteroaryldihydropyrimidine (hap) and sulfamoylbenzamide (sba) inhibit hepatitis b virus replication by different molecular mechanisms discovery and pre-clinical characterization of third-generation -h heteroaryldihydropyrimidine (hap) analogues as hepatitis b virus (hbv) capsid inhibitors a novel inhibitor of dengue virus replication that targets the capsid protein sodium taurocholate cotransporting polypeptide is a functional receptor for human hepatitis b and d virus. elife : e hepatitis b and d viruses exploit sodium taurocholate co-transporting polypeptide for species-specific entry into hepatocytes reversal of fulminant hepatic failure using an extracorporeal liver assist device prevention of hepatitis b virus infection in vivo by entry inhibitors derived from the large envelope protein dna polymerase kappa is a key cellular factor for the formation of covalently closed circular dna of hepatitis b virus ifn-alpha inhibits hbv transcription and replication in cell culture and in humanized mice by targeting the epigenetic regulation of the nuclear cccdna minichromosome alpha-interferon suppresses hepadnavirus transcription by altering epigenetic modification of cccdna minichromosomes capsid assembly modulators have a dual mechanism of action in primary human hepatocytes infected with hepatitis b virus in hepatocytes infected with duck hepatitis b virus, the template for viral rna synthesis is amplified by an intracellular pathway formation of hepatitis b virus covalently closed circular dna: removal of genome-linked characterization of the intracellular deproteinized relaxed circular dna of hepatitis b virus: an intermediate of covalently closed circular dna formation hepadnavirus envelope proteins regulate covalently closed circular dna amplification sulfamoylbenzamide derivatives inhibit the assembly of hepatitis b virus nucleocapsids conditional replication of duck hepatitis b virus in hepatoma cells hepatitis b virus covalently closed circular dna formation in immortalized mouse hepatocytes associated with nucleocapsid destabilization secretion of genome-free hepatitis b virus -single strand blocking model for virion morphogenesis of para-retrovirus discovery and mechanistic study of benzamide derivatives that modulate hepatitis b virus capsid assembly preclinical characterization of gls , an inhibitor of hepatitis b virus core particle assembly weak protein-protein interactions are sufficient to drive assembly of hepatitis b virus capsids capsid proteins of enveloped viruses as antiviral drug targets superinfection with woodchuck hepatitis virus strain whvny of livers chronically infected with strain whv superinfection exclusion in duck hepatitis b virus infection is mediated by the large surface antigen formation of the pool of covalently closed circular viral dna in hepadnavirus-infected cells production and function of the cytoplasmic deproteinized relaxed circular dna of hepadnaviruses the innate immune response to hepatitis b virus infection: implications for pathogenesis and therapy maturation-associated destabilization of hepatitis b virus nucleocapsid differential assembly of hepatitis b virus core protein on single-and double-stranded nucleic acid suggest the dsdna-filled core is spring-loaded duck hepatitis b virus nucleocapsids formed by n-terminally extended or c-terminally truncated core proteins disintegrate during viral dna maturation the structural biology of hiv assembly capsid phosphorylation state and hepadnavirus virion secretion hepatitis b virus core protein phosphorylation sites affect capsid stability and transient exposure of the c-terminal domain complete and incomplete hepatitis b virus particles: formation, function, and application mita/sting and its alternative splicing isoform mrp restrict hepatitis b virus replication inhibition of hepatitis b virus replication by activation of the cgas-sting pathway the cyclic gmp-amp synthetase-sting signaling pathway is required for both the innate immune response against hbv and the suppression of hbv assembly viral dna-dependent induction of innate immune response to hepatitis b virus in immortalized mouse hepatocytes lack of immunological dna sensing in hepatocytes facilitates hepatitis b virus infection activation of sting in hepatocytes suppresses the replication of hepatitis b virus hepatitis b virus evades innate immunity of hepatocytes but activates cytokine production by macrophages trex regulates lysosomal biogenesis and interferon-independent activation of antiviral genes inducible expression of human hepatitis b virus (hbv) in stably transfected hepatoblastoma cells: a novel system for screening potential inhibitors of hbv replication the amount of hepatocyte turnover that occurred during resolution of transient hepadnavirus infections was lower when virus replication was inhibited with entecavir interferons accelerate decay of replication-competent nucleocapsids of hepatitis b virus interferon induction of ifitm proteins promotes infection by human coronavirus oc hepatitis b virus e antigen production is dependent upon covalently closed circular (ccc) dna in hepad cell cultures and may serve as a cccdna surrogate in antiviral screening assays assembly of hepatitis delta virus: particle characterization, including the ability to infect primary human hepatocytes effect of alpha interferon on the hepatitis c virus replicon we thank dr. eain a. murphy for critical reading and comments on the manuscript. conceptualization: fang guo, jinhong chang, ju-tao guo. key: cord- - ycgd h authors: markosyan, ruben m.; miao, chunhui; zheng, yi-min; melikyan, gregory b.; liu, shan-lu; cohen, fredric s. title: induction of cell-cell fusion by ebola virus glycoprotein: low ph is not a trigger date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: ycgd h ebola virus (ebov) is a highly pathogenic filovirus that causes hemorrhagic fever in humans and animals. currently, how ebov fuses its envelope membrane within an endosomal membrane to cause infection is poorly understood. we successfully measure cell-cell fusion mediated by the ebov fusion protein, gp, assayed by the transfer of both cytoplasmic and membrane dyes. a small molecule fusion inhibitor, a neutralizing antibody, as well as mutations in ebov gp known to reduce viral infection, all greatly reduce fusion. by monitoring redistribution of small aqueous dyes between cells and by electrical capacitance measurements, we discovered that ebov gp-mediated fusion pores do not readily enlarge—a marked difference from the behavior of other viral fusion proteins. ebov gp must be cleaved by late endosome-resident cathepsins b or l in order to become fusion-competent. cleavage of cell surface-expressed gp appears to occur in endosomes, as evidenced by the fusion block imposed by cathepsin inhibitors, agents that raise endosomal ph, or an inhibitor of anterograde trafficking. treating effector cells with a recombinant soluble cathepsin b or thermolysin, which cleaves gp into an active form, increases the extent of fusion, suggesting that a fraction of surface-expressed gp is not cleaved. whereas the rate of fusion is increased by a brief exposure to acidic ph, fusion does occur at neutral ph. importantly, the extent of fusion is independent of external ph in experiments in which cathepsin activity is blocked and ebov gp is cleaved by thermolysin. these results imply that low ph promotes fusion through the well-known ph-dependent activity of cathepsins; fusion induced by cleaved ebov gp is a process that is fundamentally independent of ph. the cell-cell fusion system has revealed some previously unappreciated features of ebov entry, which could not be readily elucidated in the context of endosomal entry. ebola virus (ebov) outbreaks continually occur and up to % of those infected die; currently there are no approved vaccines or antiviral therapeutics against the virus [ , ] . ebov initiates infection by fusion from within endosomes. experimentally, endosomal interiors are difficult to control, but systems that track the entry of several other viruses into cells have been developed and employed [ , , , ] . historically, these methods have relied on fusion of infectious virus or pseudovirus within cells; cell-cell fusion has not been among the systems in use for ebov. it is surprising that a cell-cell fusion system has not been developed, as the processing of the ebola fusion protein, gp, and other conditions necessary for fusion have been elaborated [ ] . (some years ago there was an isolated report of ebov gp-mediated cell-cell fusion, but this study has not been followed up by any other laboratory, including the original [ ] ). cellcell fusion has several important advantages over intracellular fusion assays, including complete control of the aqueous solution bathing the ectodomain of the fusion protein. in the present study we describe a direct and sensitive system to measure ebov gp-mediated cell-cell fusion with high time resolution, thereby providing fusion kinetics. the system exhibits the well-known central properties of ebov entry, providing strong support for the utility of the cell-cell fusion system to explore mechanisms of ebov entry that are not possible or practical with whole infectious virus. ebov gp is a prototypic class i viral fusion protein [ ] . it is synthesized as a homotrimer; each monomer is cleaved into gp -gp subunits by proteases within the golgi apparatus [ , ] . the gp subunit is responsible for binding to the intracellular receptor niemann pick type c , (npc ) and possibly to other molecules [ ] , and the gp subunit is responsible for membrane fusion [ , , , , , , ] . the two subunits of each monomer remain linked through a disulfide bond and a multitude of weak interactions [ , , , ] . after endocytosis of the virus, the gp subunit is cleaved by the endosomal proteases cathepsin b and/or l [ , , , , ] , while remaining attached to gp [ ] , and then binds to npc [ , ] . the low ph within endosomes is necessary for viral fusion. but it has not been known whether low ph directly triggers fusion by causing conformational changes in gp or whether it augments fusion by increasing the activities of the cathepsins [ , ] . after developing our system, we discovered that an ebov gp-induced fusion pore that connects two plasma membranes does not readily enlarge over time, in contrast to the pores of other viral fusion proteins. this anomalous lack of growth may be the reason cell-cell fusion has not been successfully observed in many prior attempts that used less sensitive assays to detect fusion. on the question of low ph, we found that activation of cathepsins by acidity is the sole cause for augmentation of fusion: if ebov gp on the cell surface is artificially cleaved by thermolysin in the presence of cathepsin inhibitors, the extent of fusion is independent of ph. ebov gp mediates cell-cell fusion mediated fusion is much longer than for some viral fusion proteins, such as iav ha [ ] , but comparable to others, such as hiv env in some studies [ ] . we thus tested, at various times, whether some of the cell pairs that had not yet fully fused had hemifused: the addition of . mm cpz to cell pairs ruptures hemifusion diaphragms that have formed between cell pairs, and this is a standard means to test for hemifusion [ , , ] . we used thermolysin-treated effector cells to maximize cleavage of ebov gp and found that adding cpz either , , or min after reneutralization did not induce any dye spread above that already observed, strongly indicating that a negligible percentage of cells were hemifused, but not fused, at any given time. npc is an intracellular receptor for ebov gp [ , ] . we compared extents of fusion for target parental hek t cells versus target hek t cells that stably overexpressed npc . effector cells that were not treated with thermolysin yielded fusion at ph . ( fig a, bar ) , and a greater extent of fusion after a -min acidic ph . pulse (fig a, bar ) . the extent of calcein spread was greater for target cells overexpressing npc (fig a, bars and ) than for parental t cells (fig a, bars and ) for matching conditions. fusion was still ph-dependent for target cells overexpressing npc : calcein spread was greater hr after a -min ph (merged) . the viral proteins expressed by transfecting effector cells are shown to the right of the images. cells expressing ebov gp were treated with μg/ml thermolysin for min; fusion was augmented with a -min ph . pulse. for cells expressing jsrv env, a -min ph . pulse was used to trigger fusion. effector cells expressing influenza virus (iav) ha were treated with trypsin and neuraminidase as described [ ] , bound to hek t cells, and fusion was triggered with a -min ph . pulse. for mock-transfected effector cells, a -min ph . pulse was employed. fused cells are marked by arrowheads. for this set of experiments, the extent of fusion hr after reneutralization was about % for ebov gp, % for jsrv env, and % for iav ha. thermolysin treatment results in greater extents of fusion between cells. (a) schematic of the experimental protocol is shown above the bar graph. e, effector cells; t, target cells, th, thermolysin. bar graph: fusion of thermolysin-treated effector cells expressing ebov gp (columns and , dark red) was greater than for untreated cells (columns and , dark yellow). for both thermolysin-treated and non-treated cells, a -min ph . pulse applied at room temperature augmented fusion, measured after an additional h incubation at neutral ph. for each condition, at least experiments were performed. typical images used to obtain the data of the bar graph are shown on the right: in top images, cells were treated with μg/ml thermolysin; in bottom images, cells were not treated. cells that have fused are marked by arrows. (b) the kinetics of fusion for thermolysin-treated (dark red squares) and untreated (dark yellow circles) effector cells. cleaving ebov gp by thermolysin speeds fusion kinetics, but extents of fusion are the same for treated and untreated cells after a ph . pulse at °c is followed by a h reneutralization. * p < . ; *** p < . . doi: . /journal.ppat. .g . pulse (fig a, bar ) than in the absence of the pulse (fig a, bar ) . we confirmed that fusion was dependent on the presence of npc by generating and purifying a recombinant soluble protein consisting of domain c of npc fused to gst (denoted snpc ). the purity and size of snpc was confirmed (fig b, inset) . we added snpc to the external solution and found that the extent of fusion increased monotonically with the amount of snpc added ( fig b) , in accord with the prior demonstration that by binding npc , ebov gp undergoes conformational changes favorable for fusion [ ] . the augmentation of fusion by snpc indicated that, although there was a sufficient amount of npc on cell surfaces to stimulate fusion, this amount was relatively small and fusion was consequently limited. npc is an endosomal protein [ ] , but a small fraction of npc may be present on the plasma membrane of a cell. we assessed this possibility by using flow cytometry to measure overexpression of npc (second set of two bars) led to greater fusion with effector cells than did mock-transfected target cells (first set of bars). for these experiments, the effector cells were not thermolysin-treated (i.e., these experiments relied on endogenous levels of gp cleavage). for each set of experiments, a -min ph . pulse (labeled "pulse ph +") led to more fusion than when ph was never lowered (-). for each condition, n = . binding with an antibody against npc (from lifespan biosciences) on parental cells; shrna that targeted npc was stably expressed in one set of these cells, and npc was overexpressed in another set (fig c and d ). the level of binding of the secondary fitclabeled antibody against endogenous npc (as measured by mean fluorescence intensity, mfi) was -fold greater than in the absence of the primary ab ( fig c, bar vs. bar , and fig d) . expression was reduced for cells in which npc was knocked down by shrna (bar ), and was greater for cells in which npc was overexpressed (bar ). these results demonstrate that copies of npc reside in the plasma membrane of the cells we used as targets in cellcell fusion experiments. ebov gp is certainly cleaved within endosomes as part of viral infection [ ] . because we observed cell-cell fusion at acidic ph without adding thermolysin, it is extremely likely that a fraction of gp on the cell surface was cleaved into a fusion-competent form. an antibody that only recognizes cleaved gp has not been reported, so we had to devise an alternate means to quantitatively measure the extent of cleavage. we were able to distinguish between the two forms of gp by using the property that npc binds to cleaved, but not uncleaved, ebov gp. we used a snpc to examine cleaved gp by flow cytometry; in parallel, we measured the total amount of gp on cell surfaces by using an anti-flag antibody that bound to the flag tag on our gp construct. we also created a gp construct that was intrinsically more likely to be cleaved on the cell surface: we inserted the furin recognition site rrkr at amino acids - of gp (referred to as gp furin ), the putative cleavage site for catl in gp [ , ] . we reasoned that because exogenous expression of furin facilitates cleavage at this inserted site, generating the fusion-active - kda subunit, the extent of cleavage of gp on the plasma membrane as well as the extent of cell-cell fusion would be greater for this construct than for wt. we experimentally confirmed our expectations: we determined the amount of cleaved gp on the cell surface by adding snpc (fused with gst) to cells expressing either ebov gp or gp furin , and measuring their binding to an anti-gst antibody. this antibody was detected by a fitc-conjugated secondary antibody (fig ) . the fraction of wt gp cleaved on parental cells ( fig a, bar ) was the same for cells that were transfected with both gp and furin (bar ). the specificities of snpc and antibody binding were confirmed by the - fold higher fluorescence than was seen for cells that did not express gp (bar ). it is notable that cotransfection of cells by gp furin and furin resulted in greater cleavage (bar vs bar ). we found that the expression of total wt gp as measured by the anti-flag antibody was not significantly altered by coexpression of furin ( fig b, columns and ), but cells that coexpressed gp furin and furin consistently showed a decreased total gp (compare bar and ), possibly due to non-specific degradation of gp furin . to determine the relative percentage of cleaved gp, we normalized cleaved gp by total gp. (these are relative and not absolute percentages because different antibodies were used to detect cleaved vs. total gp.) we found that a higher percentage of gp on the plasma membrane was cleaved for cells coexpressing gp furin and furin than for cells expressing wt gp or gp furin alone ( fig c) . western blot analyses, using an anti-flag or an anti-gp antibody (kind gift of james cunningham), showed that the addition of furin increased the amount of cleaved gp furin construct as compared to gp furin alone (fig d, lanes and in left and right panels). furin did not cleave any wt gp (lanes ). we used these constructs to verify that an increased cleavage of ebov gp led to a greater extent of fusion ( fig e) . cotransfecting cells with gp and furin (bar ) led to the same extent of fusion as did transfection of gp alone (bar ). in contrast, cotransfecting with gp furin and furin led to more fusion (bar ) than transfecting only gp furin (bar ). control experiments of transfecting only furin showed that furin, per se, did not promote fusion (bar ). these experiments, taken together, establish that ebov gp does appear on the cell surface, that some of it is cleaved, and that for the gp furin construct, cleavage is augmented by coexpression of furin. to further confirm that the observed fusion was indeed mediated by ebov gp, we utilized mutations that had previously been shown to greatly reduce viral infection [ ] . we used mlv pseudovirus expressing gp, and observed that, indeed, the level of infection caused by the point mutant w a (fig , bar ) , the double mutant g a/g a (bar ), and the point mutant i a (bar ) were all substantially less than for wt gp ( bar ) . we then measured the extents of cell-cell fusion mediated by each of the mutant proteins. the extent of fusion in absence of thermolysin treatment supported by all three of the mutants (fig b, bars , , and ) was much less than for wt gp (bar ). flow cytometry measurements, using the same cells as for fusion experiments, showed that each of the mutant gps was well expressed on the cell surface ( fig c) . these experiments provide support that reduced infectivity by ebov correlates with reduced gp-mediated fusion. we next tested . , a small molecule inhibitor against npc , which prevents ebov entry, as well as testing a neutralizing antibody (kz ) against ebov gp. we found that both significantly reduced ebov gp-mediated cell-cell fusion (fig a and b ). the inhibitor . greatly reduced ebov gp-mediated fusion but did not significantly alter cell-cell fusion induced by either semliki forest virus (sfv) e /e or iav ha (fig a, . at μm). similarly, the neutralizing antibody kz , which recognizes the interface between gp and gp [ ] , reduced ebov gp-mediated fusion, but not sfv-e /e or iav ha-induced fusion ( another central fingerprint of gp-mediated fusion is inhibition of ebov infectivity by bafilomycin a (bafa ). by neutralizing endosomes, bafa inhibits infection, at least in part, by reducing cathepsin activity which in turn results is reduced cleavage of gp . we found that addition of bafa ( or nm) reduced the amount of cleaved gp that appeared on the cell surface ( fig c, bar vs bar ). this occurred despite a consistently greater amount of total gp in the plasma membrane after the addition of bafa (fig d) . (this greater amount was unexpected. possibly, bafa prevented lysosomal degradation of gp.) normalizing the amount of cleaved gp by the total shows that cleavage of cell surface gp was significantly reduced by bafa ( fig e) . thus, all data support the conclusion that the aqueous dye spread we observe is due to fusion induced by ebov gp. many of the effects of ph on kinetics and extents of ebov gp-induced fusion we found were unexpected and quite different than those of ph-dependent fusion for other viral proteins. notably, the extents of fusion did not monotonically increase as ph was progressively lowered, and the apparent ph dependence qualitatively varied with the times of reneutralization (fig ) . after a ph . pulse, the extents of fusion were always greater than those achieved after more acidic pulses; following a ph . pulse (at short incubation times (i.e., min) after the shift to neutral ph), more fusion was observed than for a less acidic pulse ( fig a) . however, for ph pulses of . and above, as the reneutralization time was increased, the extents of fusion became effector cells were labeled with dio and target cells with dii. both dyes were excited by a nm laser; dio emission was detected at nm and dii emission was recorded for nm. for facs measurements, trypsin and edta were added to cells prior to assaying; this treatment separates bound cells back into individual cells, but does not separate fused cells. representative data for lipid dye mixing is shown for a -min ph . pulse (left panel), and a ph . pulse (right panel). (e) average percentage of fusion as determined by lipid dye mixing as a function of ph (bars , , and ). lipid dye spread was negligible when using effector cells that were mock-transfected (bar ). thresholds for dio and dii were the same for all experiments and indicated on the two panels. fusion was scored as the percentage of fluorescent particles above both thresholds (i.e., the third quadrant). error bars are sem (n = , for each bar) and extents of fusion were statistically compared to the extent for a ph . pulse. * p < . ; *** p < . . doi: . /journal.ppat. .g less dependent on ph; fusion was independent of ph for . and above after a h reneutralization ( fig a) . in contrast, effector cells expressing iav ha showed the typical and expected response of greater extents of fusion for lower ph values at all times after reneutralization; fusion events reached their full extents after a min reneutralization (fig b, using the same protocol as for ebov gp experiments). thus, iav ha induces fusion more rapidly than does ebov gp. in separate experiments, we compared extents of ebov gp-mediated fusion after a h and h reneutralization that followed min, room temperature, acidic ph pulses ( fig c) . after the h reneutralization, fusion was relatively independent of the acidity of the ph pulse, and a low ph pulse did not greatly augment fusion (compare filled bars to open bars, fig c) . equality in final extents of fusion at ph . and . could be a consequence of all cell pairs quickly fusing at low ph, thereby eliminating the possibility of further fusion, although we consider this unlikely. in addition to single cell measurements of aqueous dye transfer, we also monitored lipid dye continuity between effector cells (treated with thermolysin) and target cells. we labeled effector cells with the lipophilic fluorescent dye dio and labeled target cells with dii and determined extents of fusion by flow cytometry (facs). the double positive cells (i.e., the third quadrant) are clearly products of hemifusion or cell-cell fusion ( fig d) . for effector cells treated with thermolysin, the percentage of fusion for the representative experiment was . % at ph . , the optimal ph for fusion ( fig d, second panel) and only . % at ph . (first panel). averaging six separate experiments for each condition, after a -h reneutralization, lipid mixing was greatest for a -min ph . pulse, and less for a ph . pulse than for cells maintained at neutral ph ( fig e) . the approximately two-fold greater fusion determined by flow cytometry at ph . than at . is also in agreement with the data for spread of calcein (fig ) . for mocktransfected effector cells, virtually no lipid dye spread was observed between effector and target cells (fig e) , in agreement with the aqueous dye spread measurements. therefore it is clear that ebov gp mediates a considerable amount of cell-cell fusion, and does so at an optimal ph of . . once calcein movement from effector to target cell commenced, it continued for ebov gpmediated fusion, but at an extremely slow rate. in general, the fluorescence due to calcein never equalized between target and effector cells for ebov gp-induced fusion (fig ) . in contrast, for fusion pores created by other viral fusion proteins [ , ] , such as jsrv env ( fig a, upper images) , the fluorescence did equalize. it is possible that the ebov gp pores eventually closed, preventing calcein from attaining the same concentration in effector and target cells. we therefore quantified the rate of transfer of calcein by plotting calcein fluorescence of effector and target cells as a function of time ( fig b) . for ebov gp-induced pores (red curve), the transfer occurred over a time course of tens of minutes, and over this period the increasing fluorescence of a target cell never equalized the decreasing fluorescence of an effector cell ( fig b) . the fluorescence of the effector and target cells, on the other hand, equalized within a minute or so for jsrv env-mediated pores (fig b, blue curve) . the exceedingly slow transfer of calcein is a further indicator that ebov gp-mediated pores remained extremely small. the fact that calcein transferred, albeit slowly over long times, shows that the fusion pores did not irreversibly close (or if they did, new pores opened) within tens of minutes of formation. as a control, we added saponin to effector cells and measured release of calcein to be sure that the dye did not become compartmentalized and therefore failed to transfer for reasons unrelated to the size of the fusion pore. release was fast from the saponin-treated cells and was almost complete within s, demonstrating that the overwhelming majority of calcein was, in fact, free and mobile. we further studied the size and rate of growth of ebov gp-mediated fusion pores by assessing the size of dyes that can permeate these pores over time. we loaded effector cells with cmtmr in addition to calcein. cmtmr forms disulfide bonds with the tri-peptide glutathione, and these complexes are somewhat larger than calcein. the complexed glutathione can also form disulfide bonds with cytosolic proteins and hence cmtmr fluorescently labels proteins that are much larger than calcein. as a consequence, the size distribution of molecules labeled by cmtmr is expected to be quite diverse, some only somewhat larger than calcein and others very much larger. we found that cmtmr transferred for only - % of the cell pairs for which calcein exchange occurred (fig c) . the relative inability of cmtmr to spread indicates that fusion pores typically did not enlarge sufficiently to allow passage of a molecule of the size of the nucleocapsid of ebov. in actual viral infection, factors absent in our model the small difference in fluorescence of effector and target cells connected by the jsrv env pore indicates that a small percentage of calcein is not free to transfer, possibly because it is bound to cellular elements. (c) images of effector cells loaded with calcein am (green) and cmtmr (red) and target cells loaded with cmac (blue) taken after a hr reneutralization at °c that followed a -min ph . pulse. calcein has transferred (white arrows, left panel) from most of the effector cells in contact with target cells. cmtmr (middle panel) mixed with cmac for only one cell (brown arrow). an overlay of calcein and cmtmr is shown in the third panel, with calcein (white arrows) and cmtmr (brown arrow) transfer shown. doi: . /journal.ppat. .g system are probably promoting expansion of the fusion pore connecting an envelope and endosomal membrane. we used electrical capacitance measurements to directly and quantitatively assess fusion pore size. the slow time course for ebov gp-mediated fusion necessitated that the tight electrical seal between the patch pipette and plasma membrane be maintained for long times. this proved difficult in practice. we were able, however, to electrically observe pores between cell pairs in three cases, and in these cases the pores never enlarged within s of formation and generally fluctuated within small values of conductance (fig a) . the conductance of the fluctuating pores did not return to baseline, showing that the pores did not close, but instead remained restricted to a small size. by way of comparison, it can be readily seen from representative traces of electrically measured fusion pores created by other viral proteins (fig b) that fusion pores generally significantly enlarge over time. the absence of pore enlargement for ebov gp suggests that many of the prior attempts at monitoring cell-cell fusion mediated by this fusion protein did not succeed because the reporter molecules that needed to permeate the fusion pore for detection of fusion were too large to pass through the pore. although only three pores were electrophysiologically measured, the finding that each of them did not exhibit increased conductance over time implies that the slow passage of fluorescent dyes through them was not due to structures that prevent their access to the pores. slow pore enlargement could be due to a number of factors, including slow recruitment and incorporation of additional copies of cleaved gp into the wall of the pore, or slow accumulation of lipids into the wall. we added nh cl to external media to test whether acidic intracellular compartments were essential for ebov gp-mediated cell-cell fusion. the addition of mm nh cl greatly reduced fusion after a -min ph . pulse in the absence of thermolysin treatment, so as to avoid activating uncleaved ebov gp on the cell surface (fig a) . in contrast, the addition of mm nh cl did not affect fusion induced by an optimal ph pulse for either sfv e /e or iav ha (fig a) . similarly, μm chloroquine inhibited cell-cell fusion mediated by the fusion protein of ebov, but not by the proteins from either sfv or iav (fig a) . the elimination of fusion by the addition of mm nh cl (bar ; same conditions as in fig a) was most likely caused by reducing cathepsin activity through neutralization of intracellular compartments: it was largely reversed by adding a recombinant cathepsin b to the external solution ( fig b, bar ) . because the normally acidic intracellular compartments were neutralized by nh cl, the pool of ebov gp on the cell surface that was previously uncleaved must have been cleaved by the added membrane-impermeant recombinant protease. the dose-response curves for inhibition of fusion by chloroquine (fig c) or nh cl (fig d) verified that inhibition of fusion is increased with increasing concentration of the neutralizing agent. therefore, even if the external solution is acidified, ebov gp-mediated cell-cell fusion does not occur unless the acidity of intracellular organelles is maintained. we conclude that ebov gp present on the cell surface requires an intracellular compartment for cleavage, as is consistent with previous reports. it is possible, however, that there are copies of cathepsins in the plasma membrane, and acidification of the external solution activates them to cleave ebov gp. proteinase k (pk) has proved useful for assessing conformational changes that viral proteins undergo at different stages of fusion [ , ] . we found that ebov gp was pk-sensitive for all steps of the fusion process (s text and s a fig) , that fusion was restored over time after removing pk (s b fig), and that normal cellular trafficking of protein led to replacement of proteolytically digested gp with newly delivered intact gp (s fig). we also used brefeldin a (bfa, μm)-an inhibitor of trafficking from endoplasmic reticulum to golgi-to further characterize the consequences for fusion of altering intracellular trafficking of ebov gp. here we found that treatments expected to increase the amounts of cleaved ebov gp on the cell surface led to greater extents of fusion (s text and s fig). ph-dependent cathepsin activity is essential for gp-mediated fusion for virus internalized in endosomes, ebov gp is believed to be cleaved by cathepsins b and l, but not by cathepsins a or d. we prevented cathepsin-induced cleavage by treating bound effector and target cells with a cathepsin b inhibitor (ca- ) or a cathepsin l inhibitor (z-fy-cho). in the absence of thermolysin treatment, the inhibitors led to significantly reduced fusion at both neutral and low ph (fig a, compare "untreated" and "treated": as always, changes of solutions containing membrane-impermeant buffers were used to control ph). using inhibitors against cathepsin a (lactacystin) or cathepsin d (pepstatin a)-neither of which is thought to cleave ebov gp-did not lead to reduced fusion using the same protocol as for the cathepsin b and l inhibition experiments (fig a) . these results provide strong support that fusion observed in our experiments in the absence of thermolysin treatment is due to, at least in part, copies of ebov gp on the cell surface that have their gp subunits cleaved by cathepsins. these results also document that neither cathepsin a or d cleaves ebov into a fusion-competent form. from the results as a whole, it is clear that low ph does not induce fusion unless the gp subunit has been cleaved. it is known that cathepsin activity is increased by acidity. we suggest that low ph acts, at least in part, by augmenting cathepsin activity on the cell surface. the same pattern of ph-dependence of fusion was observed for effector cells treated with thermolysin while cathepsin activity was continually inhibited: fusion was dependent on ph and was significantly reduced by the cathepsin b inhibitor or the cathepsin l inhibitor (fig a, thermolysin-treated, bars and of each set of columns), but was relatively unaffected by cathepsin a or d inhibitors (bars and ). cell-cell fusion exhibits a maximum at ph . (column compared to column - ). several cathepsins exhibit maximal activity in the ph range of . to . [ ] , so the maximum extent of fusion at ph . would likely be due to the ph dependence of cathepsin activity on the cell surface. control experiments provide additional support for the conclusion that cathepsins aid ebov gp-mediated fusion between cells. blocking cathepsin b (by adding the cathepsin b inhibitor) immediately after application of an acidic ph pulse resulted in a substantial . for effector cells not thermolysin-treated, fusion experiments were performed in the absence of a low-ph pulse (first set of columns) and with a ph . pulse (second set of columns). for cells treated with thermolysin, cathepsin inhibitors were added prior to the thermolysin treatment and maintained throughout the experiments. for thermolysin-treated cells, fusion was measured for the case without (third set of bars) and with a low ph pulse (fourth set). only inhibitors of cathepsin l or b diminished fusion, and they did so for all conditions. each of the four cathepsin inhibitors was added (separately) at the time of mixing effector and target cells. the cathepsin l and cathepsin b inhibitors reduced fusion to the same extent at ph . and ph . . the cathepsin b and d inhibitors were without effect. fusion was measured hr after the low ph pulse. the concentration of all inhibitors was μm, a high concentration to ensure maximal inhibition. (b) in contrast to the experiments in (a), the cathepsin b inhibitor was added subsequent to the low ph . pulse (second bar). less fusion occurred than for control (filled bar). (c) a recombinant human cathepsin b (rh catb, μm) was added to effector cells that had not been treated with thermolysin. the addition of rh catb (hashed bar) led to substantially increased fusion (filled bar, control). a -min ph . pulse was applied to promote fusion. the extents of fusion were measured after a -hr reneutralization for all experiments of this figure. error bars are sem. (d) t cells were co-transfected with plasmids encoding ebov gp and cathepsin b. cleaved gp on the plasma membrane was measured by flow cytometry using snpc , and the cleaved form was normalized by the total gp (measured by anti-flag), as described in fig . alternatively, gpexpressing cells were treated with thermolysin, and cleaved gp was measured and normalized as described. **p< . ; ***p< . . reduction in the extent of fusion after a -h reneutralization (fig b, effector cells were thermolysin-treated). the reduction from the control was~ -fold; a -fold reduction also occurred when the cathepsin b inhibitor was constantly present (see fig b) . the nearly equal percentages of inhibition of fusion are expected, since in the presence of the cathepsin inhibitor, uncleaved copies of ebov gp would not be cleaved during the period of reneutralization. thus, low ph appears to promote cleavage of ebov gp by cathepsins on the cell surface. incubating effector cells that were not treated with thermolysin with a recombinant human cathepsin b (rhcat b) (fig c, bar ) increased fusion significantly over the control (bar ). the simplest explanation for this increase is that the recombinant protein led to a higher level of gp cleavage than that induced by endogenous cellular cathepsins. to explicitly test whether increasing the activity of cathepsin increased the likelihood that gp on the cell surface was cleaved, we cotransfected cells to express gp and cathepsin b and used snpc to measure the percentage of gp in the plasma membrane that was cleaved (as described for fig ) . this percentage was greater (fig , column ) than the control (column ) in the presence of cathepsin b transfection. using the same techniques, we also showed that adding thermolysin to solution did indeed increase cleavage of cell surface gp (fig d) . does low ph directly cause conformational changes in ebov gp to induce fusion, or does it work via increasing the activity of cathepsins, or both [ , ] ? we were able to approach these questions by using the ability of bfa to effectively block delivery of ebov gp to the cell surface and, independently, by using cathepsin inhibitors to prevent gp cleavage. we incubated effector cells with bfa for min to prevent further delivery of ebov gp to the plasma membrane prior to a thermolysin-treatment, and maintained the presence of the drug during all solution changes. the extent of fusion was independent of ph, and considerably less than when the trafficking inhibitor was not employed (fig a) . the clear conclusion is that, with bfa present, all fusion was caused by copies of ebov gp that had been cleaved by thermolysin and that remained on the cell surface. the finding that ph pulses did not affect the extent of fusion at all shows that acidity did not promote the conformational changes in cleaved ebov gp that would lead to fusion. we inhibited cathepsin activity to further test the conclusion that once cleaved, ebov gp no longer requires low ph to induce fusion. we performed experiments in which ca- , a cathepsin b inhibitor, was continually present (fig b) . the inhibitor was added to isolated effector cells and maintained for min to ensure that all ebov gp delivered to the plasma membrane was not cleaved. effector cells were then thermolysin-treated, always maintaining the inhibitor. these cells were bound to target cells, and the external solution was acidified to ph . ; after reneutralization, cells were maintained for h at °c, with all manipulations performed in the presence of the inhibitor (fig b, experimental protocol illustrated on top) . the extent of fusion was greater when the inhibitor was not added (control): this indicates that delivery to the cell surface of ebov gp cleaved by endosomal cathepsin (subsequent to thermolysin treatment) significantly contributes to fusion. more importantly-and central to the mechanism of ebov gp-mediated fusion-the extent of fusion was independent of ph. this finding strongly implies that acidic conditions have no direct effect on ebov gp-mediated fusion. the ph dependence of fusion is solely due to the ability of cathepsin to cleave ebov gp; once cleaved, acidic conditions directly induce conformational changes in cleaved ebov gp that lead to fusion. using both cytoplasmic and membrane dye transfer assays, we established that known properties of ebov fusion occurring within endosomes are replicated by our cell-cell fusion system and that specific inhibitors of ebov infection-the small molecule inhibitor . and a neutralizing antibody kz -block fusion. the inhibition of ebov gp-mediated cell-cell fusion (but not iav ha or sfv e /e fusion) by the lysosomotropic agents nh cl and chloroquine is expected: ebov gp cleavage is eliminated because cathepsin activity is greatly reduced by neutralization of endosomes; inhibiting cathepsin activity reduces cleavage of ebov gp [ , ] . we also showed that copies of npc reside in the target membrane and some gp resides in the plasma membrane, and that a fraction of the gp is properly cleaved. it is virtually certain that the cell-cell fusion process investigated in the present study is mediated by ebov gp. it is likely that past lack of success in observing cell-cell fusion is attributable to the fact that the fusion pore mediated by ebov gp on the cell surface remains small. over the time scales of electrical measurements, the pore does not enlarge at all. based on fluorescence dye spread measurements, it enlarges more slowly and to a lesser extent than any other pore mediated by a viral fusion protein of which we are aware, and it may even tend to close. the ebov gp fusion pore is large enough to allow the passage of calcein, but just barely. there is little doubt that the fluorescent dye cmtmr does not permeate the pore because virtually all of it complexes with proteins; the complex becomes permeable only after a pore enlarges. electrical measurements directly demonstrate that the ebov gp-induced pore remains small. over the course of time in our cell-cell fusion experiments, ebov gp-mediated fusion pores do not significantly enlarge. the question now becomes: how readily does a fusion pore enlarge when connecting an ebov envelope with an endosomal membrane? this fusion pore must expand to sizes that permit passage of the large viral nucleocapsid. four major possibilities present themselves: (i) the necessary enlargement is extremely slow for the endosome-viral pores; (ii) elements engaging plasma but not endosomal membranes, such as cytoskeleton, retard the growth of fusion pores; (iii) a protein (such as the two-pore calcium channel, present in the endosomal compartments that support ebov fusion [ ] ) is required for pore enlargement; (iv) control of calcium concentrations (e.g., through the two-pore channels) regulates fusion pore formation or enlargement in endosomes. methods to monitor the formation and growth of fusion pores of ebov gp-bearing viral particles within endosomes will be needed to answer these questions [ ] . we have unambiguously shown that a fraction of ebov gp on the cell surface is cleaved. by using the gp furin construct we also demonstrated that increased cleavage correlates with greater fusion. in addition, we functionally evaluated the cleavage status of ebov gp on the cell surface by adding a water-soluble recombinant cathepsin b or thermolysin to solution and found that these proteases promoted fusion. late endosomes and lysosomes are generally thought to be the cellular site of cleavage of ebov gp by cathepsins [ ] ; it is likely that a fraction of ebov gp is cleaved within endosomes and then recycled to the plasma membrane where it mediates cell-cell fusion, independent of ph (fig ) . we suggest that uncleaved ebov gp that reaches the surface is cleaved upon acidification of the external solution by cathepsins within the plasma membrane. thermolysin cleaves uncleaved copies of ebov gp that are delivered to the cell surface, accounting for the enhancement of fusion by the addition of the protease. regardless of the site of gp cleavage, an appreciable fraction of the gp subunit is indeed cleaved into its fusion-competent form after the addition of thermolysin. npc serves as receptor for ebov gp in endosomes, and is essential for the virus to infect a cell. we have now shown that npc is not confined only to intracellular membranes, but rather that some copies reside in plasma membranes. our finding that snpc promotes ebov gp-mediated cell-cell fusion suggests that domain c of npc alone is sufficient to induce the needed conformational changes in the fusion protein. it is well established that the extents of cell-cell fusion correlate with the levels of viral fusion protein expression on cell surfaces [ , ] . thus, it is not surprising that the extents of cell-cell fusion induced by ebov gp are affected by its delivery to, and loss from, plasma membranes. for some viral fusion proteins, such as the paramyxovirus hendra and nipah virus f proteins, and sars coronavirus s protein, cell-cell fusion is sensitive to protein cycling [ , , ] . these proteins require acidic intracellular compartments for cleavage: endosomes for nipah virus [ ] , and endosomes and the trans-golgi network for hendra virus [ ] . but the dependence of cell-cell fusion on protein trafficking is unusual for typical ph-dependent viral fusion proteins; for these proteins acidity does not promote cleavage, but instead directly induces conformational changes [ ] . once activated, these typical low-ph-dependent fusion proteins quickly inactivate if they do not promote fusion [ ] . hence, protein delivered to the cell surface subsequent to an acidic pulse will not be able to promote fusion. in contrast, proteins that induce fusion at neutral ph will promote fusion once they are delivered to the cell surface. our results show that ebov gp cleaved by endosomal cathepsins are no longer sensitive to ph and therefore can induce fusion once they arrive at the plasma membrane. infectivity is subject to processes other than fusion, and so infectivity need not always correlate with extents of cell-cell fusion. for example, it has recently been shown that the activity of two-pore calcium channels in endosomes is required for ebov infection [ ] , and that ebov infects by fusing to endosomal membranes that contain both npc and the two-pore channel [ ] . tetrandrine blocks these channels and inhibits ebov infection. we found that tetrandrine ( nm) did not affect ebov gp-mediated cell-cell fusion. it is notable that the extent of fusion that occurs after h at neutral ph was roughly equal to the extent that follows a ph . pulse. because fusion induced by cleaved gp is ph-independent, we interpret the continual increase in fusion at neutral ph over time to be a consequence of intracellular trafficking: new copies of ebov gp continually replace or supplement old copies and these new/supplemented, cleaved copies can cause fusion between cells that had not previously fused. thus, acidification likely promotes more fusion at early times through activation of surface cathepsins that cleave ebov gp. however, it is not presently clear why fusion kinetics is faster after a ph . pulse than after a ph . (or more acidic) pulse. the ph dependence of cathepsin activity is complicated [ ] . while activity generally increases with acidification, some cathepsins exhibit the same activity in the range of ph as at lower values of ph [ ] . for others, activities are maximal at an intermediate ph, such as . [ ] . also, the ph dependence of cathepsin activity varies with environment and conditions, such as redox potentials on each side of the membrane in which a cathepsin resides [ , ] . any relevant cathepsins (e.g., b or l) on the cell surface can, at their optimal ph, cleave ebov gp. a direct test of whether ebov gp on the cell surface is maximally cleaved by cathepsins at ph . will require methods to measure the percentage of ebov gp that is cleaved as well as cathepsin activity at the cell surface. the role of acidic ph in ebov fusion has been debated in the field [ , , ] . our data unambiguously show that cell-cell fusion is regulated by extracellular ph. the acidity of the extracellular solution can, in principle, augment both the activity of cathepsins that reside in the plasma membrane and directly promote conformational changes of cleaved ebov gp on the cell surface. (although cathepsins are regarded as endosomal membrane proteins, some copies also likely reside in plasma membranes from which many endosomes derive [ ] ). the great reductions in fusion caused by inhibition of cathepsin activity and recovery of fusion by addition of a recombinant cathepsin establish that cathepsins' activity at the cell surface is consequential. independent experiments in which cathepsin activity was inhibited or delivery of protein to cell surface was blocked show that the ph-dependence of fusion is eliminated once ebov gp is cleaved. this demonstrates that fusion mediated by the cleaved form is intrinsically ph-independent. that is, cleaved ebov gp is essentially a neutral ph fusion-inducing protein; all the experimentally observed and biological relevant ph-dependence is a consequence of cathepsin activity. the faster kinetics of cell-cell fusion after a ph . pulse than for pulses at higher values can be accounted for by greater cleavage of ebov at the cells surface at ph . . a previous study used model peptides to mimic the six-helix bundle of ebov gp and found that low ph increased bundle stability [ ] . the stage of fusion in which bundle formation occurs has not been identified for ebov gp. it may be, for example, that the bundles form subsequent to pore formation, as occurs for hiv env [ ] , and that increased bundle stability aids pore enlargement, but not fusion itself [ ] . alternatively, the model peptide may not mimic bundle stability within a full length, structurally intact gp. as a general rule, fusion kinetics for viral proteins that induce fusion at neutral ph are slower than for proteins that utilize low ph as a trigger. this could explain the slow fusion kinetics of ebov gp mediated fusion, despite classification as a low ph-requiring process. alternatively, a need to continually deliver ebov gp could be the reason ebov gp-mediated cell-cell fusion is slow. the development of an experimentally convenient system of ebov gpmediated fusion should make it possible to determine molecular mechanisms by which ebov releases its genome into infected cells. our results and conclusions are diagrammatically summarized in fig . npc is an intracellular receptor for ebov gp within endosomes. but, as we have shown, npc can also reside in the plasma membrane. endosomal cathepsins cleave ebov gp, and any cathepsins that reside in plasma membranes will also cleave surface gp upon acidification of the external solution. both cleaved and uncleaved copies of ebov gp are continually delivered to and retrieved from the surface, and hence intracellular trafficking contributes to extents and kinetics of fusion. but binding of ebov gp to the target membrane should inhibit endocytotic retrieval. consequently, ebov gp (both cleaved and uncleaved) should accumulate at potential fusion sites, leading to more fusion over time. preventing acidification of endosomes to block cleavage of ebov gp, or inhibiting delivery of the protein to the cell surface, greatly reduces fusion. acidification of the external solution to ph . increases the activity of cathepsins that reside in the cell surface, and this results in additional cleavage of ebov gp. the addition of thermolysin converts all surface gp to the cleaved form, thereby resulting in the maximal extent of fusion. that cell-cell fusion induced by cleaved ebov gp does not depend on ph, provides critical insight into the mechanism of ebov entry and infection. . for this work, we mainly used an n-terminal flag-tagged, mucin-deleted ebov gp construct by replacing the signal peptide of gp with that of preprotrypsin followed by a flag sequence. all gp mutants, including gp furin , were made by overlapping pcr using the flag-tagged gp construct as the template. the plasmid to express jsrv env has been described [ ] . to express iav ha for dye spread experiments, we used the x strain (plasmid provided by judith white, university of virginia, charlottesville, va). a standard calcium phosphate method was used to express sfv e /e via transfection of the pcb -wt vector, plasmid provided by margaret kielian, albert einstein college of medicine, bronx, ny [ ] . a small molecular inhibitor, . , was a gift of james cunningham (harvard medical school, boston, ma). hek t and cos cells employed have been previously described [ ] . the hek t cells stably expressing were generated by transducing cells with pbabe retroviral vector expressing npc (gift of kartik chandran) followed by puromycin selection (sigma, μg/ml). all cells were grown in dulbecco's modified eagle's (dmem) medium, supplemented with . % penicillin/streptomycin plus % fetal bovine serum (fbs). schematic diagram illustrating control of fusion by cleavage of ebov gp and its transport to the cell surface. ebov gp is synthesized in er, transported to golgi complexes where it is processed into gp and gp subunits, and ultimately targeted to the plasma membrane. ebov gp can undergo endocytosis from the plasma membrane and eventually reach late endosomes and lysosomes. it is then further cleaved by cellular cathepsins, bound by npc , and recycled back to plasma membrane. alternatively, ebov gp is directly cleaved by cathepsins on the plasma membrane or by thermolysin treatment. ebov gp proteins do not permanently remain on the surface, but rather undergo continual delivery and removal. cleaved gp accumulates at potential fusion sites, leading to the observed increased fusion over time. solid arrows denote pathways definitively established in the present study. dashed arrows denote pathways that are likely to occur based on data of the present study. light dashed arrows denote pathways suggested by data of the present study. for all experiments using ebov gp, cos cells were maintained in eagle's medium with glucose, l-glutamine, and sodium pyruvate, supplemented with % cosmic calf serum (hyclone, logan, utah), pen strep (gibco), and . mg/ml g sulfate (cell gro, manassas, va), and transfected to express ebov gp by a standard calcium phosphate procedure [ ] . about~ x cells were loaded with . μm calcein-am as previously described [ ] and sometimes coloaded with μm -(and- )-((( -chloromethyl)benzoyl)amino)tetramethylrhodamine) (cmtmr) [ ] . if these effector cells were thermolysin-treated to cleave ebov gp, μg/ml thermolysin was incubated with the cells for min at room temperature. exchanging the solution with dmem removed thermolysin; residual thermolysin was further removed by spinning down the cells three times and replacing the aqueous solution. hek t cells were maintained in the same media and in the same way as cos cells and were used as targets.~ x cells were loaded with μm cmac. effector cells were mixed, including a gentle vortex-in a tube containing either pbs ++ (sometimes supplemented with mg/ml bsa) or dmem-with the labeled target hek t cells. the cells were added into polylysine-coated ( mg/ml) wells of an -well slide (thermo fisher) [ ] and allowed to settle and adhere to the bottom for min at room temperature. the ph was lowered (or not) for min at room temperature to the indicated value (ph . unless stated otherwise, using an exchange solution consisting of mm nacl, . mm kcl, . mm mgcl , . mm cacl , mm mes), the solution was then reneutralized to ph . by an exchange of solutions, and the temperature raised to °c. after this reneutralization for the indicated time, generally h, fusion was scored as a function of time by the transfer of calcein into target cells, as described [ ] . for fusion experiments utilizing iva ha as control, the expressed ha in effector cells were was cleaved into ha -ha subunits with trypsin, as previously described [ ] . to label effector cells,~ x cells/ml were incubated with μm dio for min at °c. the day before an experiment, target cells were split and plated on glass cover slips placed in culture dishes so as to allow convenient transfer. these target cells were labeled by μm dii for min at °c. labeled effector cells were thermolysin treated ( μg/ml) and added above labeled target cells. binding was allowed to occur for min at room temperature before washing out unbound effector cells. the solutions bathing the cover slips (one cover slip per culture dish) was acidified to the indicated ph for min at room temperature, and the culture dish placed in a °c incubator for h. cells were detached from cover slips by adding μg/ml trypsin to a divalent-free solution containing . mm edta for min at room temperature, followed by vigorous, repeated pipetting to dissociate bound (i.e., neither hemifused or fused) cells. colocalization of the two lipid dyes was monitored by flow cytometry (guava easy cite, guava technology, millipore), using two channels emission, one for each dye ( nm for dio and nm for dii; both excited by a nm laser). the same protocols were followed for mock-transfected cos cells; this data was subtracted from data obtain for cos cells expressing ebov gp to obtain percentages of fused cells. the domain c of npc was cloned into a pgex- t vector that had a gst tag on the n-terminus (ge healthcare life sciences, pittsburgh, pa). the expression of fusion protein was induced in e. coli. by iptg ( . mm) and purified by glutathione sepharose b (ge healthcare life sciences). protein was quantified by a bradford assay and used for cell-cell fusion and for measurements of cleaved gp. the expression of npc on t cell surfaces was determined by using anti-npc (against n-terminus - aa; lifespan biosciences). determination of ebov gp cleavage. hek t cells were transfected with ebov gp or gp furin in the presence or absence of a plasmid that encodes furin (kind gift of paul bates, university of pennsylvania). transfected cells were detached by pbs containing mm edta. one portion ( million cells) was used to measure the cleaved gp by incubating cells with μg snpc for h on ice, followed by adding a mouse monoclonal anti-gst antibody; the fluorescence signal was quantified by adding a fitc-conjugated secondary anti-mouse antibody using flow cytometry. another portion of the transfected cells (also million) was used to measure the total gp expression on the cell surface, using an anti-flag antibody. the total gp, as determined by mean fluorescence intensity (mfi), was used to determine the percentage of gp on the plasma membrane that was cleaved: the amount of cleaved normalized by total gp. cleavage of gp furin in cell lysates was determined by western blotting using anti-flag or an anti-gp antibody. production of mlv retroviral pseudotypes bearing ebov gp and viral infection were as described previously [ ] . briefly, gp/lapsn packaging cells stably expressing mlv gag-pol and alkaline phosphatase (ap) were transfected with plasmids encoding the ebov gp or mutants, and the viruses produced were used to infect htx cells (a subclone of ht ). viral infectivity was determined by counting ap + foci h after infection. we proteolytically determined the stages of fusion at which ebov gp was proteinase k-sensitive by treating cells with . mg/ml proteinase k for min, and maintaining or removing it as indicated, at various points in our protocol (s fig). we cleaved ebov gp with thermolysin and used our standard protocol (a -min ph . pulse), incubated the cells for h at ph . , and then measured fusion. all experiments were performed in parallel on the same days; as controls, proteinase k was not employed and extents of fusion were measured. brefeldin a ( μm) was used to inhibit anterograde protein trafficking as described in the experiments of s fig. we determined the latency between lowering ph and fusion by using cells mixed within a tube, placed over poly-lysine coated cover slips within a culture dish maintained at °c and allowed to settle for min. the ph was lowered to the indicated value for min at room temperature through an exchange of solutions. the cover slips were then transferred into dishes at °c, neutral ph, for indicated times. the time of transfer is defined as time = . for thermolysin-treated cells, the effector cells were treated prior to binding to target cells. the extents of fusion were quantified at varied times by spread of calcein. we used the rate of accumulation of calcein into the target cell and its depletion in effector cells to access the size of the fusion pore as a function of time [ ] . the fluorescence of both effector and target cells was proportional to calcein concentration, as verified by the procedure detailed in [ ] . neutralizing intracellular compartments nh cl ( mm unless otherwise noted) was added to the bathing solution and maintained throughout the course of an experiment, including during any low ph pulses, to obtain neutralization. the same procedure was used with chloroquine ( μm unless stated otherwise) or bafa ( or nm) to cause endosome neutralization. the ionic and buffering contents of the nh cl-containing low ph solutions were pre-adjusted to the required ph so as to maintain osmotic strength at mosm during low ph pulses. to monitor ebov gp expression and its recovery after proteinase k treatment, cells were treated with . mg/ml of the protease for min at room temperature, and the staining protocol now described was used immediately or after cells were maintained for h in dmem at °c, as indicated. ebov gp-expressing cos cells were transferred to ml tubes and incubated for h at room temperature with a primary antibody (human anti-ebov gp-kz stock at . mg/ml, ibt bioservices, gaithersburg, md) that was diluted : in pbs ++ that was supplemented with % fetal bovine serum. after three washes with the % fbs-pbs ++ solutions, a secondary fitc-conjugated goat anti-human antibody (fisher scientific) was added at a final concentration of μg/ml and maintained for min at room temperature in the dark. after cells were washed twice, they were added to -well slides that had been treated with poly-l-lysine (m.w. , - , , sigma aldrich, st. louis, mo). the cells were then fixed for min at room temperature with % paraformaldehyde, and washed twice with dmem. pore size over time was also quantified by using patch clamp time-resolved admittance measurements [ ] , often referred to as capacitance measurements. fusion was promoted by a -min ph . pulse at room temperature. because the fusion pore took considerable time to form, and a seal between the patch pipette and cell could only be maintained once solutions and temperature were established, temperature was raised to °c for~ min before attempting electrical measurements. (this procedure reduced the time between establishing the seal and fusion pore formation). the cover slip was then placed in a temperature-controlled chamber on a microscope stage, and the seal established. the external solution consisted of mm n-methyl-glucamine aspartate- mm mgcl - mm hepes (ph . ); the solution within the patch pipette was mm cesium glutamate- mm mgcl - mm bapta [ , -bis(o-aminophenoxy)ethane-n,n,n ,n -tetraacetate]- mm hepes (ph . ). to electrically characterize fusion pores created by iav ha, aslv env, and hiv env, we used cell lines that stably express the fusion protein and target cells that stably express the cognate receptor: hab cells that express iav ha as effectors and t cells as targets [ ] ; nih t enva cells that express aslv env and t tva cells as targets [ , ] ; and tf cells that express hiv env and hela t cells as targets [ ] . pair-wise student t-tests were used to compare the outcome of a manipulation on fusion as compared to the control. in figures, unless otherwise indicated, a single asterisk ( à ) denotes p < . , two asterisks ( Ãà ) denotes p < . , and three asterisks ( ÃÃà ) denotes p < . . the periods in which proteinase k (pk, μg/ml) was present are marked in the schematic protocol, and numbers correspond to bar numbers below. regardless of whether pk was present prior (bar ) or subsequent (bar ) to treating effector cells with thermolysin, the presence of pk virtually eliminated fusion. similarly, adding and then maintaining pk to effector cells as they were bound with target cells led to greatly reduced fusion (bar ). but adding pk immediately after the low ph pulse (bar ) hardly affected fusion. (b) ebov gp-mediated fusion recovered over time after proteinase k treatment: left-hand bars of each pair denote that a ph pulse was not applied; a ph . pulse was applied for the right hand bars. adding proteinase k and washing out immediately prior to thermolysin treatment virtually abolished fusion (first set of two bars). allowing hr between proteinase k removal and thermolysin treatment restored most of the fusion (second set of bars). waiting h completely restored fusion (third set of bars). the effect of pk treatment on ebov gp expression was assessed using volocity imaging software (perkin elmer). integral fluorescence per field ( image fields per datum point) was calculated after subtracting the fluorescence background determined from the mock-transfected images. this quantification shows that expression of ebov gp was greatly reduced by the proteinase k treatment and significantly recovered after the protease was absent for h. this demonstrates that the ebov gp expression levels were steady over time. (tif) s fig. inhibitors of trafficking show that ebov gp is dynamically exchanged between plasma and intracellular membranes. the presence of brefeldin a (bfa, μm) at all points of the fusion protocol that utilizes thermolysin-treated effector cells and a ph . pulse reduced fusion greatly (bar ) compared to the control (bar , bfa was not included). washing out bfa and immediately treating effector cells with thermolysin led to greater fusion (bar ). waiting min after the washout before thermolysin treatment led to fusion (bar ) comparable to control. adding and maintaining bfa after binding effector and target cells, but before applying a low ph pulse led to substantially reduced fusion (bar ). applying bfa after the low ph pulse led to less fusion than the control (bar ), but to greater fusion than when the drug was added prior to the low ph pulse (bar , extent of fusion higher than for bar ). thermolysin was used to cleave ebov gp just prior to measuring fusion for all conditions of, allowing meaningful comparisons. (tif) ebola haemorrhagic fever progress of vaccine and drug development for ebola preparedness similar uptake but different trafficking and escape routes of reovirus virions and infectious subvirion particles imaged in polarized madin-darby canine kidney cells visualizing infection of individual influenza viruses hiv enters cells via endocytosis and dynamin-dependent fusion with endosomes viral membrane fusion and nucleocapsid delivery into the cytoplasm are distinct events in some flaviviruses cathepsin cleavage potentiates the ebola virus glycoprotein to undergo a subsequent fusion-relevant conformational change detection of cell-cell fusion mediated by ebola virus glycoproteins the primed ebolavirus glycoprotein ( -kilodalton gp , ): sequence and residues critical for host cell binding a system for functional analysis of ebola virus glycoprotein endoproteolytic processing of the ebola virus envelope glycoprotein: cleavage is not required for function differential requirements for clathrin endocytic pathway components in cellular entry by ebola and marburg glycoprotein pseudovirions ebola virus glycoprotein : identification of residues important for binding and postbinding events ebola virus entry requires the cholesterol transporter niemann-pick c small molecule inhibitors reveal niemann-pick c is essential for ebola virus infection biochemical and structural characterization of cathepsin l-processed ebola virus glycoprotein: implications for viral entry and immunogenicity comprehensive analysis of ebola virus gp in viral entry ebola virus entry requires the host-programmed recognition of an intracellular receptor characterization of the receptor-binding domain of ebola glycoprotein in viral entry ebola virus glycoprotein needs an additional trigger, beyond proteolytic priming for membrane fusion cell entry of enveloped viruses biochemical analysis of the secreted and virion glycoproteins of ebola virus zaire ebola virus entry into human dendritic cells is insensitive to cathepsin l inhibition proteolysis of the ebola virus glycoproteins enhances virus binding and infectivity role of endosomal cathepsins in entry mediated by the ebola virus glycoprotein endosomal proteolysis of the ebola virus glycoprotein is necessary for infection structure and function of the complete internal fusion loop from ebolavirus glycoprotein evolution of intermediates of influenza virus hemagglutinin-mediated fusion revealed by kinetic measurements of pore formation evidence that the transition of hiv- gp into a six-helix bundle, not the bundle configuration, induces membrane fusion the pathway of membrane fusion catalyzed by influenza hemagglutinin: restriction of lipids, hemifusion, and lipidic fusion pore formation inner but not outer membrane leaflets control the transition from glycosylphosphatidylinositol-anchored influenza hemagglutinin-induced hemifusion to full fusion a point mutation in the binding subunit of a retroviral envelope protein arrests virus entry at hemifusion single residues in the surface subunits of oncogenic sheep retrovirus envelopes distinguish receptor-mediated triggering for fusion at low ph and infection the niemann-pick c protein resides in a vesicular compartment linked to retrograde transport of multiple lysosomal cargo chinese hamster ovary cell lines selected for resistance to ebolavirus glycoprotein mediated infection are defective for npc expression studies of the "chain reversal regions" of the avian sarcoma/leukosis virus (aslv) and ebolavirus fusion proteins: analogous residues are important, and a his residue unique to enva affects the ph dependence of aslv entry structure of the ebola virus glycoprotein bound to an antibody from a human survivor fusogenicity of jaagsiekte sheep retrovirus envelope protein is dependent on low ph and is enhanced by cytoplasmic tail truncations intermediates in influenza virus pr/ haemagglutinininduced membrane fusion structural and functional aspects of papain-like cysteine proteinases and their protein inhibitors ebola virus (ebov) infection: therapeutic strategies characterization of ebola virus entry by using pseudotyped viruses: identification of receptor-deficient cell lines ebola virus. two-pore channels control ebola virus host cell entry and are drug targets for disease treatment the ebola virus glycoprotein directs fusion through npc + endolysosomes ebola virus and severe acute respiratory syndrome coronavirus display late cell entry kinetics: evidence that transport to npc + endolysosomes is a rate-defining step fusion of influenza hemagglutinin-expressing fibroblasts with glycophorin-bearing liposomes: role of hemagglutinin surface density the initial fusion pore induced by baculovirus gp is large and forms quickly a mature and fusogenic form of the nipah virus fusion protein requires proteolytic processing by cathepsin l cathepsin l is involved in proteolytic processing of the hendra virus fusion protein inhibitors of cathepsin l prevent severe acute respiratory syndrome coronavirus entry the nipah virus fusion protein is cleaved within the endosomal compartment subcellular localization and calcium and ph requirements for proteolytic processing of the hendra virus fusion protein mutations in the fusion protein cleavage site of avian paramyxovirus serotype increase cleavability and syncytium formation but do not increase viral virulence in chickens acid-induced membrane fusion by the hemagglutinin protein and its role in influenza virus biology the lysosomal cysteine proteases exocytosis of active cathepsin b enzyme activity at ph . , inhibition and molecular mass cathepsin b responsiveness to glutathione and lipoic acid redox cathepsin b stability, but not activity, is affected in cysteine:cystine redox buffers a new player in the puzzle of filovirus entry properties of a plasma membraneassociated cathepsin b-like cysteine proteinase in metastatic b melanoma variants designed protein mimics of the ebola virus glycoprotein gp alpha-helical bundle: stability and ph effects hiv- envelope proteins complete their folding into sixhelix bundles immediately after fusion pore formation structural basis for differential neutralization of ebolaviruses the transmembrane domain and acidic lipid flip-flop regulates voltage-dependent fusion mediated by class ii and iii viral proteins effects of membrane potential and sphingolipid structures on fusion of semliki forest virus enzootic nasal tumor virus envelope requires a very acidic ph for fusion activation and infection two retroviral entry pathways distinguished by lipid raft association of the viral receptor and differences in viral infectivity we thank kartik chandran, james cunningham, david sanders, and gary kobinger for provision of reagents that made this work possible. key: cord- -ul q ebx authors: dutch, rebecca ellis title: entry and fusion of emerging paramyxoviruses date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: ul q ebx nan hendra virus and nipah virus are the only identified zoonotic members of the paramyxovirus family, and both are highly pathogenic in humans [ ] . hendra virus infection has resulted in multiple horse and four human fatalities since its emergence in australia in , with outbreaks in and leading to rising concern in the australian horse breeding industry. nipah virus emerged in malaysia in , causing an outbreak of viral encephalitis that led to human fatalities out of reported cases. containment of the nipah virus epidemic required the sacrifice of more than million swine. continued nipah outbreaks have occurred in southeast asia, with mortality rates of up to % and suspected human-to-human transmission. numerous molecular features have led to the placement of hendra and nipah viruses within a new genus in the paramyxovirus family, the henipaviruses ( figure ). the principal reservoir species for both viruses is thought to be pteropus fruit bats, but a number of other species have been shown to be susceptible to infection [ ] . human metapneumovirus (hmpv) was first identified in , but unlike hendra and nipah, hmpv is not a new human virus resulting from zoonotic transmission. instead, hmpv is a longterm human pathogen that was only identified by careful analysis of samples from children with respiratory tract disease for which an etiological agent had not been identified [ ] . subsequent studies indicate that hmpv is a major causative agent of respiratory tract infections worldwide, causing between % and % of lower respiratory tract infections in young children [ ] . hmpv has been circulating in the human population since at least [ ] . sequence analysis places hmpv in the pneumovirinae subfamily, along with rsv. to enter host cells, paramyxoviruses must go through the key steps of viral attachment to the target cell, followed by fusion of the viral membrane to a host cell membrane [ ] . two major viral glycoproteins promote these events: the attachment protein facilitates primary receptor binding of the virus to the target cell, while the f protein promotes the subsequent membrane fusion events. both events are hypothesized to occur at the cell surface in a neutral ph environment. interactions between the f protein and the homotypic attachment protein are hypothesized to control initiation of the fusion process for most paramyxoviruses, though the mechanistic details of triggering control remain elusive. once begun, fusion is promoted by a series of conformational changes in the f protein that first lead to insertion of a hydrophobic region (termed the fusion peptide) into the target membrane, forming a protein bridge between the two membranes. additional conformational changes lead to formation of a helical bundle, formed by interactions between two heptad repeat regions that do not interact in the prefusion form of the protein [ ] , and subsequent membrane fusion. a number of factors point to an overall conserved mechanism of fusion promotion among the paramyxovirus f proteins. while there is considerable heterogeneity at the amino acid level, f proteins from both established and newly identified paramyxoviruses display conserved positioning of cysteine, glycine, and proline residues, suggesting an overall conservation of structure. f proteins also contain similarly placed fusion peptide and heptad repeat regions. peptides corresponding to the f protein heptad repeat regions have been shown to block fusion and entry for previously studied paramyxoviruses, and similar peptides inhibit hendra, nipah, and hmpv fusion and entry, indicating that the requirement for formation of the final helical bundle is a conserved feature [ , ] . like previously identified members of the family, fusion activity of the hendra and nipah f proteins requires the presence of a viral attachment protein, though either the hendra or nipah attachment protein can be used interchangeably [ ] . as was seen with measles virus, recent evidence suggests that fusion activity for the hendra and nipah f proteins is inversely proportional to the strength of the f attachment protein interactions, in contrast to results from other paramyxovirus systems such as newcastle disease viruses [ ] , suggesting slightly different mechanisms of control of fusion initiation. initial characterization of the activity of the hendra and nipah attachment proteins indicated interaction with a protein receptor, as is the case for measles virus attachment protein, rather than the sialic acid binding observed for most paramyxovirus attachment proteins [ ] . further study identified ephrinb as the receptor for the hendra and nipah viruses [ , ] , with ephrinb later shown to serve as an additional receptor for both viruses. structural analysis of nipah g alone or in complex with ephrin b interestingly showed little conformational change upon receptor binding, suggesting that only subtle alterations in the attachment protein lead to f protein activation [ ] . ephrinb and b serve as ligands for the eph tyrosine receptor family, and their cellular expression in neurons, arterial endothelial cells, and smooth muscle is consistent with the tissue distribution observed during hendra and nipah infection [ ] . ephrinb and b are also highly conserved between species, fitting with the large number of species shown to be infected by these pathogens. ephrinb and b from multiple infectable species, including human, horse, pig, cat, dog, and bat, have been shown to serve as functional receptors for hendra and nipah [ ] , suggesting that the conserved expression of this receptor plays an important role in the unusually broad host range of these pathogens. interestingly, murine ephrinb can serve as a functional receptor for these viruses, but mice are resistant to henipavirus infection, indicating that additional factors modulate overall host range. like other paramyxovirus f proteins, the hendra and nipah virus f proteins are initially synthesized as a precursor (f ) that must be proteolytically processed to two subunits (f and f ) to be fusogenically active (figure a) . for the majority of f proteins, this critical proteolytic processing event is promoted by furin, a cellular protease present primarily in the trans-golgi network. interestingly, the mechanism for proteolytic activation of the henipavirus f proteins is completely novel. furin is clearly not involved, as there is no furin consensus at the cleavage site, furin inhibitors have no effect on henipavirus f processing, and processing occurs efficiently in furinnegative cell lines [ ] . instead, inhibitors or shrna knock-downs of the cellular endosomal protease cathepsin l were shown to inhibit cleavage of the hendra and nipah f proteins, and in vitro studies confirmed proteolytic cleavage of the henipavirus f proteins at a single specific site by purified cathepsin l [ , ] . to facilitate this key interaction with cathepsin l, endocytosis of the hendra f protein [ ] and the nipah f protein [ ] must occur, followed by a retrafficking event to the cell surface after proteolytic processing ( figure b ). as cleaved f protein is present within the packaged virion [ ] , this complex trafficking of the henipavirus f proteins through the endosomal pathway occurs prior to viral assembly. interestingly, the hendra g attachment protein does not follow this complicated trafficking pathway, indicating that the critical attachment protein: fusion protein interactions needed for fusion occur only after f protein endocytic trafficking and proteolytic cleavage [ ] . the reason for this novel activation pathway is unclear, though it is intriguing to note that ebola virus and sars coronavirus also have a role for cathepsin l at some point during the viral life cycle, and like hendra and nipah virus, the reservoir species for these important pathogens is thought to be bats. future studies on protease profiles in bat cells may shed light on the reason for the unusual role of cathepsins in the life cycles of these pathogens. while analysis of henipavirus entry mechanisms has broadened diversity related to receptor usage and proteolytic activation, study of hmpv entry has further illuminated differences between the paramyxovirinae and pneumovirinae sub-families ( figure ). specific attachment protein:fusion protein interactions are needed for fusion promoted by paramyxovirinae glycoproteins, with the exception of the parainfluenza virus f protein, which can promote fusion in the absence of its attachment protein, though at a significantly decreased level [ ] . in contrast, a recombinant rsv lacking the attachment protein can replicate in some types of cultured cells [ ] , indicating a significantly decreased requirement for this protein during attachment and entry. the altered role of the attachment protein is even more striking in hmpv, as recombinants lacking the attachment protein are competent for replication in african green monkeys, though there is decreased replication in the lower respiratory tract [ ] . studies of cell-cell fusion found that the hmpv f protein by itself was capable of promoting both binding and fusion, and no stimulation by the attachment protein was observed [ ] . combined with the viral studies, these results suggest that hmpv f can interact with a receptor(s) on the target cells, though the identity of the f receptor remains to be defined. these findings also raise the important question of what triggers the hmpv f protein to initiate fusion, as in this case fusion initiation is clearly not controlled by interactions with the attachment protein. interestingly, recent studies indicate that low ph can serve as a fusion trigger [ ] for at least a portion of the hmpv strains [ ] , and specific residues that could promote low-ph-induced conformational change through an electrostatic repulsion mechanism have been identified [ ] . in addition, endocytosis has very recently been implicated in both hmpv [ ] and rsv [ ] entry, indicating that the virus will come in contact with the acidic ph of the endosomes during entry. overall, the study of entry and membrane fusion of recently identified paramyxoviruses has broadened the paradigms of receptor usage, f protein proteolytic activation, and membrane fusion triggering. future work will continue to define how these variations modulate infectivity and pathogenicity in this important viral family. paramyxoviridae: the viruses and their replication hendra and nipah viruses: different and dangerous fields virology: lippincott, williams and wilkins a newly discovered human pneumovirus isolated from young children with respiratory tract disease respiratory syncytial virus and metapneumovirus viral entry mechanisms: the increasing diversity of paramyxovirus entry a novel receptor-induced activation site in the nipah virus attachment glycoprotein (g) involved in triggering the fusion glycoprotein (f) ephrin-b ligand is a functional receptor for hendra virus and nipah virus ephrinb is the entry receptor for nipah virus, an emergent deadly paramyxovirus host cell recognition by the henipaviruses: crystal structures of the nipah g attachment glycoprotein and its complex with ephrin-b functional studies of host-specific ephrin-b ligands as henipavirus receptors cathepsin l is involved in proteolytic processing of the hendra virus fusion protein endocytosis plays a critical role in proteolytic processing of the hendra virus fusion protein the nipah virus fusion protein is cleaved within the endosomal compartment differential rates of protein folding and cellular trafficking for the hendra virus f and g proteins: implications for f-g complex formation infection of nonhuman primates with recombinant human metapneumovirus lacking the sh, g, or m - protein categorizes each as a nonessential accessory protein and identifies vaccine candidates characterization of human metapneumovirus f protein-promoted membrane fusion: critical roles for proteolytic processing and low ph low ph induced membrane fusion mediated by human metapneumoviruses f protein is a rare, strain dependent phenomenon low-ph triggering of human metapneumovirus fusion: essential residues and importance in entry small interfering rna profiling reveals key role of clathrin-mediated endocytosis and early endosome formation for infection by respiratory syncytial virus we thank the members of the dutch laboratory and dr. trevor creamer for careful reading of the manuscript, and dr. matthew gentry for assistance with the figures. key: cord- -vzcfeh t authors: talbert-slagle, kristina; atkins, katherine e.; yan, koon-kiu; khurana, ekta; gerstein, mark; bradley, elizabeth h.; berg, david; galvani, alison p.; townsend, jeffrey p. title: cellular superspreaders: an epidemiological perspective on hiv infection inside the body date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: vzcfeh t nan worldwide, more than people become infected with hiv every hour [ ] , yet an individual's chance of becoming infected after a single sexual exposure, the predominant mode of hiv transmission, is often lower than one in [ ] . when sexually transmitted hiv- infection does occur, it is usually initiated by a single virus, called the founder strain, despite the presence of thousands of genetically diverse viral strains in the transmitting partner [ ] . here we review evidence from molecular biology and virology suggesting that heterogeneity among cd + t cells could yield wide variation in the capability of individual cells to become infected and transmit hiv to other cells. using an epidemiological framework, we suggest that such heterogeneity among cd + t cells in the genital mucosa could help explain the low infection-to-exposure ratio and selection of the founder strain after sexual exposure to hiv. during sexual transmission, founder viral strains preferentially infect cd + t cells using the ccr coreceptor [ , ] . at the time of initial exposure to hiv, these cd + t cells exhibit baseline heterogeneity due to stochasticity in cellular gene expression [ ] and dynamic variation in immunological status (activated, resting, etc.) [ ] . in addition, because cd + t cells are mobile, they are heterogeneously distributed in the genital mucosa, with varying degrees of clustering and contact [ ] [ ] [ ] [ ] . in other contexts, it is well-known that heterogeneity among isogeneic cells inside the body can affect many cellular behaviors and outcomes, including infection dynamics [ , ] . epidemiological analyses of disease outbreaks among people indicate that heterogeneity in the ability of individuals in a population to spread disease can have a significant impact on whether a local outbreak becomes an epidemic [ ] . heterogeneity among a population of cd + t cells may play a similarly critical role in the establishment and spread of hiv in the genital mucosa after sexual exposure. to quantify the spread of infectious disease, epidemiologists use the basic reproductive number, r , which describes the average number of secondary infections that arise from one infected individual in an otherwise totally susceptible population [ ] . the basic reproductive number can be approximated as the product of the following: ( ) the average number of susceptible individuals contacted by an infected individual during the infectious period (the ''number of contacts'') and ( ) the average probability that a susceptible individual will become infected by a single infected individual during its infectious period (the ''shedding potential''). thus, r &number of contacts |shedding potential: the number of secondary infections caused by a specific individual throughout the time that the individual is infectious is called the ''individual reproductive number'' [ ] . for any disease within a given population, there exists a distribution of individual reproductive numbers, of which r is the mean [ ] . in populations of homogeneous individuals, the distribution of individual reproductive numbers will be clustered around the population average value of r , and thus, this average value will more accurately predict the likelihood of transmission from each infected to each susceptible individual. if r . , then an outbreak is likely to become an epidemic, and if r , , then an outbreak will not spread beyond a few initially infected individuals [ , ] . in heterogeneous populations, however, the population average value of r is less predictive of transmission dynamics [ ] . for example, in populations with highly right-skewed distributions of individual reproductive numbers, most individuals infect few, if any, others, but a few individuals infect many others. in such populations, there is a high probability that a disease outbreak will not be sustained in the population and will instead go extinct [ ] . in some cases, however, those rare individuals in the tail of the distribution with a much higherthan-average individual reproductive number while they are infected, known as ''superspreaders'' [ ] , can have a significant impact on whether an outbreak becomes an epidemic or goes extinct. epidemiological outbreak investigations, which track the spread of disease by a technique called contact tracing, have identified the existence of superspreaders in many well-known infectious disease outbreaks, including typhoid fever, measles, smallpox, ebola, and severe acute respiratory syndrome (sars) [ , , ] . these rare individuals often make a significant, sometimes deciding, contribution to the dynamics of disease spread (table ) . during the sars outbreak in singapore, for example, the majority of individuals who became infected spread the virus either to no one else or to only one other [ ] . five infected individuals, however, were superspreaders, each infecting at least others (figure ) [ ] . in this example, r , which is an average population value, did not adequately describe the dynamics of sars because it did not capture the heterogeneity among individuals in their ability to spread disease or the key contribution made by superspreaders to establishment and spread of the virus [ ] . for a population of hiv-infected cells inside the body, the basic reproductive number, r (a population average), has been quantified [ ] . as with individual humans inside a population, however, empirical evidence indicates that individual human cells inside a population are also heterogeneous [ ] , varying in their contact with one another, their ability to become infected (permissivity), and also in whether and to what extent they transmit infectious virus to other cells during their infectious period. such heterogeneity among cd + t cells in the genital mucosa of a single individual could generate a skewed distribution in the individual cellular reproductive number, or icrn, in the context of hiv infection. here we review evidence for heterogeneity among cd + t cells that could lead to wide variation in icrn and possibly give rise to cellular superspreaders. cd + t cells exhibit considerable heterogeneity in activation status (e.g., resting or activated) and expression of surface molecules important for hiv infection (including the hiv coreceptors ccr and cxcr ) in human penile [ ] , foreskin [ , ] , cervical [ , ] , and rectal [ ] tissue. in addition, various studies that stain for cd + t cells in uninfected genital mucosal tissue, such as cervical tissue [ ] and human foreskin [ ] , indicate that t cells vary in their spatial distribution and the extent to which they form clusters. cell density and spatial arrangement have been identified as important sources of heterogeneity among cells that can affect virus spread in vitro [ , [ ] [ ] [ ] . indeed, imaging studies exploring the dynamics of virus spread in a model of sexual transmission to female nonhuman primates indicate that virus spreads unevenly among clusters of cells in the endocervix [ ] . cells in these clusters tend to be in close proximity, and if cell-tocell transmission is far more efficient than cell-free transmission, as some studies suggest [ ] , then a cell that is physically touching its neighbors could generate more secondary infections than a cell that is not close enough to others to transmit virus by direct contact [ ] . thus, heterogeneity in cell distribution and clustering inside the body could generate wide variation in the efficiency of virus transmission from cell to cell and in icrn. transmission of virus from an infected to a susceptible cell also depends on a cell's permissivity to productive infection. the level of surface expression of cd and ccr (the predominant coreceptor utilized during acute infection [ ] ) varies widely among cd + t cells [ , ] , even in a single individual [ ] , and affects cellular permissivity to hiv [ , ] . indeed, low expression of cd or ccr can completely inhibit infection of cd + t cells by certain viral strains [ ] . a recent multiparameter analysis of hiv entry efficiency at the level of single cells indicated large cell-to-cell variation in expression of cd , ccr , and the coreceptor cxcr , which subsequently influenced permissivity of individual cells to hiv binding and entry [ ] . in addition, cd + t cells isolated from rectal and cervical tissue exhibit considerable heterogeneity in expression of the surface integrin a b , which can specifically bind the v loop of the hiv envelope protein gp and may improve cell-to-cell spread by activating other cellsurface molecules in the viral synapse [ ] . experiments in vitro indicate that hiv preferentially infects cells expressing high levels of ccr and that infection can be further enhanced by high levels of surface a b expression in some individuals [ , ] . heterogeneity among cd + t cells in expression of specific cell surface receptors can thus affect permissivity and cell-to-cell transfer of hiv infection in the genital mucosa. permissivity of cells to productive hiv infection can also be affected by intracellular proteins called restriction factors, which block the progress of hiv through the cell [ ] . expression of some cellular restriction factors has been shown to vary among different human populations [ ] and even between different types of cd + t cells within the same individual [ ] . notably, an intracellular host restriction factor known as samhd (sterile alpha motif [sam] and histidine/aspartic acid [hd] domain-containing protein ) blocks reverse transcription in resting-but not activated-cd + t cells, inhibiting hiv replication. these findings help to explain the inability of resting cd + t cells to produce infectious virus [ ] . hiv combats the effects of some cellular restriction factors with its own viral proteins, but the success of these proteins in overcoming cellular resistance and facilitating virus production depends on their quantity within the cell, which can also vary depending on viral gene expression levels [ ] . together, these data suggest that among cd + t cells in the genital mucosa, significant heterogeneity may exist in the number of contacts that become infected by any given infected cell even within the same host, potentially leading to a skewed distribution of icrn. release of infectious viral particles from an infected cd + t cell, or shedding potential, can be influenced by many factors in both the cell and the virus. variation in virus gene expression at the level of individual cells has been demonstrated in vivo in a mouse model of cytomegalovirus infection [ ] , and heterogeneity among individual cells in the production of virus particles, or virus shedding, has been shown through analysis of cells isolated from simian immunodeficiency virus (siv)-infected nonhuman primates, in which rare activated cd + t cells were shown to individually release large quantities of virus [ ] . here we focus on variability in gene expression and its potential impact on an individual cell's capability to release infectious virus as a possible source of heterogeneity in shedding potential. stochasticity in cellular gene expression is a common phenomenon [ ] and has also been observed in viral gene expression, including hiv. in populations of genetically identical cells infected with hiv, viral genes tended to be expressed at either high or low levels but were also rarely expressed at intermediate levels, varying from cell to cell [ ] . the site of virus integration also affects viral gene expression. hiv viral dna preferentially integrates at sites of active cellular gene expression although not at any specific site or in any specific gene [ ] [ ] [ ] . some viral integration events occur at sites of much higher gene expression than others [ ] , and gene expression can vary significantly depending on the site of integration, even in genetically identical cells [ ] . since the majority of hiv-infected cells in lymph nodes and peripheral blood contain a single virus [ ] , while cells in splenic tissue contain one to eight integrated proviruses (mean . ) [ ] , viral integration site can be an important factor in determining viral gene expression and likely also subsequent virus shedding potential from any given cell. within the population of cd + t cells in the genital mucosa, therefore, a wide range of icrn may exist due to stochastic and/or infection-driven variations in viral gene expression and viral particle production, arising from potentially wide heterogeneity in virus shedding potential among infected cells. experiments in a nonhuman primate model of hiv infection have demonstrated that the vast majority of cd + t cells in the genital mucosa of a healthy, uninfected individual are resting cells, which outnumber activated cells : (figure a ) [ ] . activated cd + t cells express higher levels of ccr [ ] and a b [ ] than do resting cells. in addition, experiments in nonhuman primates indicate that infected, activated cells contain five times more viral rna, release -fold more viral particles to their surrounding environment, and tend to form larger cell clusters than resting cells [ ] . notably, although the nonhuman primate model has long been used to study many facets of hiv infection [ ] , the virus used in these experiments, siv, expresses a protein that allows it to productively infect resting cd + t cells. in contrast, hiv- does not express this protein and is thus unable to generate productive infection in resting cd + t cells [ ] . resting human cd + t cells in the human genital mucosa are therefore even less likely to be able to produce and spread hiv than are resting cd + t cells in a nonhuman primate model. together, these data suggest that upon infection with hiv the majority of cd + t cells in the genital mucosa would spread the virus to few if any others and that only rare cells would have the capacity to release large quantities of hiv to their nearest neighbors. these rare cells may be activated cd + t cells, which have been described as ''amplifiers'' that can cause additional cells to become infected with hiv [ , ] . such rare cells may exhibit a specific set of traits that facilitate establishment of hiv inside the body. for example, experiments done in vitro indicate that cd + th cells, which express high levels of ccr as well as the chemokine receptor ccr and the integrin a b , are preferentially targeted during early infection, and analysis of samples from female sex workers who are infected with hiv indicate that cd + th cells are selectively depleted from figure . contact tracing of sars in singapore showed that most people (gray circles) transmitted the virus to very few others, while a few individuals acted as ''superspreaders,'' infecting many more people than average. patient numbers corresponding to those individuals who were identified as superspreaders are shown. all cases trace back to patient [ ] . doi: . /journal.ppat. .g the cervix during hiv infection [ , [ ] [ ] [ ] . in addition, given that the majority of hiv infections begin with a single founding strain [ ] and most infected cells in peripheral blood and lymph node tissue contain a single copy of hiv dna (although these may or may not be representative of hiv integration in mucosal lymphatic cells) [ ] , infection of a single cell has the potential to establish hiv infection inside the genital mucosa after a given exposure. infection of a rare cd + t cell with very high icrn could thus be the superspreading event that both establishes hiv infection in the genital mucosa and selects a single founder strain ( figure b) . such a founding superspreader infection event would parallel, for example, the dynamics that governed the sars outbreak in singapore, giving rise to an epidemic despite a low individual reproductive number for most individuals. as shown in figure , infection of a single superspreading individual (labeled as '' '') triggered the sars outbreak in singapore in . a highly skewed distribution of individual reproductive numbers in a population, in which most individuals infect few if any others but a tiny minority are superspreaders, has two important implications when applied to cd + t cells in the genital mucosa. first, since the majority of the cells would have a low icrn, most, if not all, of the viral strains that successfully overcome physiological barriers during exposure are likely to infect cells that have an icrn less than one. provided that none of these cells becomes latently infected, which has been shown to occur within days after infection in vitro [ , ] but has yet to be confirmed in vivo, a ''local outbreak'' inside the body would go extinct. in this case, infection of most cells immediately after sexual exposure would not lead to sustained infection, which could explain the very low infection-toexposure ratio for sexual exposure to hiv. second, such short-lived ''local outbreaks'' of hiv within an individual could still yield a low level of virus production, even if the initial outbreak of hiv infection ultimately goes extinct. among a group of nonhuman primates exposed to a low physiological dose of siv, some animals experienced initial low levels of viral replication and immune response without ever proceeding to full infection or seroconversion, a phenomenon called occult infection [ ] [ ] [ ] . in addition, some hiv-exposed, seronegative humans who continue to engage in high-risk sexual behavior exhibit immunological markers that indicate a prior immune response to hiv infection even though they remain seronegative, supporting the idea that local infections may have occurred in these patients [ ] [ ] [ ] . finally, analysis of the unsuccessful hiv vaccine step trial suggested that a large portion of the exposed individuals may have experienced occult infection, implying that this phenomenon could be more widespread than previously suspected [ ] . there is, however, no direct experimental evidence for occult infection in humans. if infection of a rare superspreader cd + t cell establishes the founder strain, then the few features of founder viral strains that have been observed might in fact confer a selective advantage during early infection. these features include shorter envelope glycoproteins with fewer n-linked glycosylation sites [ , ] as well as preferential infection of cd + t cells expressing high levels of ccr . some founder viral strains have also exhibited high affinity for the a b integrin receptor [ , ] though this has not been universally observed [ , ] . the founder virus, which more closely resembles the ancestral founder strain than it does the predominant strain in the transmitting partner [ ] , may be selected by its ability to infect a subtype of cd + t cell: a cellular superspreader. this specific efficiency could explain why hiv founder strains have not shown a consistent infectivity advantage in cd + t cells over strains from chronic infection in vitro [ , , ] , as these experiments likely do not fully replicate the cellular heterogeneity of the in vivo environment [ , , ] , where the founder viruses might have an advantage in infecting the rare superspreader cells. here we have applied two key concepts from epidemiology: r , which is the approximated product of number of contacts and shedding potential throughout the infectious period, and individual reproductive number, to suggest that a skewed distribution of individual cellular reproductive number among cd + t cells in the genital mucosa gives rise to cellular superspreaders that may drive establishment of hiv infection inside the genital mucosa after sexual transmission. the definition of r provided here implicitly integrates transmission over the time that an index cell was infected, meaning that we have incorporated duration of infectiousness into our overall definition of r . thus, a cd + t cell could theoretically become a superspreader either by spreading a large amount of infectious virus to other cells in a short amount of time, or by spreading a smaller amount of virus to other cells for a comparatively longer period of time, or through some combination of the two. though any of these mechanisms are possible in early infection [ ] , studies in nonhuman primates suggest that establishment of infection in the genital mucosa typically occurs within - days after male-to-female sexual exposure [ , ] . the lifespan of a productively infected cd + t cell is on average . days [ ] . thus, in the specific case of hiv infection in the genital mucosal tissue, if infection becomes established via a superspreading event, it is likely to occur within the first few days of exposure and be driven by a relatively short-lived, productively infected cell that generates a much higher-thanaverage number of secondary infections due to a high shedding rate, or a high contact rate, or both. notably, as in other superspreading events, establishment of hiv by infection of a cellular superspreader could occur even if the basic reproductive number for the entire population of susceptible cells is low. if cellular hiv superspreaders do exist, and if they are the cellular culprits driving the establishment of hiv infection inside the body, then the most successful strategy for preventing the infection from becoming established in the body is to block or remove these cells before or shortly after infection in order to drive a local, withinhost outbreak to extinction [ , ] . such cells may have specific traits, such as high expression of surface receptors including ccr and possibly also a b , that allow them to be identified and targeted by novel therapies to prevent establishment of infec-tion. several recent studies suggest that th cells, a subset of cd + t cells, are preferentially infected by early viral strains and selectively depleted from the cervix during hiv infection [ , ] . we are not aware of in vivo data that explicitly support the existence of cellular superspreaders; nevertheless, the data reviewed here suggest their existence, warranting further empirical research. identification and targeting of cells most likely to become superspreaders could facilitate the development of preexposure or immediate postexposure therapies that could prevent a local outbreak of hiv inside the genital mucosa from becoming a within-host epidemic that spreads throughout the body [ , ] . in this review, we have applied epidemiological concepts of disease spread specifically to explore unsolved questions regarding establishment of the hiv founder strain and the low infection-to-exposure ratio of infection after sexual transmission. we suggest that these concepts may also be applied more broadly to explain the documented existence of hiv founder strains after transmission via injection drug use and from mother to child [ ] since cd + t cells in the blood of healthy, uninfected individuals are also heterogeneous, with only a very small subset exhibiting an activated or replicating phenotype [ ] . since heterogeneity among cells has been acknowledged as an important factor in a variety of cellular processes, including certain viral infections [ ] , we suggest that the epidemiological framework described here may also be applicable to the establishment and spread of other cellular diseases inside the body, including not only infections but perhaps also certain cancers. the impact of cellular heterogeneity may be particularly profound if the distribution of individual cellular reproductive numbers is highly skewed, yielding cellular superspreaders. global report: unaids report on the global aids epidemic . edition. online: joint united nations programme on hiv/aids heterosexual risk of hiv- infection per sexual act: systematic review and meta-analysis of observational studies barriers to mucosal transmission of immunodeficiency viruses high multiplicity infection by hiv- in men who have sex with men phenotypic properties of transmitted founder hiv- nature, nurture, or chance: stochastic gene expression and its consequences heterogeneity of cd + memory t cells: functional modules for tailored immunity the clustering of infected siv cells in lymphatic tissue glycerol monolaurate prevents mucosal siv transmission roles of substrate availability and infection of resting and activated cd + t cells in transmission and acute simian immunodeficiency virus infection heterogeneity and plasticity of t helper cells cellular heterogeneity: do differences make a difference? quality and quantity: mucosal cd + t cells and hiv susceptibility superspreading and the effect of individual variation on disease emergence infectious diseases of humans: dynamics and control modeling infectious diseases in humans and animals largest measles epidemic in north america in a decade-quebec, canada, : contribution of susceptibility, serendipity, and superspreading events super-spreaders in infectious diseases severe acute respiratory syndrome-singapore estimation of the initial viral growth rate and basic reproductive number during acute hiv- infection hiv infection and immune defense of the penis differential compartmentalization of hiv-targeting immune cells in inner and outer foreskin tissue susceptibility to human immunodeficiency virus- infection of human foreskin and cervical tissue grown in explant culture hiv- sexual transmission: early events of hiv- infection of human cervico-vaginal tissue in an optimized ex vivo model characterization of a human cervical cd + t cell subset coexpressing multiple markers of hiv susceptibility hiv- pathogenesis differs in rectosigmoid and tonsillar tissues infected ex vivo with ccr -and cxcr -tropic hiv- immunological microenvironments in the human vagina and cervix: mediators of cellular immunity are concentrated in the cervical transformation zone spatiotemporal dynamics of hiv propagation population context determines cell-to-cell variability in endocytosis and virus infection single-cell analysis of population context advances rnai screening at multiple levels cell-to-cell transmission of viruses hiv- transmission biology: selection and characteristics of infecting viruses quantification of cd , ccr , and cxcr levels on lymphocyte subsets, dendritic cells, and differentially conditioned monocyte-derived macrophages mechanisms of nonrandom human immunodeficiency virus type infection and double infection: preference in virus entry is important but is not the sole factor differences in cd dependence for infectivity of laboratory-adapted and primary patient isolates of human immunodeficiency virus type transmitted/founder and chronic hiv- envelope proteins are distinguished by differential utilization of ccr a quantitative affinityprofiling system that reveals distinct cd /ccr usage patterns among human immunodeficiency virus type and simian immunodeficiency virus strains an expanded model of hiv cell entry phenotype based on multiparameter single-cell data hiv- envelope protein binds to and signals through integrin alpha beta , the gut mucosal homing receptor for peripheral t cells the integrin alpha beta forms a complex with cell-surface cd and defines a t-cell subset that is highly susceptible to infection by hiv- the restriction factors of human immunodeficiency virus analysis of human apobec h haplotypes and anti-human immunodeficiency virus type activity intracellular detection of differential apobec g, trim alpha, and ledgf/p protein expression in peripheral blood by flow cytometry restrictions to hiv- replication in resting cd (+) t lymphocytes single cell detection of latent cytomegalovirus reactivation in host tissue stochastic gene expression in a lentiviral positive-feedback loop tat fluctuations drive phenotypic diversity hiv- integration in the human genome favors active genes and local hotspots retroviral dna integration: aslv, hiv, and mlv show distinct target site preferences transcription start regions in the human genome are favored targets for mlv integration hiv promoter integration site primarily modulates transcriptional burst size rather than frequency single cell analysis of lymph node tissue from hiv- infected patients reveals that the majority of cd + t-cells contain one hiv- dna molecule recombination: multiply infected spleen cells in hiv patients update on animal models for hiv research detection of plasma viremia in human immunodeficiency virus-infected individuals at all clinical stages intravaginal inoculation of rhesus macaques with cell-free simian immunodeficiency virus results in persistent or transient viremia susceptibility of human th cells to human immunodeficiency virus and their perturbation during infection memory ccr +cd + t cells are preferential targets for productive hiv type infection regardless of their expression of integrin beta preferential hiv infection of ccr + th cells is associated with higher levels of virus receptor expression and lack of ccr ligands hiv transmission. cold spring harb perspect med : a a double-fluorescent hiv- reporter shows that the majority of integrated hiv- is latent shortly after infection determinants of the establishment of human immunodeficiency virus type latency propagation and dissemination of infection after vaginal transmission of simian immunodeficiency virus occult systemic infection and persistent simian immunodeficiency virus (siv)-specific cd (+)-t-cell proliferative responses in rhesus macaques that were transiently viremic after intravaginal inoculation of siv a period of transient viremia and occult infection precedes persistent viremia and antiviral immune responses during multiple low-dose intravaginal simian immunodeficiency virus inoculations epidemiologic and biologic characterization of a cohort of human immunodeficiency virus type highly exposed, persistently seronegative female sex workers in northern thailand. chiang mai heps working group new insights into hiv- specific cytotoxic t-lymphocyte responses in exposed, persistently seronegative kenyan sex workers cervical hiv-specific iga in a population of commercial sex workers correlates with repeated exposure but not resistance to hiv hiv vaccine development in the aftermath of the step study: re-focus on occult hiv infection? characterization of glycosylation profiles of hiv- transmitted/founder envelopes by mass spectrometry recurrent signature patterns in hiv- b clade envelope glycoproteins associated with either early or chronic infections the genotype of early-transmitting hiv gp s promotes alpha ( ) beta( )-reactivity, revealing alpha ( ) beta( ) +/cd + t cells as key targets in mucosal transmission hiv- envelope replication and alpha beta utilization among newly infected subjects and their corresponding heterosexual partners early infection hiv- envelope v -v genotypes do not enhance binding or replication in cells expressing high levels of alpha beta integrin previously transmitted hiv- strains are preferentially selected during subsequent sexual transmissions transmitted/founder and chronic subtype c hiv- use cd and ccr receptors with equal efficiency and are not inhibited by blocking the integrin alpha beta application of a case-control study design to investigate genotypic signatures of hiv- transmission evaluation of cervical mucosa in transmission bottleneck during acute hiv- infection using a cervical tissue-based organ culture generation of transmitted/founder hiv- infectious molecular clones and characterization of their replication capacity in cd t lymphocytes and monocytederived macrophages stochastic theory of early viral infection: continuous versus burst production of virions sexual transmission and propagation of siv and hiv in resting and activated cd + t cells hiv- dynamics in vivo: virion clearance rate, infected cell life-span, and viral generation time immediate antiviral therapy appears to restrict resting cd + cell hiv- infection without accelerating the decay of latent infection early events in sexual transmission of hiv and siv and opportunities for interventions circulating cd (+) and cd (+) t cells are activated in inflammatory bowel disease and are associated with plasma markers of inflammation the basic reproductive number of ebola and the effects of public health measures: the cases of congo and uganda the reemergence of ebola hemorrhagic fever, democratic republic of the congo an epidemic of measles in southern greenland, ; measles in virgin soil. ii. the epidemic proper an explosive point-source measles outbreak in a highly vaccinated population. modes of transmission and risk factors for disease epidemiologic determinants for modeling pneumonic plague outbreaks primary pneumonic plague in mukden, , and report of cases with three recoveries dynamically modeling sars and other newly emerging respiratory illnesses: past, present, and future evidence of airborne transmission of the severe acute respiratory syndrome virus sars reference investigation of a nosocomial outbreak of severe acute respiratory syndrome (sars) in toronto transmission potential of smallpox in contemporary populations smallpox and its eradication. geneva: world health organization. xvi we thank pradeep uchil, walther mothes, and daniel dimaio for helpful discussions and maggie zhou for contributions to the manuscript revision. key: cord- - twx y c authors: abraham, jonathan; kwong, jo ann; albariño, césar g.; lu, jiajie g.; radoshitzky, sheli r.; salazar-bravo, jorge; farzan, michael; spiropoulou, christina f.; choe, hyeryun title: host-species transferrin receptor orthologs are cellular receptors for nonpathogenic new world clade b arenaviruses date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: twx y c the ability of a new world (nw) clade b arenavirus to enter cells using human transferrin receptor (tfr ) strictly correlates with its ability to cause hemorrhagic fever. amapari (amav) and tacaribe (tcrv), two nonpathogenic nw clade b arenaviruses that do not use human tfr , are closely related to the nw arenaviruses that cause hemorrhagic fevers. here we show that pseudotyped viruses bearing the surface glycoprotein (gp) of amav or tcrv can infect cells using the tfr orthologs of several mammalian species, including those of their respective natural hosts, the small rodent neacomys spinosus and the fruit bat artibeus jamaicensis. mutation of one residue in human tfr makes it a functional receptor for tcrv, and mutation of four residues makes it a functional receptor for amav. our data support an in vivo role for tfr in the replication of most, if not all, nw clade b arenaviruses, and suggest that with modest changes in their gps the nonpathogenic arenaviruses could use human tfr and emerge as human pathogens. arenaviruses are enveloped viruses that carry single-stranded, bi-segmented, rna genomes [ ] . the family arenaviridae comprises a single genus (arenavirus) that contains at least members [ ] [ ] [ ] . arenaviruses have been classified into two antigenically and geographically distinct groups, the lassa-lymphocytic choriomeningitis serocomplex (''old world arenaviruses'') and the tacaribe serocomplex (''new world arenaviruses'') [ ] [ ] [ ] . the new world (nw) arenavirus cluster is subdivided into clades a, b, and c. in addition, several north american viruses are recombination products of clades a and b (a/b) ( figure ). all nw arenaviruses that cause hemorrhagic fever in humans are in the b clade. these include the junín (junv), machupo (macv), guanarito (gtov), and sabiá (sabv) viruses, which respectively cause argentine, bolivian, venezuelan, and brazilian hemorrhagic fevers with high ( - %) case fatality rates [ , , ] . a novel nw clade b arenavirus, chapare virus, was recently isolated from a fatal case of hemorrhagic fever in bolivia [ ] . in addition to these pathogens, there are clade b viruses that do not cause disease in humans. these include the amapari (amav), cupixi (cpxv), and tacaribe (tcrv) viruses, although tcrv may have caused a febrile illness with mild central nervous system symptoms in a laboratory worker [ ] . pathogenic and nonpathogenic nw arenaviruses do not cluster independently on phylogenetic analysis; tcrv is closely related to macv and junv, while amav shares highest sequence similarity with gtov ( figure ). the zoonotic reservoir of a given nw arenavirus has a unique geographical distribution, and each arenavirus prefers its own host [ ] . most of the natural hosts for nw arenaviruses belong to the subfamily sigmodontinae in the family cricetidae, which contains nw mice and rats [ , ] . the host animals for macv, junv and gtov are calomys callosus (large vesper mouse), calomys musculinus (drylands vesper mouse), and zygodontomys brevicauda (short-tailed cane mouse), respectively [ ] [ ] [ ] [ ] . the natural reservoirs of sabv and chapare virus have not been identified. amav was isolated from neacomys spinosus (bristly mouse) and neacomys guianea (guiana bristly mouse), and tcrv is suggested to have a non-rodent host, artibeus species fruit bats in trinidad and tobago [ , [ ] [ ] [ ] . the arenavirus envelope glycoprotein (gp), the sole protein found on the surface of virions, is processed into three associated subunits: the stable signal peptide (ssp), gp , and gp [ , ] . ssp is a unique component of the arenaviral fusion machinery and plays a role in the transport, maturation, and ph dependent membrane fusion activity of the gp complex [ ] [ ] [ ] [ ] . the gp subunit engages a cellular receptor(s), and gp mediates phdependent membrane fusion after viral particles are internalized into acidified endosomes [ , [ ] [ ] [ ] [ ] . two cell surface molecules have been implicated as cellular receptors for arenaviruses. old world arenaviruses and nw clade c arenaviruses oliveros and latino use a-dystroglycan as an obligate receptor, while the pathogenic nw clade b arenaviruses macv, junv, gtov and sabv use human transferrin receptor (tfr ) to infect cells [ ] [ ] [ ] [ ] . the ability of nw clade b arenaviruses to cause disease in humans correlates with the utilization of human tfr , a molecule that has several properties favorable to arenaviral replication and viral hemorrhagic fevers; it is rapidly endocytosed into acidic compartments, expressed on endothelial cells, and upregulated on rapidly dividing cells including activated lymphocytes [ ] [ ] [ ] [ ] [ ] . we have previously shown that recombinant retroviral particles pseudotyped with the gps of macv, junv, or gtov efficiently use the tfr ortholog of the rodent hosts of these viruses [ ] . a number of studies have suggested that amav and tcrv do not use human tfr [ ] [ ] [ ] [ ] . blocking human cells with inactivated amav significantly reduces the infection of these cells with amav pseudovirus, but does not interfere with the infection of macv, junv or gtov pseudoviruses [ ] . it remains to be shown, however, if these nonpathogenic nw arenaviruses could use the tfr orthologs of their principal host animals. here, we confirm that amav and tcrv do not use human tfr , and show that they nonetheless efficiently use the tfr orthologs of their respective animal hosts, n. spinosus and a. jamaicensis. we also show that mutation of one residue converts human tfr into an efficient receptor for tcrv, and replacement of four residues with those found in n. spinosus tfr converts human tfr into an efficient receptor for amav. these data show that tfr has an important role in the replication of nonpathogenic nw arenaviruses, and suggest that subtle changes in the gps of tcrv and amav might adapt them to use human tfr . we confirmed that human tfr is not involved in the entry of amav and tcrv into human cells by examining whether an ahuman tfr antibody could inhibit the infection of hek t cells mediated by the gps of amav and tcrv. we generated recombinant moloney murine leukemia virus (mlv) expressing green-fluorescent protein (gfp), pseudotyped with the gps of amav, tcrv, macv and gtov. cells were exposed to these recombinant viruses in the presence or absence of a control antibody (a-hla), or an a-human tfr antibody previously shown to inhibit infection of macv and junv pseudoviruses [ ] . infection levels were assayed forty-eight hours later by flow cytometry (figure ). the a-human tfr antibody inhibited infection by gtov and macv pseudoviruses, but had no effect on the entry of amav and tcrv pseudoviruses. the entry of all four pseudoviruses was unaffected by the a-hla antibody. these results are in agreement with previous studies showing that human tfr is not involved in the entry of the nonpathogenic nw clade b arenaviruses into human cells [ ] . animal orthologs of tfr support entry of amav and tcrv pseudoviruses phylogenetic analysis reveals that gtov gp shares greater sequence similarity with amav gp than it does with the gps of the other pathogenic nw arenaviruses ( figure ). amav, however, does not use human tfr as a cellular receptor. we sought to determine if animal orthologs of tfr could support entry mediated by the gps of amav and tcrv. chinese hamster ovary (cho) cells were transfected with plasmids expressing the human, mouse, rat, feline, canine, calomys callosus (cc ; macv host), calomys musculinus (cm ; junv host), or viruses that cause hemorrhagic fever in humans, which are only found in clade b (encircled), are labeled with an asterisk. viruses known to use human tfr are boxed in orange, and the nonpathogenic arenaviruses investigated in this study are boxed in cyan [ , ] . doi: . /journal.ppat. .g several arenaviruses found in the new world cause hemorrhagic fever when they are transmitted from their natural reservoirs to humans. these pathogenic arenaviruses use human transferrin receptor (tfr ), a protein involved in cellular iron uptake, to infect human cells. the nonpathogenic new world arenaviruses characterized thus far do not use human tfr . we show here that two of these nonpathogenic viruses, amapari and tacaribe, can use animal orthologs of tfr to infect cells. we observe that recombinant viruses coated with the entry proteins of amapari and tacaribe viruses use the tfr orthologs of their natural reservoirs. modest alteration of human tfr converts it to an efficient receptor for amapari and tacaribe viruses. our findings provide insight into the potential of amapari and tacaribe viruses to adapt to use human tfr and perhaps emerge as human pathogens. zygodontomys brevicauda (zb ; gtov host) orthologs of tfr . tfr expression was assessed by flow cytometry using an antibody directed against a flag tag added to the c-terminus of each ortholog. in parallel, aliquots of the same cells were infected with amav or tcrv pseudoviruses. as these viruses readily enter cho cells using a tfr -independent pathway, pseudoviruses were diluted to minimize background entry. pseudovirus bearing the gp of lymphocytic choriomeningitis virus (lcmv) was used as a control. as shown in figure a , infection by amav and tcrv pseudoviruses was significantly enhanced by the introduction of zbtfr into cho cells, while amav pseudovirus entry was enhanced to a lesser degree by cctfr . the entry of amav pseudovirus was also enhanced by feline tfr expression. we and others have previously shown that this tfr ortholog is also used efficiently by the pathogenic viruses macv, junv and gtov [ , ] . the entry of lcmv pseudovirus was unaffected. we repeated these experiments using hek t cells, and obtained similar results ( figure b ). these data show that although amav and tcrv pseudoviruses do not use human tfr , they nonetheless use the tfr orthologs of several mammalian species. we next investigated whether amav and tcrv gp could also use the tfr orthologs of their host species, the small rodent n. spinosus and the fruit bat a. jamaicensis, respectively. we cloned the genes of their tfr orthologs by rt-pcr from frozen tissues of n. spinosus (nstfr ) and a. jamaicensis (ajtfr ). in order to determine the ability of the amav and tcrv gp proteins to bind these tfr orthologs, hek t cells were transfected with plasmids expressing human, ns, or ajtfr . these cells were incubated with ig-fusion proteins comprising the gp subunits of amav or tcrv (amav-gp d-ig and tcrv-gp d-ig, respectively). the binding of these proteins to transfected cells was measured by flow cytometry using an a-human igg antibody. the expression levels of tfr orthologs were assessed in parallel using an a-flag antibody. as shown in figure a , while the expression levels of all tfr orthologs were comparable (left panels), amav-gp d-ig bound cells expressing nstfr , but did not bind cells expressing ajtfr . tcrv-gp d-ig bound cells expressing ajtfr and also cells expressing nstfr . neither ig-fusion protein bound cells expressing human tfr . although both amav and tcrv pseudoviruses enter hek t cells using a tfr -independent pathway, indicating the presence of an alternative receptor, we did not observe binding of amav or tcrv-gp d-ig to these cells (data not shown). this observation is consistent with an entry process dependent on a lower affinity receptor. to determine if amav and tcrv pseudoviruses could use nstfr and ajtfr to infect cells, hek t cells were transfected with plasmids expressing human tfr , zbtfr , nstfr , ajtfr , or vector alone, and then incubated with amav and tcrv pseudoviruses. pseudovirus bearing the g protein of vesicular stomatitis virus (vsv) was used as a control. consistent with the gp d-ig binding data, amav pseudovirus used only nstfr , while tcrv pseudovirus efficiently used both ajtfr and nstfr ( figure b ). we next assessed whether nstfr and ajtfr could also serve as receptors for the pathogenic nw clade b arenaviruses. as the pathogenic nw arenaviruses efficiently enter hek t cells using endogenous human tfr [ ] , cho cells were transfected with plasmids expressing human tfr , ajtfr , nstfr , zbtfr , or vector alone, and subsequently infected with macv, junv, or gtov pseudoviruses. as shown in figure c , macv and junv, like tcrv, used both nstfr and ajtfr , while gtov, like the closely related amav, efficiently used nstfr but not ajtfr . we confirmed these findings using replication-competent tcrv and amav viruses. hek cells were transfected with plasmids expressing human tfr , ajtfr or nstfr , and subsequently infected with replication-competent tcrv or amav. as shown in figure , the infection of tcrv was enhanced on cells expressing ajtfr and nstfr , while that of amav was only enhanced on cells expressing nstfr . a loop (residues - ) between b strands and in the apical domain of human tfr is a likely interaction site for the gps of the nw hemorrhagic fever arenaviruses [ , ] . this loop includes tyrosine , which is essential for macv, junv, and gtov to use human tfr [ ] . we compared the sequences of zbtfr , nstfr , and ajtfr in this region with that of human tfr , and found several differences between human and animal tfr ( figure a and b). we sought to determine whether replacing the human tfr residues with their equivalents from animal tfr orthologs would allow the modified human tfr to serve as a receptor for amav and tcrv pseudoviruses. figure a shows a panel of eleven human tfr variants that were examined. in the first two variants (zh and zh ), residues from human tfr were replaced with those from zbtfr . four variants (ah through ah ) include residues from ajtfr , and five variants (nh , nh , nh , nh and nh ) include nstfr sequence. these variants were expressed in hek t cells, and the cells were incubated with tcrv or amav pseudoviruses. a variant (zh ) containing two alterations -r g and dv -was an efficient receptor for tcrv ( figure c ). as expected from comparison with zh , a variant (ah ) containing three ajtfr residues in place of human tfr residues - (r g, l a, and v g) was a functional receptor for tcrv ( figure d ). when mutations at r and v were individually introduced into human tfr , the variant containing v g (ah ) supported tcrv pseudovirus entry efficiently, while the variant containing r g (ah ) was a less efficient receptor. deletion of v (ah ), however, did not convert human tfr into a receptor for tcrv. a slightly larger alteration in human tfr was necessary for amav pseudovirus infection; nh , which contains four mutations -d n, k s, r g, and dv -or nh , which contains five mutations, were efficient receptors for amav ( figure e ). the entry of vsv pseudovirus was unaffected by the introduction of any of the variants. thus, only minimal alteration of human tfr was sufficient for it to serve as an efficient receptor for the nonpathogenic viruses amav and tcrv. five nw arenaviruses have been shown to cause hemorrhagic fever in humans. we have previously shown that four of these viruses -macv, junv, gtov, and sabv -require tfr to enter human cells [ ] . we have also shown that these viruses utilize the tfr orthologs of their rodent hosts efficiently and with specificity [ ] . a recently identified fifth hemorrhagic fever nw arenavirus, chapare virus, remains to be characterized. collectively, these data indicate a central role for tfr in the replication of nw clade b arenaviruses that cause hemorrhagic fever in humans. closely related to these pathogenic viruses are three clade b arenaviruses that do not cause disease in humans, namely tcrv, amav, and cpxv, although tcrv may have caused mild disease in a laboratory worker [ ] . here we have studied the two best characterized of these viruses, tcrv and amav. the gp sequences of these viruses are more similar to those of the pathogenic viruses than they are to each other (figure ). in spite of the similarity of amav and tcrv to the other members of their respective subclades, these nonpathogenic viruses do not utilize human tfr . in contrast, every clade b virus shown to use human tfr causes a south american hemorrhagic fever [ , [ ] [ ] [ ] [ ] . there is thus a close correlation between use of human tfr and the ability of a nw arenavirus to cause severe human disease. a possible exception is the whitewater arroyo virus (wwav), a nw clade a/b arenavirus that does not appear to use human tfr , but is tentatively associated with three human fatalities in - [ , ] . follow up studies by the cdc, however, failed to find any evidence of wwav infection in the human tissues mentioned in the original article (nichol, personal communication). in addition, there have been no further reports of wwav infection being associated with human disease. this correlation suggests that tfr use is a key determinant in the virulence of clade b viruses, which raises an important question: how easily might clade b viruses circulating in animals gain the ability to use human tfr ? our findings shed light on this issue; tcrv and amav pseudoviruses efficiently utilize the tfr orthologs of several species including those of their native hosts, a. jamaicensis and n. spinosus, respectively (figures and ) . as with the pathogenic clade b viruses, amav and tcrv utilized most efficiently the tfr ortholog of their natural hosts. it is therefore likely that tfr is also central to the replication of these nonpathogenic viruses in their native hosts. to investigate the possibility that gain-of-use of human tfr may not require extensive alteration of the tcrv and amav gps, we sought to identify differences between human tfr and host animal tfr orthologs that determine the ability of these molecules to support infection by tcrv and amav pseudoviruses. we swapped residues in a loop in the apical domain of tfr that is a key determinant of host specificity ( figure ) . surprisingly, alteration of a single residue (v g, ah ) allowed human tfr to efficiently support the entry of tcrv pseudovirus, while replacement of four residues in this loop with the corresponding residues of nstfr (nh ) was necessary to support efficient infection by amav pseudovirus (figure d and e). although these alterations are made to the receptor, it is reasonable to infer that similarly modest changes in the arenaviral gps would also permit efficient use of human tfr . for example, in order to bind human tfr , tcrv gp would only need to acquire mutations that accommodate v , present in human tfr but absent or replaced by a glycine in animal tfr orthologs. our data, although limited, suggest that small alterations in the arenaviral gp could convert these nonpathogenic nw arenaviruses into human pathogens. this possibility has precedent in other emerging viruses. for example, alteration of only a few residues in the spike protein of the severe acute respiratory syndrome coronavirus (sars-cov) was sufficient for this glycoprotein, derived from an animal isolate of the virus, to efficiently use the human ortholog of its receptor, angiotensin converting enzyme (ace ) [ ] . similarly, the adaptation of avian influenza viruses to humans is thought to require as few as two amino acid substitutions in the influenza hemagglutinin glycoprotein (ha). these mutations allow ha to bind its human receptor, a , -linked sialosides [ , ] . structural studies of the sars-cov and influenza a virus entry proteins in complex with their respective receptors have helped to identify and predict entry protein alterations that contribute to the ability of these viruses to cause human disease [ , ] . similar studies of arenanaviral gp molecules bound to tfr are likely to further elucidate the risk posed by amav, tcrv, and perhaps other nonpathogenic arenaviruses. of note in this regard, bats are increasingly recognized as reservoirs for viruses that can cross species barriers to infect humans [ ] . nipah, hendra, and perhaps sars-cov and ebola viruses, are on the list of emerging viruses that involve amplification in bat hosts [ ] . although tcrv was isolated from bats [ ] , the suggestion that artibeus species bats serve as the natural hosts for tcrv has been questioned [ ] . our observation that ajtfr is an efficient receptor for tcrv, and also for junv and macv, supports a role for bats in sustaining the replication of arenaviruses. ecological surveys of bats for novel arenaviruses are thus warranted, and may afford further insight into the diversity of arenaviruses. in addition, although cats experimentally infected with macv do not become ill [ ] , viral titers in these animals were not determined. the finding that feline tfr supports the entry of amav and the pathogenic clade b arenaviruses thus highlights a possible role for cats in serving as intermediate hosts in the transmission of nw arenaviruses to humans [ , ] . despite the inability of amav and tcrv to cause disease in humans, their gp proteins mediate entry into human cells through a pathway independent of human tfr or a-dystroglycan ( figure ) [ , ] . this observation suggests that these viruses can use more than one receptor, and raises the possibility that the pathogenic clade b arenaviruses may also use an additional receptor [ ] . indeed, certain strains of the old world arenavirus lymphocytic choriomeningitis virus (lcmv) use both a-dystroglycan dependent and independent pathways to infect cells [ ] . an alternative receptor may substitute for tfr , or function downstream of tfr in a manner analogous to the hiv- coreceptors. identification of such a receptor on human cells will likely broaden our understanding of the entry mechanisms of a number of nw arenaviruses. clustalw was used to align the gp sequences of the nw arenaviruses and for phylogenetic calculations, and the cladogram was plotted using drawtree [ , ] . hek t cells (human embryonic kidney, atcc, crl- ) were maintained in dulbecco's modified eagle's medium, and cho cells (chinese hamster ovary epithelial, atcc, ccl- ) in f medium. both cell lines were grown in the presence of % fetal bovine serum. plasmids encoding human, mouse, rat, c. callosus (large vesper mouse), and c. musculinus (drylands vesper mouse), z. brevicauda (short-tailed cane mouse) tfr are previously described [ ] . the plasmids expressing the tfr orthologs of f. domesticus (cat) and c. familiaris (dog) were generously provided by colin parrish (cornell university, ithaca, ny). tfr genes for neacomys spinosus and artibeus jamaicensis were cloned from frozen heart and kidney, and liver tissues, respectively. deposited sequences in genbank (fj and fj ) cross-reference to catalog numbers of the museum of southwestern biology, university of new mexico from where these specimens were obtained. rna was isolated from these tissues using rnaqueous (ambion), and cdna was generated by superscript iii first-strand synthesis system for rt-pcr (invitrogen). pcr primers used to amplify the genes for both nstfr or ajtfr orthologs are: sense atgatggatcaagccagatcagcawt-ctct and anti-sense aaaytcattgtcaatgtcc-caaaygtcacca . these genes, fused with a c-terminal flag tag, were cloned into the pcdna . expression vector (invitrogen). amino acid substitutions in human tfr were performed using the quikchange method for site-directed mutagenesis (stratagene). amav gp d-ig and tcrv gp d-ig were generated by pcr-amplification of amav gp residues - and tcrv gp residues - , with boundary residues selected according to a similar construct of macv gp previously shown to bind most efficiently to vero cells [ ] . these fragments were cloned into a previously described pcdm -based plasmid containing the cd signal sequence and the fc region of human igg [ ] . macv (carvallo strain), junv (mc ), and gtov (inh- ), gp-expressor plasmids have been described previously [ ] . genes for amav and tcrv gp are gifts from stefan kunz (university of lausanne, switzerland); they were pcr amplified from infected vero cells, and cloned into pcaggs expression vector. the sequence of amav gp is the same as bean strain (genbank af ), and that of tcrv has a deletion spanning amino acid residues - , and substitutions at three residues, i a, g s and e r, compared to genbank nc_ . amav gp d-ig and tcrv gp d-ig binding to various tfr orthologs was detected by flow cytometry. ig-fusion proteins were produced in free style hek expression medium (invitrogen) from hek t cells transfected with the appropriate plasmid, purified by binding to protein-a sepharose and eluted with m mgcl , dialyzed in pbs, and concentrated using a ym filter unit (millipore). for binding assays, hek t cells were transfected with the various tfr -encoding plasmids. after hr, cells were detached by mm edta/pbs and incubated in ul of pbs- % goat serum containing . ug of either an aflag m monoclonal antibody (sigma) or the indicated ig-fusion protein. cells were then stained with a-mouse igg, or with ahuman igg (fc-specific) antibodies conjugated with phycoerythrin (jackson immuno laboratories), respectively. to generate recombinant retroviruses pseudotyped with arenaviral gp molecules, hek t cells were transfected at a : : ratio with plasmids encoding the respective arenaviral gp, the mlv gag and pol genes, and the pqcxix retroviral vector (bd biosciences) expressing gfp, as previously described [ ] . the structure of the human tfr dimer is shown, oriented with the cell membrane at the bottom. the apical, protease-like, and helical domains are indicated in green, red, and yellow, respectively, on one monomer. the other monomer is shown in cyan. in the right panel, the tfr apical domain is enlarged; a loop comprising residues - , implicated as a site of interaction with the gps of nw clade b arenaviruses, is shown. the side chains of residues d , k , r , v , and y are colored yellow. the image was rendered using pymol [ ] . (b) sequence alignment of residues through of human tfr with analogous sequences of the tfr orthologs of z. brevicauda (zbtfr ), a. jamaicensis (ajtfr ), and n. spinosus (nstfr ). variants of human tfr containing sequence from z. brevicauda (zh and zh ), a. jamaicensis (ah through ah ), and n. spinosus (nh , nh , nh , nh , and nh ) tfr were generated based on this sequence alignment. zb, aj, and nstfr sequences are shown in green, blue, and yellow, respectively. the right panel summarizes the entry data. nd = not determined. (c-e) hek t cells were transfected with plasmids encoding human, zb, aj, or nstfr along with the zh variants (c), the ah variants (d), or the nh variants (e). the expression level of each tfr variant was assessed as in figure . in parallel, cells were infected with amav, tcrv, or vsv pseudoviruses. the expression levels of the various tfr orthologs were normalized to that of human tfr (aflag, left panels), and infection levels were normalized to that of mock-transfected cells. doi: . /journal.ppat. .g biology and pathogenesis of lymphocytic choriomeningitis virus infection arenaviruses other than lassa virus chapare virus genetic identification of kodoko virus, a novel arenavirus of the african pigmy mouse (mus nannomys minutoides) in west africa the phylogeny of new world (tacaribe complex) arenaviruses molecular phylogeny of the arenaviruses phylogeny and evolution of old world arenaviruses new arenavirus isolated in brazil description of guanarito virus (arenaviridae: arenavirus), the etiologic agent of venezuelan hemorrhagic fever arenavirus diseases. london: chapman & hall medical phylogenetic analysis of the arenaviridae: patterns of virus evolution and evidence for cospeciation between arenaviruses and their rodent hosts mammalian reservoirs of arenaviruses natural rodent host associations of guanarito and pirital viruses (family arenaviridae) in central venezuela isolation of machupo virus from wild rodent calomys callosus chronic infection of rodents by machupo virus junin virus activity in rodents from endemic and nonendemic loci in central argentina serological evidence of infection of tacaribe virus and arboviruses in trinidadian bats serologic survey of neotropical bats in guatemala for virus antibodies tacaribe virus, a new agent isolated from artibeus bats and mosquitoes in trinidad, west indies site protease is required for proteolytic processing of the glycoproteins of the south american hemorrhagic fever viruses junin, machupo, and guanarito cell entry by human pathogenic arenaviruses role of the stable signal peptide of junin arenavirus envelope glycoprotein in ph-dependent membrane fusion the signal peptide of the junin arenavirus envelope glycoprotein is myristoylated and forms an essential subunit of the mature g -g complex distinct requirements for signal peptidase processing and function in the stable signal peptide subunit of the junin virus envelope glycoprotein identification of lassa virus glycoprotein signal peptide as a trans-acting maturation factor viral membrane fusion structural characterization of viral fusion proteins receptor structure, binding, and cell entry of arenaviruses genetic analysis of heptad-repeat regions in the g fusion subunit of the junin arenavirus envelope glycoprotein identification of alpha-dystroglycan as a receptor for lymphocytic choriomeningitis virus and lassa fever virus molecular analysis of the interaction of lcmv with its cellular receptor [alpha]-dystroglycan transferrin receptor is a cellular receptor for new world haemorrhagic fever arenaviruses new world arenavirus clade c, but not clade a and b viruses, utilizes alphadystroglycan as its major receptor transferrin receptors in human tissues: their distribution and possible clinical relevance transferrin receptor on endothelium of brain capillaries arenaviruses. ii. the molecular pathogenesis of arenavirus infections. introduction pathology of bolivian hemorrhagic fever in the rhesus monkey the transferrin receptor part i: biology and targeting with cytotoxic antibodies for the treatment of cancer receptor determinants of zoonotic transmission of new world hemorrhagic fever arenaviruses new world clade b arenaviruses can use transferrin receptor (tfr )-dependent and independent entry pathways, and glycoproteins from human pathogenic strains are associated with the use of tfr differences in tropism and ph dependence for glycoproteins from the clade b arenaviruses: implications for receptor usage and pathogenicity receptor use by pathogenic arenaviruses characterization of the cellular receptors for the south american hemorrhagic fever viruses junin, guanarito, and machupo crystal structure of the ectodomain of human transferrin receptor fatal illnesses associated with a new world arenavirus-california receptor use by the whitewater arroyo virus glycoprotein receptor and viral determinants of sars-coronavirus adaptation to human ace x-ray structures of h avian and h swine influenza virus hemagglutinins bound to avian and human receptor analogs glycan microarray analysis of the hemagglutinins from modern and pandemic influenza viruses reveals different receptor specificities structure of sars coronavirus spike receptor-binding domain complexed with receptor bats: important reservoir hosts of emerging viruses bats as a continuing source of emerging infections in humans arenaviridae: the viruses and their replication epidemiology of machupo virus infection. ii. ecological and control studies of hemorrhagic fever differences in affinity of binding of lymphocytic choriomeningitis virus strains to the cellular receptor alpha-dystroglycan correlate with viral tropism and disease kinetics phylip-phylogeny inference package (version . ) clustal w: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice angiotensinconverting enzyme is a functional receptor for the sars coronavirus the pymol molecular graphics system we thank joseph a. cook, museum of southwestern biology, division of genomic resources, university of new mexico, and robert j. baker, natural science research laboratory, the museum of texas tech university, for kindly providing tissues for artibeus jamaicensis and neacomys spinosus. we thank stephen c. harrison for manuscript editing and helpful discussion. cell supernatants were harvested hr post-transfection, and filtered through a . mm filter. hek t cells or cho cells were transfected with plasmids expressing various tfr orthologs, and at hr, the cells were replated for flow cytometry and infection. at hr post-transfection, tfr expression levels were assessed by flow cytometry using an a-flag m antibody. in parallel, cells were infected with the indicated pseudoviruses for hours. twenty-four (hek t cells) or forty-eight (cho cells) hours after infection, the cells were detached with trypsin and the gfp expression levels were measured by flow cytometry, and normalized to that of mock-transfected cells. values are the average of - duplicated experiments. infection of hek cells with replication-competent tcrv (trvl ) or amav (bean ), was performed in the bsl laboratory at the special pathogens branch, centers for disease control and prevention, atlanta. infections and immunofluorescence staining were performed as described previously [ ] . briefly, hek cells growing on gelatin-treated glass coverslips were transfected with plasmids encoding ajtfr , nstfr , or human tfr . thirty-six hours later, these cells were infected with tcrv or amav at a multiplicity of infection of . . at hr post-infection, the cells were washed, fixed with % formaldehyde, permeabilized with . % triton x- and incubated with mouse hyperimmune sera specific for nw arenaviruses. cells were then washed and incubated with a fitc-conjugated secondary antibody. key: cord- -w xuf h authors: galluzzi, lorenzo; brenner, catherine; morselli, eugenia; touat, zahia; kroemer, guido title: viral control of mitochondrial apoptosis date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: w xuf h throughout the process of pathogen–host co-evolution, viruses have developed a battery of distinct strategies to overcome biochemical and immunological defenses of the host. thus, viruses have acquired the capacity to subvert host cell apoptosis, control inflammatory responses, and evade immune reactions. since the elimination of infected cells via programmed cell death is one of the most ancestral defense mechanisms against infection, disabling host cell apoptosis might represent an almost obligate step in the viral life cycle. conversely, viruses may take advantage of stimulating apoptosis, either to kill uninfected cells from the immune system, or to induce the breakdown of infected cells, thereby favoring viral dissemination. several viral polypeptides are homologs of host-derived apoptosis-regulatory proteins, such as members of the bcl- family. moreover, viral factors with no homology to host proteins specifically target key components of the apoptotic machinery. here, we summarize the current knowledge on the viral modulation of mitochondrial apoptosis, by focusing in particular on the mechanisms by which viral proteins control the host cell death apparatus. the sacrifice, via programmed cell death (pcd), of infected cells represents the most primordial response of multicellular organisms to viruses. this response is common to all metazoan phyla, including plants (which lack an immune system based on mobile cells) (text s , [s ]). in mammals, microbial invasion does not only trigger pcd of infected cells but also elicits an immune reaction. this is hierarchically organized in a first-line response provided by innate immune effectors (e.g., infiltrating phagocytes and natural killer cells) [s ], followed by the activation of adaptive immunity, mediated by t and b lymphocytes [s ] . importantly, other layers of defense exist to prevent viral replication and spread [s ] . for instance, in invertebrates like drosophyla melanogaster (as well as in plants), a prominent antiviral mechanism is provided by rna interference (rnai) [s ] . although the rnai pathway is preserved in mammals, it has presumably been superseded in its antiviral role by the extremely potent interferon system, as well as by a number of additional mechanisms [s ] . such a multivariate antiviral response is designed to recognize virions, virus-infected cells, and virus-induced signals of stress (including cell death) to eliminate the pathogen (together with the host cell) and to elicit immunological memory [s ] . thus, the co-evolution between host and virus has forced the latter to develop strategies for modulating host cell pcd and/or for avoiding immunogenic cell death. apoptosis is an active mode of pcd exhibiting a series of morphological and biochemical changes by which it can be distinguished from other cell death subroutines [s ] . at a morphological level, these modifications include a dramatic reduction in cell volume (cell shrinkage), nuclear pyknosis (chromatin condensation), and karyorrhexis (nuclear fragmenta-tion). eventually, dying cells break down into small, discrete bodies known as apoptotic bodies [s ] . the morphological appearance of apoptosis is accounted for by an ensemble of biochemical events that include, but are not limited to: ( ) loss of the structural integrity and bioenergetic functions of mitochondria, ( ) cascade activation of a specific set of catabolic enzymes (e.g., proteases of the caspase family, nucleases), ( ) exposure of phosphatydylserine on the outer leaflet of plasma membrane and, finally, ( ) loss of the barrier function of the plasma membrane [s ] . apoptosis can be triggered by two fundamentally distinct signaling cascades, namely the extrinsic and intrinsic (or mitochondrial) pathways (for a recent review, see [ ] ) ( figure ). the extrinsic pathway is started by the ligand-induced oligomerization of specific cell surface receptors, such as fas/cd and the tumor necrosis factor receptor (tnfr). this induces the intracellular assembly of the death-inducing signaling complex (disc), a molecular platform for the activation of the caspase cascade that emanates from caspase- and results in the activation of effector caspases and nucleases (e.g., caspase- , - , and - , caspase-activated dnase) ( figure ) [s ,s ]. in contrast, the intrinsic pathway is controlled by mitochondria, which collect and integrate pro-and antiapoptotic signals incoming from other organelles as well as from the extracellular microenvironment. notably, proapoptotic stimuli as diverse as dna damage, endoplasmic reticulum (er) stress, lysosomal stress, reactiveoxygen species (ros), and calcium (ca + ) overload are able to activate the intrinsic pathway of apoptosis by favoring mitochondrial membrane permeabilization (mmp) (figure ) [s ,s ]. in some cells, mitochondrial apoptosis may ensue the activation of death receptors, due to the mmp-promoting activity of the bh only protein bid, which can be proteolytically activated by caspase- [s ,s ]. mmp culminates in the loss of mitochondrial transmembrane potential (dy m ), an arrest of mitochondrial bioenergetic and biosynthetic functions, and in the release of mitochondrial intermembrane space (ims) proteins into the cytosol. these proteins include caspase activators like cytochrome c (cyt c) [s ,s ] and smac/diablo [s ], as well as caspaseindependent death effectors such as apoptosis-inducing factor (aif) [s ,s ] and endonuclease g (endog) [s ]. thus, mmp activates caspase-dependent and/or -independent mechanisms to execute cell death [ ] . impaired mmp is associated with multiple pathological conditions, including autoimmune diseases and cancer [s ]. conversely, unscheduled mmp contributes to the development of diseases characterized by an excess of cell death, such as ischemia/ reperfusion injuries, trauma, toxic/metabolic syndromes as well as chronic neurodegenerative conditions like amyotrophic lateral sclerosis or alzheimer, parkinson, and huntington diseases [s ]. mmp is regulated by a complex network of signaling pathways that involves both endogenous (e.g., pro-and antiapoptotic bcl during the last decade, major efforts have been dedicated to the elucidation of the mechanisms underlying mmp in health and disease. according to current beliefs, mmp is executed via either of two distinct, yet partially overlapping and non-mutually exclusive, mechanisms. these two routes to mmp are initiated at different mitochondrial subcompartments (notably at the mitochondrial outer or inner membrane, i.e., om and im) and each relies on a specific set of factors. nonetheless, they both lead to a functional and structural collapse of mitochondria that commits the cell to death [ ] . notably, mmp is associated with dramatic changes in the mitochondrial network as well as in mitochondrial ultrastructure, concerning both matrix volume and cristae organization [s -s ]. how these structural modifications of mitochondria might impact on viral infection, however, remains to be elucidated. the abundant presence of the voltage-dependent anion channel (vdac) renders om freely permeable to solutes and small metabolites up to approximately kda. this cutoff ensures that soluble proteins are retained in the ims under normal circumstances. the apoptosis-associated drastic increase in om permeability may originate at the om itself by means of multiple mechanisms, including ( ) the assembly of large homo-or heteromultimeric channels, allowing for the release of ims proteins, by proapoptotic pore-forming proteins of the bcl- family (e.g., bax, bak) [ , ;s ,s ] ; ( ) the destabilization of the lipid bilayer mediated by proapoptotic bcl- family members (e.g., bax, figure . the extrinsic and the intrinsic (mitochondrial) pathways of apoptosis. the extrinsic apoptotic pathway involves the activation of death receptors at the cell surface, followed by a caspase cascade that eventually leads to the execution of cell death. in contrast, different proapoptotic stimuli initiate the intrinsic pathway by triggering mitochondrial membrane permeabilization (mmp). following mmp, intermembrane space proteins are released into the cytosol, the mitochondrial transmembrane potential (dy m ) is dissipated, and the bioenergetic and redoxdetoxifying functions of mitochondria are compromised. the resulting bioenergetic and redox crises, associated with the activation of both caspasedependent and -independent executioner mechanisms, commit the cell to death. the two pathways are interconnected by the bh -only protein bid, whose truncated form (tbid) is generated by caspase- and can target mitochondria to trigger mmp. for a more detailed description of the intrinsic and extrinsic pathways of apoptosis please refer to the introduction and to [ ] . disc, death-inducing signaling complex; er, endoplasmic reticulum. doi: . /journal.ppat. .g truncated bid, i.e., tbid), which results in the priming of mitochondria for the release of ims proteins [s -s ]; and ( ) the induction of the so-called mitochondrial permeability transition (mpt) at the im, following the interaction between bax (or tbid) and components of the permeability transition pore complex (ptpc) at the om [ ;s -s ]. in this latter case, mmp begins and ends at the om, yet is mediated by an event taking place mainly at the im, i.e., mpt (see the section ''mmp regulation by the ptpc'' for further details). independently from the specific mechanisms that activate mmp, the bcl- family of proteins exerts a major regulation of this process [s ]. the bcl- family is composed of antiapoptotic multidomain members (e.g., bcl- , bcl-x l , mcl- ), which contain four bcl- homology (bh) domains (bh - ) [s ], proapoptotic multidomain proteins (e.g., bax, bak) [s ], which contain three bh domains (bh - ), and pro-apoptotic bh -only proteins (e.g., bid, bad) [ ] . due to an additional c-terminal domain, some members of all the subgroups share the ability to insert into the om and other intracellular membranes (e.g., er) [ ] .the specific set of bh domains contained in each bcl- family member determines its profile of activity [s -s ]. in this context, early structure-function studies identified bh , bh , and bh as the major antiapoptotic determinants of bcl- [s -s ]. conversely, the presence of the bh domain was found to suffice for apoptosis induction by bax (as well as for heterodimerization with bcl- /bcl-x l ) [s -s ]. later, numerous reports showed that the conserved transmembrane (tm) domain and less conserved, unstructured loops between bh domains also contribute to define the functional profile of bcl- proteins, either by acting as targeting signals for subcellular compartments (e.g., mitochondria, er) or by modulating the overall tertiary structure [s -s ]. bcl- /bcl-x l stabilizes mitochondrial membranes via multiple mechanisms, including ( ) the sequestration into inactive complexes of its proapoptotic counterparts, bax, bak, and bh only proteins (e.g., bid) (for review: [s ,s ]), ( ) inhibitory interactions with ptpc constituents, in particular with vdac and the adenine nucleotide translocase (ant) [ , ] , ( ) an enhancement of cyt c oxidase activity and mitochondrial respiration [s ], and/or ( ) indirect effects on intracellular ca + stores of the er [s ,s ]. while bax/bak execute mmp by one (or more) of the aforementioned mechanisms, bh -only proteins exhibit indirect proapoptotic effects [ ;s ,s ]. thus, ''activator'' bh -only proteins (e.g., bid) would directly interact and activate bax/bak, whereas the ''derepressors'' (e.g., bad) would rather disrupt bcl- /bcl-x l inhibitory complexes, thus allowing for the release of bax/bak [ ,s ] . in healthy cells, inactive bak is constitutively associated with mitochondria, while bax is found as a cytosolic monomer [s ]. upon apoptosis induction, both undergo a conformational modification to become activated, and bax translocates to om in the form of an active dimer [s ,s ]. when bax or bak induce mmp, the release of specific ims proteins occurs via large pores in the om and may precede dy m loss. in some instances of bax-mediated apoptosis, indeed, mitochondrial membrane permeabilization (momp) occurs without discernable dy m alterations [ ] (figure ). large pores formed by the oligomerization of proapoptotic bcl- proteins (e.g., bax, bak) and/or the voltage-dependent anion channel (vdac) may promote selectively mitochondrial outer membrane permeabilization (momp). in this case, specific intermembrane space (ims) proteins are liberated in the cytosol, but the mitochondrial transmembrane potential (dy m ) is (at least initially) retained (a,b). on the contrary, some proapoptotic stimuli, such as calcium (ca + ) overload, reactive oxygen species (ros), and the lipid second messenger ceramide, favor mmp by inducing the permeabilization of the inner mitochondrial membrane (im) via the activation of the permeability transition pore complex (ptpc). when the ptpc opens, dy m is immediately lost and an unregulated entry of solutes and water into the mitochondrial matrix occurs. this results in the osmotic swelling of mitochondria, followed by rupture of both mitochondrial membranes and the unspecific release into the cytosol of ims proteins (a,c) (please refer to the sections ''mmp regulation by bcl- family proteins'' and ''mmp regulation by the ptpc'' for additional details). notably, antiapoptotic proteins from the bcl- family play a role in both models. aif, apoptosis-inducing factor; ant, adenine nucleotide translocase; ck, creatine kinase; cypd, cyclophilin d; cyt c, cytochrome c; hk, hexokinase; oxphos, oxidative phosphorylation complexes; pbr, peripheral-type benzodiazepine receptor. doi: . /journal.ppat. .g despite their structural similarity, each bh -only protein presents a specific mechanism of activation (either at a transcriptional level or mediated by post-translational modifications), and acts as a sensor of a particular type of cell stress [ ,s ] . for instance, the bh -only proteins puma and noxa are activated by dna damage via p dependent transactivation [ ;s ] , bim and bmf are released from cytoskeletal structures upon c-jun n-terminal kinase (jnk)-mediated phosphorylation [s -s ], and bid is proteolytically processed by caspase- following the activation of the extrinsic pathway of apoptosis [s ,s ]. following caspase- -mediated cleavage, a glycine residue of tbid is exposed, allowing for post-translational (rather than usual cotranslational) n-myristoylation. this modification has been shown to act as an activating switch and to enhance tbid-induced cyt c release and cell death [s ] . interestingly, tbid mitochondrial targeting [s ] and proapoptotic activity [s ] have been associated with cardiolipin, a mitochondrial lipid particularly abundant in the im. tbid-cardiolipin interaction requires three ahelical domains (a -a ) of tbid and occurs prominently at the contact sites between the om and the im [s ]. cardiolipin might also be implicated in the dissociation of bid fragments (tbid and nbid), which would rather occur during the targeting of tbid to mitochondria than immediately after caspase- -mediated cleavage [s ] . although bcl- family proteins exert their apoptosismodulatory functions mainly at mitochondria, extra-mitochondrial activities contribute to their effects. for instance, bcl- /bcl-x l localize at the er and decrease luminal ca + concentration, thus protecting against ca + -dependent death stimuli [s ,s ,s ]. conversely, bax/bak favor the transfer of ca + from the er to mitochondria and cell death [s ,s ]. moreover, bax / /bak / mefs show an impaired mobilization of er ca + following numerous proapoptotic stimuli, which can be partially restored by overexpressing the sarco/endoplasmic reticulum ca + atp-ase (serca) [ ] . taken altogether, these observations point to the bcl- system as a prominent pharmacological target for the modulation of mitochondrial apoptosis (for a review, see [ ] ). mmp may originate at the im due to the activation of the ptpc, a large multiprotein structure assembled at the contact sites between om and im. this applies in particular to cell death models characterized by enhanced ca + fluxes and disproportionate ros generation [s ] . ptpc activation provokes a sudden increase in the im permeability to solutes of low molecular weight (i.e., mpt), which leads to the unregulated entry of water and osmotic swelling of the mitochondrial matrix. in turn, this may result in the physical rupture of the om, because the surface area of the im (with its folded cristae) largely exceeds that of the om [s ,s ,s ]. in the context of mpt-derived mmp, dy m dissipates before om is permeabilized and ims are released ( figure ) [s ]. although its exact molecular composition remains elusive, numerous independent studies suggest that the ptpc might result from the association of multiple proteins, including ant (in the im) and vdac (in the om), in the context of a dynamic interaction with mitochondrial matrix proteins (e.g., cyclophilin d [cypd]), ims proteins (e.g., creatine kinase [ck]), om proteins (e.g., peripheral-type benzodiazepine receptor [pbr]), as well as with cytosolic factors (e.g., hexokinase isoforms) (for recent reviews, see [s ,s ,s ]). nevertheless, genetic studies performed in the murine system suggest that all the aforementioned components of the ptpc, most of which exist in multiple isoforms, are either dispensable for cell death or preferentially participate in necrotic pathways (rather than in apoptosis) [ - ;s ]. in addition, controversial views remain about the mechanisms by which the ptpc promotes mpt and therefore mmp. some authors have proposed that in physiological conditions vdac would be found within the ptpc in a state of low conductance, rapidly switching between the open and closed conformations [s ]. in this configuration, the ptpc would ensure the normal exchange of metabolites between the mitochondrial matrix and the cytosol. following proapoptotic stimuli, a state of high conductance for vdac would be favored, resulting in longlasting openings of the ptpc and mpt [ ;s ] . alternatively, it has been suggested that the high conductance state of vdac would serve to its physiological functions, whereas cell death would result from a closed conformation, favoring a transient hyperpolarization of the mitochondrial matrix, followed by osmotic imbalance, swelling, and eventually mmp [s -s ]. several reports indicate ptpc components as targets for the apoptosis-modulatory activity of both pro-and antiapoptotic bcl- family members. in this context, it has been demonstrated that bax and bak accelerate the opening of vdac in reconstituted proteoliposomes, and that vdac-deficient mitochondria do not exhibit bax/bak-induced cyt c release and dy m dissipation occurring in vdac-proficient control mitochondria [ ;s ]. in the same model, recombinant bcl- /bcl-x l as well as synthetic peptides corresponding to their bh domains were shown to prevent vdac opening, cyt c release, and dy m dissipation [s ,s ]. in addition, the bh -only proteins bid and bim have been reported to interact directly with vdac, the latter interaction being remarkably enhanced during apoptosis [s ,s ]. bax and bcl- /bcl-x l also modulate ptpc activity by binding to ant. as demonstrated by the yeast two-hybrid system, coimmunoprecipitation assays, and in artificial lipid bilayers, ant and bax directly interact and cooperate to form a channel with distinct electrophysiological properties as compared to the channels formed by bax or ant alone [ ;s ]. in artificial membranes, the presence of bcl- inhibited cooperative channel formation by bax and ant as well as the atractyloside-induced assembly of channels by ant alone, thus pointing to a direct interaction between ant and bcl- [s ,s ]. furthermore, bcl- promotes (and bax inhibits) adp/atp exchange in ant-containing proteoliposomes, isolated mitochondria, and mitoplasts [s ]. interestingly, in this system the bax-mediated inhibition of ant translocase activity could be separated from the formation of cooperative channels by bax and ant [s ]. as determined by co-immunoprecipitation and proteomics analysis, the interactome of ant undergoes major rearrangements in the course of the chemotherapy-induced apoptosis. thus, soon after the treatment with etoposide of a human tumor cell line (ht cells), the amount of bax contained within the ant interactome significantly augmented, whereas the quantity of bcl- was decreased [s ]. as previously mentioned in the section ''mmp regulation by bcl- family proteins'', bcl- family members are known to modulate luminal ca + concentration and ca + release at the er [ ;s ,s ]. in doing so, they exert an additional indirect control on the ptpc, since cytosolic ca + liberated from er stores (for instance upon the induction of the unfolded protein response) can accumulate in mitochondria and promote ptpc opening, mptdependent mmp, and cell death [s ]. thus, it appears that an intricate crosstalk for the modulation of mmp exists between mitochondria and the er, in which proteins from the bcl- family participate at the level of both organelles [s ,s ]. during the last decade, numerous viral proteins have been reported to modulate (either positively or negatively, either in a direct or indirect fashion) the apoptotic response of host cells to infection ( figure , tables and ) [ ;s ]. with regard to this, viral factors can be classified into one of the four following subgroups: proapoptotic proteins ( ) that insert into mitochondrial membranes and hence trigger mmp through the action of amphipathic a-helical domains or ( ) that promote mmp indirectly, through the activition of host-encoded factors (table ) , and antiapoptotic modulators ( ) that exhibit sequence and/or structural similarity to multidomain bh - members of the bcl- family (so-called viral bcl- proteins [vbcl- s]) or ( ) that inhibit apoptosis via other mechanisms (table ) . notably, some viral proteins exhibit mixed apoptosis-modulatory functions, and hence cannot be unambiguously classified into one of the aforementioned groups. several proteins encoded by the human immunodeficiency virus (hiv- ) exert a proapoptotic activity, thereby contributing to the hiv-induced depletion of cd + lymphocytes (for a review, see [ ] ). among these, the viral protein r (vpr) has direct mitochondrial effects in numerous cell types independently from its mode of delivery (viral infection, transfection of a vpr gene, or exogenous administration of recombinant vpr protein) [s ,s ]. the c-terminal moiety (aa - ) of vpr directly interacts with ant and vdac, thereby triggering mmp associated with dy m loss, ims proteins release, and caspase cascade activation [ ;s ,s ]. when added in vitro to purified mouse liver mitochondria, a synthetic vpr-derived peptide (vpr - ) induced large amplitude swelling (an indicator of im permeabilization and ptpc pore opening) in less than minutes. this effect could be prevented by bcl- as well as by pharmacological agents targeting ant (such as bongrekic acid [ba]) or vdac (such as , -diisothiocyanatostilbene- , disulfonic acid [dids]) [ ] . in lymphoid cells, vpr-mediated mmp and apoptosis is facilitated by bax, yet inhibited by overexpression of bcl- or addition of the ptpc inhibitor cyclosporine a (csa) [s ,s ]. recently, it has been proposed that two distinct domains of vpr (namely aa - and aa - ) would bind to a region encompassing the first ant ims loop and part of its second and third tm helices [s ]. this model may explain why vpr is able to convert ant into a non-specific pore, as this has been observed experimentally when adding vprderived peptides to ant-containing proteoliposomes [ ] . importantly, one of the vpr arginine residues that is required for the interaction with ant (r ) [s ] is frequently mutated (r q) in long-term non-progressors (i.e., hiv- -carriers who fail to develop signs of cd + t cell depletion within years after infection) [s ]. this points to the functional relevance of the ant-vpr interaction to the development of aids. nonetheless, vpr has been reported to modulate viral replication independently from its proapoptotic function, presumably via an interaction with host proteins other than ant [s ]. the minimal cytotoxic domain of vpr that binds to ant (aa - ) is structured as an amphipathic a-helix [ ] . this short cytotoxic domain (vpr [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] has been recently conjugated with a tumor blood vessel rgd-like ''homing'' motif. this hybrid peptide is highly efficient in killing human endothelial cells, presumably because it binds to the cell surface, internalizes, and finally interacts with mitochondrial membranes to trigger mmp [s ]. in contrast with vpr - , vpr - induces cell death independently from bax and bak, and efficiently overcomes bcl- -mediated apoptosis resistance [s ]. the differences in the modes of action of vpr - and vpr - remain elusive. nonetheless, the design of cell type-targeted mitochondriotoxic peptides, such as those derived from vpr, has opened tantalizing possibilities for the therapeutic induction of apoptosis (reviewed in [ ;s ,s ]). hepatitis b virus (hbv) is one of the most prominent etiological agents of chronic liver disease and persistent infection is associated with hepatocellular carcinoma [ ;s ]. hbv x protein (hbx) is implicated in viral replication and exhibits an oncogenic potential in animal models [ ;s ]. moreover, hbx sensitizes cells to tumor necrosis factor a (tnfa)-induced apoptosis [s ]. heterologous expression of hbx in cultured human hepatoma cells results in its mitochondrial accumulation, interaction with vdac , and dy m dissipation, coupled to a perinuclear redistribution of mitochondria [s ,s ]. mutagenesis studies revealed that hbx localization to mitochondria and dy m dissipation do not depend on its transactivation domain [s ,s ], but require a putative tm region (aa - ) of the protein, while two additional amphipathic helical domains (aa - and - ) provide only marginal contributions [s ]. morevoer, hbx binds to the heat shock protein of kda (hsp ), a mitochondrial matrix chaperon [s ]. interestingly, ultrastructural and functional impairment of mitochondria as well as vdac overexpression have been associated with chronic liver disease, further supporting an etiological role for hbx in this context [s ]. poliovirus (plv) infection causes paralytic poliomyelitis, an acute disease resulting in flaccid paralysis associated with caspasedependent apoptosis of motor neurons [s ]. similar to hbx, the plv viroporin b localizes to mitochondria, induces a perinuclear redistribution of these organelles and alters their morphology, suggesting that b might exert proapoptotic effects by directly promoting mmp [s ]. as discussed below, this is not the sole mechanism accounting for plv-induced neurodegeneration (see the section ''indirect mmp facilitators''). similar perinuclear clustering of mitochondria and dy m loss, followed by partial cyt c release and signs of apoptosis (e.g., phosphatidylserine exposure on the outer leaflet of the plasma membrane and chromatin condensation), is observed upon the overexpression of the orfc protein from the walleye dermal sarcoma virus (wdsv), a retrovirus causing benign tumors in fish characterized by seasonal regression [s ,s ]. orfc is a basic protein of aa that localizes to mitochondria, although it does not possess any homology to known mitochondrial targeting sequence (mts) [s ]. interestingly, regressing tumors express orfc at high levels, pointing to the involvement of orfc-mediated apoptosis in this phenomenon [s ,s ]. human t lymphotropic virus (htlv- ) infection is linked with a diverse range of neurodegenerative and lymphoproliferative disorders, notably acute t cell leukemia [ ] . the genome of this complex retrovirus codes for typical structural and enzymatic proteins but also for unique regulatory and accessory factors that are involved in both htlv- viral cycle and pathogenesis [s ,s ]. among these, p (ii) is an -aa protein that is targeted to mitochondria, where it promotes a rapid flux of k + and ca + across im together with swelling, dy m dissipation, and fragmentation [ ;s ]. p (ii) n-terminus includes a short hydrophobic leader peptide, followed by an amphipathic a-helical mts. within this region, ten residues are sufficient to target a green fluorescent protein (gfp)-p (ii) fusion to mitochondria [s ], and four arginines form a positively charged patch on one side of the a-helix, which is responsible for its amphipathic properties [s ]. p (ii) expression has been shown to enhance caspase-dependent fas-and c ceramide-induced apoptosis [s ], and to suppress the proliferative and tumorigenic potential of cells transformed by the myc and ras oncogenes [s ]. the bovine leukemia virus (blv) is an htlv- homolog causing lymphoproliferative disorders in multiple species [s ]. similarly to p (ii), blv accessory protein g is localized to both the nucleus and mitochondria, due to an mts consisting of a hydrophobic region and an amphipathic a-helix [s ]. while g is known to alter mitochondrial morphology [s ], its exact role in blv pathogenesis remains poorly understood. indeed, whereas g exhibits an oncogenic potential both in vitro and in vivo [s ,s ] and g -deleted blv variants are characterized by reduced in vivo propagation [s ], blv-infected peripheral blood mononuclear cells are protected from apoptosis independently from g expression [s ]. as a possibility, g effects on cellular transformation may rather result from its interaction with farnesyl pyrophosphate synthetase (fpps) than from the direct modulation of mmp [s ]. pb -f is an -aa protein encoded by influenza a virus (iav) that determines virulence in murine models of infection [ ;s ]. while iavs genetically engineered to lack pb -f replicated normally both in tissue cultures and in mouse lungs, their pathogenicity and associated mortality were greatly diminished as compared to wild-type strains [s ]. pb -f inserts into mitochondrial membranes via a positively charged amphipathic ahelix located in its c-terminus [ ] . there, pb -f acquires poreforming properties similar to those displayed by proapoptotic bcl- family members (e.g., bax) [s ]. the domain necessary and sufficient for mitochondrial targeting has been mapped to residues to of pb -f [s ]. this region comprises a short hydrophobic region including several basic residues followed by an amphipathic a-helix, and closely resembles the mts found in vpr, p (ii) and, g [ ] . as assessed by glutathione-s-transferase pulldown assays followed by mass spectrometry, the sole mitochondrial proteins interacting with pb -f are the isoform of vdac (vdac ) and the isoform of ant (ant ) [s ]. accordingly, ptpc blockers such as ba and csa prevent pb -f induced mmp and apoptosis [s ]. however, pb -f is capable of destabilizing lipid bilayers in the electric field, implying that the protein may promote mmp by acting directly on mitochondrial membranes [s ]. altogether, human papillomaviruses (hpvs) are responsible for a broad range of infectious diseases, ranging from anogenital warts (e.g., hpv- and - ) to progressive dysplastic-neoplastic lesions of the genital mucosa (e.g., hpv- and - ) [s ]. the hpv genome codes for a -aa protein encompassing part of the e and e open reading frames (orfs), called e e [s ]. in human mature keratinocytes (the natural host for hpv infection), e e binds to cytokeratins, thereby inducing the total collapse of the keratin (but not of tubulin and actin) cytoskeleton [ ] . thereafter (or in cells lacking cytokeratins), e e localizes to mitochondria, due to an n-terminal leucine-rich region [s ]. via a yet unidentified mechanism, e e displaces mitochondria from microtubules, promoting the aggregation of the organelles in a large perinuclear cluster, followed by dy m dissipation and apoptosis [s ]. both the structural protein vp and the non structural protein c encoded by the avian encephalomyelitis virus (aev) promote cell death [s ,s ]. in different cell lines, vp localizes to mitochondria and sets off the caspase cascade leading to apoptosis [s ]. comparable effects result from the expression of c, which is a conserved protein of picornaviruses with a role in several steps of the viral life cycle [s ]. the proapoptotic function of c is associated with an n-terminal domain of aa (from residues to ), which has a putative a-helical structure and lies in the proximity of a coiled-coiled domain [s ]. as a last example, the non structural protein a (ns a) from hepatitis c virus (hcv) localizes (at least in part) at mitochondria, thereby causing mitochondrial damage (associated with dy m dissipation and cyt c release) and impairing the intracellular distribution of these organelles [s ]. notably, acute hcv infection often progresses to chronic hepatitis, cirrhosis and hepatocellular carcinoma [s ], and hence it is not surprising that the genome of hvc encodes for several other modulators of apoptosis (see also the sections ''indirect mmp facilitators'' and ''other antiapoptotic viral strategies'') [s ,s ]. multiple proteins encoded by the hiv- genome initiate mitochondrial apoptosis in an indirect fashion (table ) , without a physical interaction with mitochondrial membrane proteins. thus, nef stimulates lysosomal membrane permeabilization resulting in the release of cathepsin d from the lysosomal lumen into the cytosol. this triggers the activation of bax (but not that of bak) and mmp-dependent cell death [s ,s ]. the hiv- envelope glycoprotein complex (env, constituted by gp and gp ) is expressed by infected cells and promotes cellto-cell fusion by interacting with its receptor/co-receptor complex (cd /cxcr or cd /ccr ) on the surface of uninfected cells. env-elicited syncytium formation is followed by mmp after a latency of at least hours [ ] . env triggers mmp through a complex signal transduction pathway that involves the sequential activation of cyclin-dependent kinase (cdk ), mammalian target of rapamycin (mtor), p map kinase, phosphorylation of p , and p -dependent transactivation of puma and bax [ , ;s ]. tat, a powerful activator of hiv- gene expression, triggers apoptosis of infected and uninfected cells, thereby contributing to the hiv- -induced neurodegeneration [ ;s -s ]. transfection with tat results in its accumulation in mitochondria followed by dy m loss, ros overproduction, and caspase activation [s ]. however, recombinant tat protein added to cultured cells fails to localize at mitochondria and primarily accumulates in the endosomal compartment, presumably due to its uptake via the endocytic pathway [s ]. tat associates with the tubulin network through a -aa subdomain of its conserved core region, thereby altering microtubule dynamics, promoting the proteasomal degradation of the microtubule-associated protein (map ), and activating a mitochondrion-dependent apoptotic pathway [ ;s ]. tat cytotoxicity relies (at least partially) on the proapoptotic activity of the bh -only protein bim. in healthy cells, bim is inactivated through its association with the microtubule-associated dynein motor complex, and tat liberates bim from this inhibition [ ;s ] . thus, bim / cells exhibit increased resistance against tat-induced cell death [ ] . nevertheless, tat may influence mitochondrial apoptosis through other mechanisms, and contradictory reports suggest that tat might regulate p activity as well as the expression levels of bax and bcl- [s -s ]. the hiv- -encoded protease is required for the viral life cycle because it processes large polypeptide precursors into mature viral proteins. due to its degenerate substrate specificity, the viral protease promotes the proteolytic activation of caspase- , as assessed both in vitro and in t cells, which leads to bid cleavage and mitochondrial apoptosis [ ] . reportedly, the hiv- protease would also favor apoptosis and viral replication via the cleavage and inactivation of bcl- , which would result in the oxidative stress-dependent activation of nf-kb, a transcription factor required for hiv- enhancer activation [s -s ]. conversely, high bcl- levels protect cells in vitro and in vivo from the viral protease and prevent apoptosis induced by hiv- infection of human lymphocytes, while reducing the yields of viral structural proteins, infectivity, as well as the secretion of tumor necrosis factor b (tnf b) [s ]. additional viral proteases have been implicated in the induction of apoptosis. thus, the hcv non structural protein (ns ) participates in a protease complex [s ], and has been reported to induce caspase- -mediated apoptosis independently from its enzymatic activity [s ]. upon expression either as single proteins or in combination, plv proteases a ( apro) and c ( cpro) activate caspase-dependent apoptosis [s -s ]. however, other mechanisms are involved in the induction of apoptosis by pvs, including ( ) modulation of antiapoptotic proteins of the bcl- family (e.g., bcl-x l ) [s ]; ( ) jnk-mediated activation of bax [s ]; and ( ) mmp promoted by viroporins b and a (see also the section ''direct inducers of mmp'') [s ]. following the infection with human adenoviruses (advs), cells exhibit an apoptotic response mediated by the expression of the viral e a protein. this lethal response can be counteracted by the vbcl- e b- k (see also the section ''viral bcl- homologs'') [ ] [ ] [ ] . notably, e a was the first viral protein found to promote apoptosis [ ] [ ] [ ] , via both p -dependent and -independent mechanisms, the latter involving additional viral factors and in particular products of the e gene that are expressed upon e amediated transactivation [s -s ]. moreover, e a sensitizes cells to apoptosis induced by multiple stimuli including death receptor agonists (e.g., fasl, tnfa, and tnf-related apoptosis inducing ligand [trail]) [s -s ] and nitric oxide (no) [s ]. recently, a prominent role for bh -only proteins (and in particular for bik) has been reported in adv-induced apoptosis [ ;s ]. bik is upregulated at the transcriptional level after adv infection, in a p -dependent fashion [s ]. moreover, during the viral life cycle, the proapoptotic activity of bik is enhanced as a result of an activating phosphorylation. accordingly, sirna-mediated depletion of bik has been shown to dramatically diminish adv-dependent cell death [s ]. e orf , a -kda protein encoded by the adenoviral gene e , can promote apoptosis by inhibiting the protein phosphatase a (pp a) [ ] . pp a inhibition prolongs the signal of dna damage emanating from phosphorylated histone h ax (ch ax), thereby leading to poly(adp-ribose) polymerase (parp) hyperactivation, aif nuclear translocation, and ultimately cell death [ ] . moreover, pp a is known to maintain mitochondrial bcl- in an hypophosphorylated form, which allows for its antiapoptotic function [s ] . in this context, pp a inhibition would also favor the phosphorylationdependent inactivation of bcl- . e and e contribute to the transforming properties of high-risk hpvs by targeting p to ubiquitin-mediated degradation [ ;s ] and by inactivating the retinoblastoma (rb) protein [ ;s ], respectively. e (alone or together with e ) has been reported to sensitize cells to different apoptotic stimuli [s -s ], via mechanisms that may depend [s ,s ] or not [s ] on p . similar to e , e has been implicated in the sensitization of cells to cell death induced by growth factor withdrawal [s ], chemotherapeutic agents [s ,s ], and ultraviolet (uv) irradiation [s ]. the vesicular stomatitis virus (vsv) belongs to the family of rhabdoviruses, and its infection is associated with the development of neurological disorders characterized by enhanced neuronal apoptosis [s ,s ]. thus, vsv-infected cells exhibit an early activation of the mitochondrial pathway of apoptosis [s ], which does not depend on de novo viral protein synthesis nor on viral replication [s ,s ]. multiple pathways are involved in vsv-induced apoptosis, including ( ) while the induction of host cell apoptosis may favor viral dissemination at late stages of infection, it is vital for viruses to inhibit pcd at early steps of the infectious cycle, thereby avoiding premature cell death and allowing the virus to replicate. thus, viruses have developed a battery of bcl- homologs by which they mimic the major antiapoptotic system of host cells (for a recent review, see [s ]). in some instances, such vbcl- s fail to show significant sequence similarity with their mammalian counterparts, yet exhibit striking structural resemblance. finally, a number of viral factors inhibit apoptosis via other mechanisms, which do not directly involve the bcl- system (table ). the ''founder'' of the viral bcl- homolog (vbcl- ) family is the -kda protein encoded by the adenoviral e b gene (e b- k) [ ;s ] . during adv infection, e b- k blocks host cell apoptosis, thereby sustaining viral replication [ ] have been originally identified thanks to their interaction with e b- k. while e b- k and bcl- exhibit limited overall sequence similarity [ ] , they share short homologous domains that can be exchanged between the two proteins without a significant loss in their antiapoptotic functions [s ,s ]. accordingly, bcl- and e b- k functionally complement each other, and in hela cells, bcl- overexpression is able to substitute for the absence of e b- k, thereby allowing for productive adv infection and inhibiting tnfr-and fas-mediated apoptosis [ ] . human cytomegalovirus (cmv) encodes several proteins that subvert host cell functions in order to favor viral propagation [ ] . one of the best characterized among these factors is the product of the cmv gene ul (pul x ), known also as viral mitochondria-localized inhibitor of apoptosis (vmia). vmia, which is required for viral replication, has been shown to inhibit apoptosis triggered by different stimuli, including ligation of death receptors and exposure to cytotoxic agents, as well as infection with a mutant adv strain lacking the antiapoptotic modulator e b- k [s ]. although vmia does not share any obvious structural similarity with bcl- nor with its viral homologs (vbcl- s), vmia exerts its antiapoptotic activity predominantly by inhibiting mmp at the level of mitochondria [ ] . deletion mutagenesis studies revealed that an n-terminal mts is necessary and sufficient for vmia mitochondrial localization, but not for its antiapoptotic activity, which requires an additional c-terminal region of the protein [ ;s ,s ]. vmia can physically interact with bax, recruit it to mitochondria, and neutralize it [ ] . since vmia effects on apoptosis are lost in bax-deficient cells, it appears that vmia exerts its antiapoptotic functions solely by neutralizing bax [ ] . the structure-function relationship of the bax/vmia interaction has been addressed by mutational and computational analyses, suggesting a model in which the overall fold of vmia closely resembles that of bcl-x l [s ]. in contrast to bcl-x l , however, it seems that vmia does not bind to the bh domain of bax but rather engages in electrostatic interactions involving a region of bax between its bh and bh domains [s ]. this region is not conserved in bak, explaining why vmia (in contrast to bcl-x l ) fails to interact with bak [ ;s ]. besides its ability to neutralize bax, vmia exerts multiple functions, not all of which are directly linked to its mmp-modulatory role. in particular, vmia ( ) interacts with ant and enhances its antiporter activity [ ] ; ( ) induces the fragmentation of the mitochondrial network, which might hamper the propagation of ca + -dependent proapoptotic signals [s ]; ( ) inhibits the atp synthasome via an interaction with the mitochondrial inorganic phosphate carrier (pic, an im protein), thus reducing mitochondrial atp generation [ ] ; and ( ) induces the release of er ca + stores into the cytosol [s ]. this mobilization of er ca + might have several consequences, including activation of the unfolded protein response, modulation of mitochondrial functions, induction of mitochondrial fission, and protection against proapoptotic signals, through an inhibition in the propagation of ca + waves [s ]. poxviruses cause several diseases of humans and domestic animals, including smallpox, cowpox, sheeppox, fowlpox, and goatpox [s ]. the genome of many poxviruses codes for proteins that, despite the lack of sequence similarity, fold like bcl- and exert similar antiapoptotic properties, thereby belonging de facto to the vbcl- family. f l from vaccinia virus (vacv) was first characterized following the observation that viral strains lacking the serpin crma (which acts as a direct inhibitor of caspases [ ] ) maintained the ability to protect cells from apoptosis [ ] . the c-terminus of f l is composed by a -aa tm domain flanked by positively charged lysines and followed by an -aa hydrophilic tail. as assessed by mutagenesis studies, this domain (which exhibits slight homology to the c-terminus of bcl- ) is required for both the mitochondrial targeting and the antiapoptotic function of f l [s ]. f l directly interacts with the bh domain of proapoptotic bcl- family members, including bak [ ;s ] and bim [s ], thereby inhibiting dy m dissipation and cyt c release induced by diverse stimuli (e.g., staurosporine, fas crosslinking) [ ] . the region of f l involved in this interaction encompasses aa - and has limited sequence similarity to known bh -binding domains [s ]. while f l has been shown to inhibit mitochondrial translocation and activation of bax, a direct interaction between f l and bax has never been detected, suggesting that f l acts upstream of bax activation [s ]. other poxviruses, including variola virus, monkeypoxvirus, and ectromelia virus, encode functional f l orthologs, all of which display % sequence homology in the c-terminus of the protein [ ] . myxoma virus (mxv) is a leporipoxvirus causing myxomatosis (a highly lethal disease) in the european rabbit. productive infection by mxv requires the expression of m l [s ], a aa protein with no defined structural motifs except a putative cterminal tm domain [s ] . within this region, six positively charged residues flanking a hydrophobic trait followed by a short positively charged tail constitute an mts, which is responsible for the targeting of m l to om during infection [s ]. this kind of mts resembles that of some members of the bcl- family [s ]. from its mitochondrial localization, m l suppresses apoptosis induced by treatment with staurosporine [s ] and the pbr ligand protoporphyrin ix [ ] , overexpression of bak [s ] , and viral infection [ ] . these effects derive from the ability of m l to constitutively interact with pbr [ ] , bak [s ], and bax [ ] , and hence to inhibit mpt-dependent mmp as well as bak/bax-mediated momp. bax-mediated apoptosis is blocked in mxv-infected cells lacking bak expression, suggesting that m l interacts with bax independently from bak [ ] . m l has no obvious sequence homology with bcl- or bcl-x l , yet adopts a virtually identical three-dimensional fold [ ] . this high level of structural homology allows m l to associate with a peptide derived from the bh domain of bak with an affinity comparable to that of bcl- and bcl-x l for the same peptide [ ] . thus, m l represent a structural mimic of bcl- and hence a bona fide vbcl- [ ] . n l is the most potent virulence factor of vacv and is required for productive replication in vivo, as assessed in murine models of mucosal and brain infection [s ,s ]. preliminary studies indicated that n l associates with several components of the i-kb kinase (ikk) complex, and hence can interfere with innate immunity signalling mediated by nf-kb and toll-like receptors (tlrs) [s ,s ]. however, cells infected with recombinant viruses with or without the n l gene exhibited no difference in nf-kb-dependent gene expression [ ] , suggesting that another function of n l underlie its important role in vivo. indeed, during viral infection, n l binds to endogenous proapoptotic bcl- family members such as bid, bak, and bax as well as to exogenous bad and bax (following transfectionenforced overexpression) [ ] . similar to m l, n l shares no sequence homology with cellular proteins yet exhibits a threedimensional structure that closely mimics that of bcl- and bcl-x l [s ]. thus, the surface of n l possesses a constitutively open groove common to other antiapoptotic members of the bcl- family, which mediates the interaction with bh -containing proteins [ ] . the examples provided by m l and n l illustrate the importance of the conservation of structure, rather than of primary amino acid sequence, for the maintenance of protein function along evolution. recently, two novel bcl- -like inhibitors of apoptosis encoded by poxviruses have been discovered: fpv from fowlpox virus (fpv) [ ] and orfv from parapoxvirus orf virus (ppvo) [ ] . in human and chicken cells, fpv localizes at mitochondria and constitutively interacts with bak, thereby suppressing apoptosis induced by tnfa and bak overexpression. moreover, fpv is able to substitute for f l in inhibiting bak activation and apoptosis triggered by staurosporine and vacv infection, confirming that fpv is a functional homolog of f l [ ] . in uv-irradiated hela cells, orfv fully inhibits dna fragmentation, caspase activation, and cyt c release, but not jnk activation, an event occurring upstream of mitochondria. the mitochondrial localization of orfv is determined by a distinctive c-terminal domain, and is required for its antiapoptotic function. as assessed by bioinformatic analyses, orfv shares sequence and structure similarities with antiapoptotic members of the bcl- family (i.e., bcl- , bcl-x l , and bcl-w). accordingly, orfv inhibited the uv-induced activation of bax and bak, strongly corroborating the idea that orfv represents a novel bona fide member of the vbcl- family [ ] . epstein-barr virus (ebv) is a prominent member of cherpesviruses, invading primary resting b lymphocytes to establish a latent infection that eventually culminates in cell transformation [s ] . the potent mitogenic effect of ebv is mediated by the coordinated expression of several gene products, including the apoptotic modulators balf and bhrf [s ,s ]. these two factors share sequence and structure homology with bcl- and belong de facto to the growing list of vbcl- s [ ;s ] . while bhrf clearly resembles bcl- in its antiapoptotic function [ ] , the role of balf (which is able to interact with bax and bak, and has been proposed as a bhrf antagonist) is controversial [ ; ] . balf is actively expressed in ebv-positive burkitt lymphoma's cell lines and nasopharyngeal carcinoma biopsies, and renders cells independent from serum [s ]. this points to a prominent antiapoptotic (rather than proapoptotic) function for balf during ebv-driven tumorigenesis [s ] . as true for other dna viruses, antiapoptotic proteins appear to be essential for the early phases of the herpesvirus life cycle. however, both proteins are neither expressed nor required once latent infection is established [s ], nor they are essential for in vitro viral replication and transformation of b cells [ ;s ] . as assessed by immunoelectron microscopy studies, bhrf colocalizes with bcl- at the om [ , ] . the c-terminal hydrophobic portion of bhrf (which is responsible for bhrf targeting to intracellular membranes) exhibits high levels of homology with several members of the bcl- family, in particular with bcl- ( %), bcl-x l ( %), and bax ( %) [ ;s ] . moreover, the threedimensional structure of bhrf closely resembles that of bcl- in thus far that it contains two central hydrophobic a-helices that are surrounded by several amphipathic a-helices. in contrast to bcl- / bcl-x l , however, bhrf is not able to sequestrate and inhibit proapoptotic bh -only proteins, presumably because it lacks an exposed, pre-formed bh binding groove [ ;s ] [ ;s ] . hhv- orf encodes the so-called kaposi sarcoma-associated bcl- (ksbcl- ), a polypeptide of residues that shares limited ( %- %) overall sequence identity with other bcl- homologs (including bcl- , bcl-x l , bax, bak, and bhrf ) [ ] . interestingly, significant amino acid identity is concentrated in the bh and bh (but not in the bh ) domains [ ] . moreover, although ksbcl- exhibits an overall fold almost identical to that of bcl- /bcl-x l , key differences exist in the lengths of helices and loops [ ] . presumably, structure and sequence dissimilarity with bcl- account for the fact that ksbcl- neither homodimerizes nor heterodimerizes with other bcl- family members, suggesting that it may have evolved to escape any negative regulatory effects mediated by proapoptotic host proteins from the bcl- family [ , ] . mutation of a conserved arginine and the two adjacent residues within the bh binding groove resulted in a correctly folded protein that failed to bind bax bh peptide and to inhibit bax toxicity in yeast. moreover, viruses harboring the same mutation exhibited impaired persistent replication and reactivation from latency in vivo [s ] . altogether, these studies point to a major role of m in vivo for the establishment of a persistent viral pool and chronic infection, rather than for viral replication and virulence during acute infection [ ] . in contrast to ksbcl- , hvs orf has been shown to interact with bax and bak to inhibit virus-induced apoptosis. while orf exhibits highly conserved bh and bh domains, it lacks the core sequence of the conserved bh domain, suggesting that this portion might be dispensable (at least in some proteins) for antiapoptotic functions [ ] . similar to other vbcl- s (i.e., bhrf and ksbcl- ), orf contains a stretch of conserved hydrophobic residues at its c-terminus, ending with basic amino acids, that may direct its (not yet demonstrated) targeting to intracellular membranes [ ] . interestingly, several herpesvirus-encoded vbcl- s cannot be converted into proapoptotic factors by activated caspases during pcd (as it occurs to their mammalian counterparts), and hence fail to display any latent proapoptotic activity [ ] . in addition to ksbcl- , hhv- codes for yet another protein with limited homology to bcl- [ ] . thus, k is a -kda glycoprotein structurally related to a splice variant of human survivin (survivindex ), a mammalian antiapoptotic factor belonging to the inhibitor of apoptosis protein (iap) family [s ,s ]. both proteins contain an mts, an n-terminal region of a baculovirus iap repeat (bir) domain, and a putative bh domain [ ] . the mts of k consists of a single tm hydrophobic region flanked by positively charged residues, and resembles that of m l from mxv [s ]. k efficiently represses apoptosis induced by activation of death receptors (e.g., fas, tnfr), bax overexpression and thapsigargin-mediated er stress [ ;s ] . similarly to other iaps, k binds to and hence inhibits caspase- via its bir domain. however, k antiapoptotic effects depend on its bh domain, which mediates the interaction of k with bcl- . thus, it seems that k exerts its functions by bridging effector caspases and bcl- , thereby enabling the latter to inhibit caspase activity [ ] . interestingly, k has also been shown to modulate intracellular ca + concentration and protein stability, by interacting with the cellular ca + -modulating cyclophilin ligand (caml) and with a regulator of the ubiquitin system, respectively [s ,s ]. whereas mutational analyses showed that the interaction between caml and k is required for its antiapoptotic activity [s ], the significance of k -mediated proteasome regulation remains to be established [s ]. due to its molecular structure, k can be considered either as a viral iap (viap) or as a vbcl- [ ] . to avoid premature apoptosis of the host cell, asfv encodes multiple antiapoptotic proteins, including the vbcl- family member a l (also known as -hl) [ ;s ,s ]. a l codes for a polypeptide of kda that contains all known domains associated with bcl- structural and functional features, including those mediating protein-binding (i.e., homo-and heterodimerization) and regulating cell death [s ]. thus, a l has been shown to suppress apoptosis induced by multiple stimuli, including growth factor deprivation [s ] and exposure to cytotoxic agents [s ]. viruses inhibit host cell apoptosis via a plethora of mechanisms other than vbcl- s. for instance, the of ul gene of cmv encodes the viral inhibitor of caspase- activation (vica), which has been shown to inhibit fas/cd -mediated apoptosis by binding to the pro-domain of caspase- and preventing its autoproteolytic processing [ ] . although this effect regards in particular the pathway of apoptosis emanating from death receptors, it also avoids the activation of the intrinsic pathway occurring along the caspase- r bid axis [ ;s ] . recently, another mechanism of cmv-mediated inhibition of mitochondrial apoptosis has been discovered [ ] . during cmv infection, a . -kilobase virally encoded rna (b . ) interacts with the mitochondrial respiratory chain complex i (reduced nicotinamide adenine dinucleotide-ubiquinone oxidoreductase) and prevents the mitochondrial release (that normally would be induced in response to apoptotic stimuli) of the complex i subunit grim- . this stabilizes dy m and results in continued atp production, hence improving the viability of infected cells and favoring the successful completion of the viral life cycle [ ] . thus, cmv employs two distinct strategies to influence mitochondrial respiration of infected cells, namely vmia-mediated inhibition of the atp synthasome and stabilization of complex i by b . rna. as a net result, this combined modulation should not lead to an increase in dy m , but to decreased mitochondrial atp generation, correlating with the documented glycolytic switch of cmvinfected cells [s ] . ebv-induced transformation of primary b lymphocytes into continuously proliferating lymphoblastoid cell lines involves proteins other than vbcl- s [s ]. among these, the epstein-barr nuclear antigens (ebna) a and c (ebna a and ebna c) but not ebna b may downregulate the bh -only protein bim, and hence reduce the propensity of host cells to undergo apoptosis [s ]. moreover, the ebna leader protein (ebna-lp) has been shown to interfere not only with host cell transcription but also with the apoptotic machinery [ ;s ]. interestingly, it has been suggested that ebna-lp would interact with bcl- though the hs -associated protein x- (hax- ) [ , and k is required for the long term survival of infected cells [ ] . thus, k promotes the activation of nf-kb via multiple, partially distinct molecular pathways [s -s ], which in turn lead to ( ) reduced sensitivity to death receptor-mediated [s ] and intrinsic apoptosis [ ] ; in addition, rid has been shown to target tyrosine kinase receptors, such as the epidermal growth factor receptor (egfr) [s ] . both rid subunits are tm proteins oriented with their ctermini in the cytoplasm [s ,s ]. ridb contains a cterminal tyrosine residue that is required for receptor internalization and inhibition of fas-and trail-induced apoptosis [s ]. rida has been reported to undergo o-glycosylation [s ] and phosphorylation on serine residues [s ]. how rida posttranslational modifications might affect rid antiapoptotic function remains to be established. mutagenesis studies revealed that the extracellular domain of rida is important for the clearance of egfr from the cell surface, but not for the internalization of death receptors like fas [s ]. interestingly, e - . k, another protein encoded by the e unit, is specifically implicated in ridmediated clearance of trail-r [s ]. this suggests that additional viral (or cellular) factors might cooperate with rid to determine its target specificity. baculoviruses infect insect cells, and possess at least two different classes of proteins by which they control the host apoptotic response [s ]. one of these is represented by p , a potent inhibitor of metazoan caspases acting via a cleavage-dependent mechanism [ ;s ]. p mechanism-based inhibition of caspases is the most broadly acting antiapoptotic system known recently, the viral mitochondrial antiapoptotic protein (vmap), encoded by the m gene of chv- , has been shown to suppress intrinsic apoptosis via a completely novel, dual mechanism [ ] . via its n-terminus, vmap is able to augment the recruitment of bcl- to mitochondria and to enhance its affinity for bh -only proapoptotic proteins, thereby suppressing bax activation. morevoer, vmap interacts with vdac via two leucine-rich motifs located in the central and c-terminal parts of the protein, thus repressing staurosporine-induced cyt c release and apoptosis. interestingly, vmap is necessary for efficient chv- lytic replication in normal cells (with an intact apoptotic apparatus), but not in bax / /ba / cells, pointing to a crucial role for apoptosis inhibition during the early steps of the viral life cycle [ ] . we have reviewed the cellular impact of viral infection on cell fate via modulation of mitochondrial apoptosis. while specific cellular and molecular mechanisms have been elucidated for a number of individual proteins (e.g., vpr, vmia), a clear scheme of the integrated effects resulting from the expression of whole virus genomes has only recently begun to emerge from transcriptomics and proteomics analyses (for a review, see [ ] ). future studies will have to take into account the variability of the host cell and its microenvironmental context (e.g., local inflammation, oxidative stress) as key factors susceptible to modulating the response to specific pathogens. this will undoubtedly be instrumental for the prediction of the general consequences of viral infections, as well as for a more accurate identification of novel therapeutic targets designed to eradicate infectious diseases. a complete list of accession numbers (uniprotkb/swiss-prot knowledgebase, http://www.expasy.org/sprot/) for the proteins discussed in this manuscript can be found online in text s . text s list of the accession numbers (uniprotkb/swiss-prot knowledgebase) of all the proteins described in this article. mitochondrial membrane permeabilization in cell death direct activation of bax by p mediates mitochondrial membrane permeabilization and apoptosis ca +-induced apoptosis through calcineurin dephosphorylation of bad requirement for gd ganglioside in cd -and ceramide-induced apoptosis disruption of mitochondrial function during apoptosis is mediated by caspase cleavage of the p subunit of complex i of the electron transport chain cell biology mitochondrion-targeted apoptosis regulators of viral origin mitochondrial apoptosis induced by the hiv- envelope mitochondria as therapeutic targets for cancer chemotherapy proapoptotic bax and bak: a requisite gateway to mitochondrial dysfunction and death bid, bax, and lipids cooperate to form supramolecular openings in the outer mitochondrial membrane bcl- family proteins regulate the release of apoptogenic cytochrome c by the mitochondrial channel vdac distinct bh domains either sensitize or activate mitochondrial apoptosis, serving as prototype cancer therapeutics the permeability transition pore complex: a target for apoptosis regulation by caspases and bcl- -related proteins differential targeting of prosurvival bcl- proteins by their bh -only ligands allows complementary apoptotic function and drug-induced apoptotic responses mediated by bh -only proteins puma and noxa bax and bak regulation of endoplasmic reticulum ca +: a control point for apoptosis pharmacological manipulation of bcl- family members to control cell death the adp/ atp translocator is not essential for the mitochondrial permeability transition pore cyclophilin d-dependent mitochondrial permeability transition regulates some necrotic but not apoptotic cell death mitochondrial apoptosis without vdac bax and adenine nucleotide translocator cooperate in the mitochondrial control of apoptosis apoptosis as an hiv strategy to escape immune attack control of mitochondrial membrane permeabilization by adenine nucleotide translocator interacting with hiv- viral protein rr and bcl- hbx gene of hepatitis b virus induces liver cancer in transgenic mice multiple viral strategies of htlv- for dysregulation of cell growth control the human t-cell leukemia virus type p ii protein: effects on mitochondrial function and cell growth a novel influenza a virus mitochondrial protein that induces cell death the influenza a virus pb -f protein targets the inner mitochondrial membrane via a predicted basic amphipathic helix that disrupts mitochondrial function specific interaction between hpv- e -e and cytokeratins results in collapse of the epithelial cell intermediate filament network apoptosis control in syncytia induced by the hiv type -envelope glycoprotein complex: role of mitochondria and caspases human immunodeficiency virus envelope glycoprotein complex-induced apoptosis involves mammalian target of rapamycin/fkbp -rapamycin-associated protein-mediated p phosphorylation nf-kappab and p are the dominant apoptosis-inducing transcription factors elicited by the hiv- envelope induction of apoptosis in uninfected lymphocytes by hiv- tat protein hiv- tat targets microtubules to induce apoptosis, a process promoted by the pro-apoptotic bcl- relative bim hiv- protease processes procaspase to cause mitochondrial release of cytochrome c, caspase cleavage and nuclear fragmentation the -kilodalton adenovirus e b transforming protein inhibits programmed cell death and prevents cytolysis by tumor necrosis factor alpha the adenovirus e a proteins induce apoptosis, which is inhibited by the e b -kda and bcl- proteins adenovirus e b -kilodalton protein overcomes the cytotoxicity of e a proteins nbk/ bik antagonizes mcl- and bcl-xl and activates bak-mediated apoptosis in response to protein synthesis inhibition the adenoviral e orf protein induces atypical apoptosis in response to dna damage the e oncoprotein encoded by human papillomavirus types and promotes the degradation of p complex formation of human papillomavirus e proteins with the retinoblastoma tumor suppressor gene product vesicular stomatitis viruses expressing wild-type or mutant m proteins activate apoptosis through distinct pathways west nile virus infection activates the unfolded protein response, leading to chop induction and apoptosis induction of apoptosis by the severe acute respiratory syndrome coronavirus a protein is dependent on its interaction with the bcl-xl protein viral homologs of bcl- : role of apoptosis in the regulation of virus infection bcl- , bcl-xl and adenovirus protein e b kd are functionally equivalent in their ability to inhibit cell death the e b k protein blocks apoptosis by interacting with and inhibiting the p -inducible and death-promoting bax protein cloning of a bcl- homologue by interaction with adenovirus e b k functional complementation of the adenovirus e b -kilodalton protein with bcl- in the inhibition of apoptosis in infected cells a cytomegalovirus-encoded mitochondria-localized inhibitor of apoptosis structurally unrelated to bcl- cytomegalovirus cell death suppressor vmia blocks bax-but not bak-mediated apoptosis by binding and sequestering bax at mitochondria cytopathic effects of the cytomegalovirus-encoded apoptosis inhibitory protein vmia viral inhibition of inflammation: cowpox virus encodes an inhibitor of the interleukin- beta converting enzyme vaccinia virus encodes a previously uncharacterized mitochondrial-associated inhibitor of apoptosis the vaccinia virus f l protein interacts with the proapoptotic protein bak and inhibits bak activation the myxoma poxvirus protein, m l, prevents apoptosis by direct interaction with the mitochondrial permeability transition pore myxoma virus m l blocks apoptosis through inhibition of conformational activation of bax at the mitochondria structure of m l: a myxoma virus structural homolog of the apoptosis inhibitor, bcl- a structural viral mimic of prosurvival bcl- : a pivotal role for sequestering proapoptotic bax and bak functional and structural studies of the vaccinia virus virulence factor n reveal a bcl- -like anti-apoptotic protein fowlpox virus encodes a bcl- homologue that protects cells from apoptotic death through interaction with the proapoptotic protein bak a novel bcl- -like inhibitor of apoptosis is encoded by the parapoxvirus orf virus epstein-barr virus encodes a novel homolog of the bcl- oncogene that inhibits apoptosis and associates with bax and bak epstein-barr virus-coded bhrf protein, a viral homologue of bcl- , protects human b cells from programmed cell death epstein-barr virus balf is a bcl- -like antagonist of the herpesvirus antiapoptotic bcl- proteins bhrf of epstein-barr virus, which is homologous to human proto-oncogene bcl , is not essential for transformation of b cells or for virus replication in vitro ultrastructural localization of bhrf : an epstein-barr virus gene product which has homology with bcl- epstein-barr virus bhrf protein protects against cell death induced by dna-damaging agents and heterologous viral infection a bcl- homolog encoded by kaposi sarcoma-associated virus, human herpesvirus , inhibits apoptosis but does not heterodimerize with bax or bak herpesvirus saimiri encodes a functional homolog of the human bcl- oncogene solution structure of a bcl- homolog from kaposi sarcoma virus identification of the in vivo role of a viral bcl- antiapoptotic herpesvirus bcl- homologs escape caspase-mediated conversion to proapoptotic proteins characterization of an anti-apoptotic glycoprotein encoded by kaposi's sarcomaassociated herpesvirus which resembles a spliced variant of human survivin african swine fever virus gene a l, a viral homologue of bcl- , protects cells from programmed cell death a cytomegalovirus-encoded inhibitor of apoptosis that suppresses caspase- activation complex i binding by a virally encoded rna regulates mitochondria-induced cell death epstein-barr virus (ebv) nuclear antigen leader protein (ebna-lp) forms complexes with a cellular anti-apoptosis protein bcl- or its ebv counterpart bhrf through hs -associated protein x- the hepatitis c virus ns protein is an inhibitor of cide-b-induced apoptosis e of hepatitis c virus inhibits apoptosis viral flice-inhibitory proteins (flips) prevent apoptosis induced by death receptors modulation of host gene expression by the k protein of kaposi's sarcomaassociated herpesvirus kshv vflip is essential for the survival of infected lymphoma cells the human herpes virus -encoded viral flice inhibitory protein protects against growth factor withdrawalinduced apoptosis via nf-kappa b activation mechanism for removal of tumor necrosis factor receptor from the cell surface by the adenovirus ridalpha/beta complex the adenovirus e / . k- . k proteins downmodulate the apoptosis receptor fas/apo- by inducing its internalization prevention of apoptosis by a baculovirus gene during infection of insect cells control of programmed cell death by the baculovirus genes p and iap baculovirus p prevents developmentally programmed cell death and rescues a ced- mutant in the nematode caenorhabditis elegans inhibition of the caenorhabditis elegans cell-death protease ced- by a ced- cleavage site in baculovirus p protein an apoptosis-inhibiting baculovirus gene with a zinc finger-like motif cloning and expression of apoptosis inhibitory protein homologs that function to inhibit apoptosis and/or bind tumor necrosis factor receptor-associated factors drosophila homologs of baculovirus inhibitor of apoptosis proteins function to block cell death iaps block apoptotic events induced by caspase- and cytochrome c by direct inhibition of distinct caspases african swine fever virus iap-like protein induces the activation of nuclear factor kappa b nuclear and cytoplasmic survivin: molecular mechanism, prognostic, and therapeutic potential a novel inhibitory mechanism of mitochondrion-dependent apoptosis by a herpesviral protein we apologize to our colleagues for not having been able to cite their work. key: cord- -anp gu authors: effantin, grégory; estrozi, leandro f.; aschman, nick; renesto, patricia; stanke, nicole; lindemann, dirk; schoehn, guy; weissenhorn, winfried title: cryo-electron microscopy structure of the native prototype foamy virus glycoprotein and virus architecture date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: anp gu foamy viruses (fv) belong to the genus spumavirus, which forms a distinct lineage in the retroviridae family. although the infection in natural hosts and zoonotic transmission to humans is asymptomatic, fvs can replicate well in human cells making it an attractive gene therapy vector candidate. here we present cryo-electron microscopy and (cryo-)electron tomography ultrastructural data on purified prototype fv (pfv) and pfv infected cells. mature pfv particles have a distinct morphology with a capsid of constant dimension as well as a less ordered shell of density between the capsid and the membrane likely formed by the gag n-terminal domain and the cytoplasmic part of the env leader peptide gp (lp). the viral membrane contains trimeric env glycoproteins partly arranged in interlocked hexagonal assemblies. in situ d reconstruction by subtomogram averaging of wild type env and of a env gp (tm)- gp (su) cleavage site mutant showed a similar spike architecture as well as stabilization of the hexagonal lattice by clear connections between lower densities of neighboring trimers. cryo-em was employed to obtain a Å resolution map of the glycoprotein in its pre-fusion state, which revealed extensive trimer interactions by the receptor binding subunit gp (su) at the top of the spike and three central helices derived from the fusion protein subunit gp (tm). the lower part of env, presumably composed of interlaced parts of gp (tm), gp (su) and gp (lp) anchors the spike at the membrane. we propose that the gp (tm) density continues into three central transmembrane helices, which interact with three outer transmembrane helices derived from gp (lp). our ultrastructural data and Å resolution glycoprotein structure provide important new insights into the molecular architecture of pfv and its distinct evolutionary relationship with other members of the retroviridae. spuma or foamy viruses (fv) are the only members of the spumaretrovirinae subfamily of the retroviridae. as such they share many similarities in their life cycle with the orthoretrovirinae as well as some features with the more distant hepadnaviridae [ ] . fvs infect nearly all mammals and the best-studied member is the prototype fv (pfv) previously called human fv (hfv), which has been isolated from infected human cells [ ] . fv infection in humans is asymptomatic [ ] but the virus can replicate very efficiently in human cell lines and is therefore an elegant model system for the study of more hazardous orthoretroviruses and a promising candidate for gene transfer therapy [ ] . the main pfv structural protein is gag ( amino acids (aa)), which is encoded as a single protein and unlike orthoretroviruses does not exist as a gag/pol fusion variant. pfv gag is also unusual in that it is not processed by the viral protease into canonical matrix (ma), capsid (ca) and nucleocapsid (nc) domains, like other retroviral gag polyproteins. the kda pfv gag (pr gag ) precursor is only partially proteolysed at its c-terminus to yield a kda (p gag ) and a kda (p gag ) peptide [ , ] . in addition, three less systematic secondary cleavage sites are located in the middle of the gag sequence (residues , and ) and have been proposed to serve capsid disassembly during virus entry [ , ] . the c-terminus of pfv gag contains a glycine-arginine rich (gr) region, which is important for interaction with the viral rna genome and is the functional equivalent to the cys-his motif found in orthoretroviruses [ ] . pfv gag lacks a membrane-binding domain and instead virus egress relies on a physical interaction between gag and the leader peptide (lp) domain of the envelope (env) protein [ , ] . the pfv env precursor comprises residues and undergoes post-translational processing by cellular proteases resulting in three distinct domains: surface (su) gp su , transmembrane (tm) gp tm and lp gp lp [ ] . mutants defective in the su/tm cleavage produce non-infectious virions [ , ] while an inactive su/lp processing inhibits budding [ ] . env forms trimers of heterotrimers (gp -gp -gp ) anchored by two transmembrane regions (per monomer) in the virus membrane, where it can further assemble into hexameric lattices [ ] . pfv capsid assembly commences at the centrosome or microtubule organizing center similar to type b/d orthoretrovirus assembly [ ] . capsids are then transported to the secretory pathway either the er or the golgi or directly to the plasma membrane where env lp interacts with the n-terminal gag region [ , , ] . budding into intracellular compartments or at the plasma membrane depends on the recruitment of the escrt machinery [ , ] , which completes budding by membrane scission [ ] . intracellular virions are most likely transported in vesicles for release at the plasma membrane (reviewed in [ ] ). in order to obtain structural insight into pfv organization at medium resolution we used cryo-electron tomography (cryo-et) and microscopy (cryo-em) to analyze isolated wild-type particles and variants with mutations in pfv gag and env proteins. these data are correlated to gag assemblies analyzed in infected cells by employing high pressure freezing, cryo-substitution and electron tomography. we further present a Å resolution structure of the glycoprotein in situ, which shows unprecedented molecular details of its membrane-anchored organization and higher order assemblies stabilized most likely by the leader peptide gp lp . in order to probe the various structures of pfv found in vivo and correlate the results with the studies on purified particles analyzed by cryo-et (see below), we prepared samples of ht cells infected with replication competent wt pfv by high pressure freezing and cryo-substitution to h post infection (see material and methods) . viruses with clear capsid at their center were observed budding from the plasma membrane (fig a and c) . viruses were often found as well into large vacuolar compartments which can either be related to plasma membrane budding or constitute an alternative budding site (fig b and d ). naked capsids were also observed in the cytoplasm ( fig b) . all these observations agree well with previous results obtained in [ ] . electron tomography of to nm thick sections of viruses budding from the plasma membrane ( fig e) show that the virions are composed of the capsid spaced from the viral membrane by an additional fainter intermediate shell of density (fig e, arrowheads) . in addition to the previous observations, we found on several occasions cells with an unusually high concentration of round objects in the cytoplasm (s fig), which are rather regular in size (r = nm) similar to the capsid size of released virions and the one observed at the plasma membrane (s fig). they are also often aligned in membrane delimited tubes (s fig and s movie). because, we did never observe such assemblies in non-infected ht cells, we speculate that they constitute assembled capsids, which accumulate in tubular membrane compartments. three different pfv viruses, wild type virus (wt), a gag mutant impaired in rna binding (inab) and an env mutant (ifuse) were purified from the supernatant of cells expressing different combinations of pfv proteins from a replication-deficient pfv vector system. in the inab mutant, arginines in the glycine/arginine rich (gr) region in the c-terminus of gag have been replaced by alanine, which results in a gag protein unable to bind nucleic acid [ ] (s fig) . virus particles are still released from cells, although less efficiently, but are non-infectious and display capsid assembly defects. the ifuse mutant is a variant of env where the furine cleavage site between the su (gp su ) and tm (gp tm ) domains of env has been mutated [ , ] (s fig) . this results in a partially processed glycoprotein as the cleavage between the lp and su domains is preserved. particles are released at nearly wild type level from cells but are non-infectious. the presence of viral proteins of wild type and mutant viruses (pr gag , p gag , gp env for the ifuse mutant, gp env and gp lp for wt and inab mutant) were confirmed by western blot analysis (s fig). when observed by cryo-et, pfv wt forms mainly near spherical particles (fig ) as previously reported in [ ] . the env glycoprotein projections on the virus surface are~ nm in length. underneath the membrane a fuzzy density not directly attached to the membrane connects to the capsid. rare oval shaped viruses also exist but no tubular, elongated or more irregular structures were observed (fig ) . although the majority of viruses are spherical, their overall sizes are variable, ranging from to nm in radius measured from the center of the particle to the outer margin of the viral membrane (r mean = . ± . nm (n = )) confirming previous observations [ ] . apart from their dimensions, pfv particles differ with respect to their internal structures. we classified pfv wt particles into four main classes (a to d) according to the variation in shape and morphology of their interior (fig ) . class a virus ( % of total particles) contains an intact core at the center of the particle (r mean = . ± . nm (n = )) consistent with previous measurement [ ] . the capsid is surrounded by a second shell of density (width~ - nm), which most likely corresponds to the gag n-terminal domain that interacts with env lp [ , ] . class b ( % of total particles) is similar to a except that the central gag core is either incomplete or disrupted resulting in an open gag structure, which generally merges with the intermediate shell. class c ( % of total particles) contains spherical particles with no regular, ordered internal structures and class d ( % of total particles) miscellaneous particles having non spherical shape or an interior morphology significantly different from class a and b. we interpret classes a, b and (most of) d as particles containing capsids. such viruses have been observed by electron microscopy of infected cells (fig ) [ , ] and by cryo-em [ , ] . they likely represent the released, mature and infectious form of pfv. whether any of the virions of class c are infectious or not remains to be determined [ , ] . for near spherical viruses (classes a to c), the distribution of particle dimensions within each group is different. class a virions are less variable in dimensions (r mean = . ± . nm, n = ) than the other classes (r mean = . ± . nm, n = and . ± . nm, n = for class b and c respectively) (s fig) (fig a) as indicated previously [ ] , which probably contribute to the stability of the virus and the narrower size distribution of class a viruses. when complete, the capsid at the center of particles has a relatively uniform mean radius (r~ nm) (fig , class a-radial plots) but few exceptions with larger and less uniform shapes exist as well (fig , class d). the population with a constant radius often has a hexagonal outline with more or less sharp vertices by cryo-et. ab initio d class averages of only the capsid, calculated from images of ifuse mutant (identical to wt in term of gag sequence) acquired by cryo-em, reveal a variety of capsid shapes ranging from hexagonal to near round ( fig d) . this could be an indication of a regular, (pseudo-)symmetrical arrangement of the capsid. the capsid shell is relatively thick (~ Å) as there are strong densities associated with the capsid outer wall while its center appears comparatively less dense (fig , plots and fig d) . the intermediate shell, which strictly follows the contour of the capsid, precludes a direct interaction of the capsid with the viral membrane. this - nm thick shell is less polyhedral (hexagonal) and appears discontinuous and structurally variable. despite that, local regular organization (spac-ing~ - nm) are visible in some tomographic slices ( fig a) [ ] . the inab mutant can't bind the viral rna or any nucleic acid. we previously showed by cryo-em that this mutant displays severe assembly defects [ ] . in the present study, we extended the analysis of inab mutant to cryo-et to unambiguously visualize the interior of the viral particles ( fig c) . as wt pfv, the inab mutant forms a majority of near spherical particles on which the glycoproteins are clearly visible. their dimensions (r mean = . ± . nm, n = ) are larger than wt pfv (r mean = . ± . nm). on the average, more deformed and irregular particles are observed compared to wt pfv. we also confirm our previous result that without the rna binding motif in gag, and hence no rna interaction, no regular capsid are assembled inside the near spherical particles ( fig c) . however, these particles are not empty and some diffuse densities are detected often in the vicinity of the viral membrane ( possibility that the intermediate shell is made only of the env gp lp cytoplasmic domain. nevertheless, the env gp lp cytoplasmic domain ( residues long) has been shown to interact with the n-terminal domain of gag [ ] and therefore it most likely contributes as well to the overall intermediate shell density of mature pfv particles (class a, b and d of fig ) . wt pfv and the inab mutant particles should have an identical env structure (same amino acid sequence, same processing by proteases), at least for the extracellular domain. the ifuse mutant lacks the gp su -gp tm cleavage, which could influence the position of the fusion peptide region, but should have otherwise a similar structure than wt. central slices through tomograms of the samples show that the glycoprotein extends around nm away from the membrane while sections perpendicular to the glycoprotein's long axis clearly confirm that they are trimeric (s fig) [ ] . the d reconstruction of pfv wt, inab and ifuse glycoproteins by subtomogram averaging at~ nm resolution (s fig) demonstrates that all three structures have a similar knob-like shape at this resolution ( fig d- f ). they are arranged such that each trimer can interact with up to three other trimers to form a network of interlocked hexagons ( fig a- c and g- i). this hexagonal network organization described previously in [ ] is not obligatory as the viral membrane is not fully covered with glycoproteins. isolated trimers were hardly found but incomplete hexagonal networks (with less than six trimers) were observed. less ordered areas are also possible as well as pentagonal arrangements (fig a and s fig) . from cryo-et reconstructions of a network of three adjacent and interlocked hexagons for wt pfv (fig g) , it appears that the trimers at the periphery (arrows in fig g) are less well defined than the central one, which indicates that the occupancy rate of the trimers within hexagons is quite low or that not all glycoproteins arrange in a hexagonal fashion or that the hexagonal packing is flexible. on the contrary, the same reconstructions of inab and ifuse mutants (fig h and i) show stronger resolved features for all trimers, including the one at the periphery. this was also confirmed directly on tomograms where slices orthogonal to the mutant's glycoprotein long axis often display more regular hexagonal lattices than the wt ( fig b and c) . thus, it appears that the two mutants have a stronger propensity to form a regular hexagonal network suitable for d reconstruction than the wt. it is not clear if this observation results directly from the mutations of the inab and ifuse samples or if the observed difference simply results from variability between virus preparations. to gain more insights into the structure of the pfv glycoprotein, we use d reconstructions of inab and ifuse env hexagonal assemblies of six trimers determined by subtomogram averaging as initial references for automatic particle selection of micrographs acquired by cryo-em (s fig) (see material and methods). the data were refined to higher resolution imposing c symmetry. once the refinement converged, the six equivalent trimers of the hexagonal assembly were -fold symmetrized to yield the final single spike structure. the inab env (a fully processed env as in wt virus) and the ifuse mutant (a gp su -gp tm cleavage site mutant) were computationally processed independently but yielded virtually identical structures at~ Å resolution at fsc = . (s fig). in the density map of the hexagonal assembly (fig a and b) , the neighboring trimers are spaced by~ Å and are interacting with each other approximately Å above the membrane level. this interaction seems to serve as a spacer for hexagonal lattice formation (fig a and b ). there is no sign of ordered densities extending from the inner leaflet of the membrane towards the virus interior, which can be attributed to the cytoplasmic domain of env gp lp or the n-terminal region of gag. the pfv env trimer can be decomposed in an upper and lower region followed by the transmembrane (tm) region ( fig c- e ). the upper part likely composed of gp su forms three arch-like structures, which join at the top and delineate a less dense area in its center (fig e) . we propose that the three central short rods of density visible at the top of the lower part and surrounded by extra density are three α-helical regions derived from the fusion protein subunit gp tm (fig c- f ). gp tm contains two potential coiled coil regions from residues to and to [ ] or residues to and to [ ] , which can correspond to heptad repeat region and , respectively, present in retroviral class i fusion proteins [ ] . based on the cryo-em map, the length of the predicted helices is approximately ± Å (fig c- e ), which is consistent with to helical turns as predicted by sequence analysis. however, due to the limited resolution of the reconstruction, the actual helices may be slightly longer. the lower part of the spike is likely composed of parts of gp su and gp tm , which anchor the extracellular domain in the membrane (fig c and d ). the interaction on the top via the coiled coil and the one close to the membrane generate a central open space (fig c- e and s fig) . each env monomer is predicted to have two transmembrane helices (tmh): one in the gp lp domain between aa - and one in the gp tm domain between aa - . we propose that the gp tm density extends into three central tmhs, which interact with three outer tmhs derived from the gp lp (fig c, d and g) . notably, the three central tmhs of gp tm are positioned such that they can interact with each other. furthermore each tmh derived from gp lp is in close contact to one gp tm tmh (fig c, d and g) . however, at the current resolution of the map, we cannot exclude the possibility that the three central helices are derived from gp lp and the external ones from gp tm the majority (~ %) of wt pfv virions have a nearly spherical shape with an average radius of nm as indicated previously in [ ] which is smaller than mature particles formed by other orthoretroviruses such as htlv- ( nm) [ ] , hiv- ( nm) [ , ] and rous sarcoma virus ( nm) [ ] . moreover, pfv viruses containing an intact capsid (class a) have an even more uniform radius. pfv budding is strictly dependent on the physical interaction between env and gag [ ] . as gag with rna forms internal structures (nucleocapsid with a surrounding shell) of homogeneous size, it is not surprising that the viral membrane that contains inserted env trimers ends up near spherical as well and follows closely the contour of the gag assembly. therefore, the narrower size distribution of pfv compared to other retroviruses could be the direct consequence of the interaction of env with a preformed nucleocapsid. we interpret the viruses containing well-defined internal gag structures (class a, b and d of fig ) as the mature pfv particle. this is supported by in vivo observation of the same viral layout in virus infected cells. this organization is quite different from the mature particles of other retroviruses, which consist of a polyhedral core of ca containing the nc/rna complex [ , , ] as well as from their immature forms [ ] [ ] [ ] [ ] . these differences in virus assembly likely relate directly to differences in posttranslational processing. gag from most retroviruses is cleaved after the release of immature virions from the cell at various positions, which triggers the reorganization of the ma, ca and nc domains inside the virus [ ] . for pfv, there is only one primary cleavage of gag by the viral protease occurring in the course of virus assembly in infected cells, which produces the two gag products p gag and p gag [ , ] . pfv virions contain an extra layer of intermediate density between the viral membrane and the capsid which we believe is derived from the gag n-terminal domain and the cytoplasmic domain of the gp lp as suggested previously [ ] . this also indicates that in mature virions, the gag n-terminal and gp lp domains are flexibly linked to the capsid core and the glycoprotein respectively. secondary cleavages have been also observed at three different positions of gag [ ] , however, their relevance for the virus life cycle needs to be established. foamy viruses also share similarities with hepatitis b virus. notably hbv contains a capsid with icosahedral symmetry twice smaller (r =~ nm) than pfv [ ] , buds intracellularly in the er and forms large amounts of capsidless subviral particles used as a decoy for the host immune system [ ] . although pfv capsids can be variable in morphology, the majority has a uniform mean radius of nm with a round to hexagonal outline. the latter are compatible with d projections of a symmetrical object including icosahedra. however, the analysis of intact capsids by either cryo-et or cryo-em did not allow defining a clear repetitive unit. the same conclusion was obtained by analyzing capsid images acquired by cryo-em of complete virions in an earlier study [ ] . therefore, more studies are required to firmly establish whether the capsid has a defined (pseudo-) symmetry or not. among the various morphology of pfv viruses, one large population (class c in fig ) representing % of the spherical particles has no sign of regular internal ordering and it is also the most variable class in terms of dimensions. the smallest members of this class cannot accommodate the nm capsid of the mature particles and may constitute env-only virus-like particles consistent with the role of env in budding [ ] . we used high pressure freezing and cryo substitution to analyze thin sections of pfv infected cells by et. we observed viruses in various states and cellular localization. the landmark for each virus is the capsid, which appears as a dense round material in the cell. capsids were present with or without viral membrane as reported before [ , ] . in addition, we found viruses budding from the plasma membrane, which have an intermediate shell of density between the capsid and the viral membrane. the same topology is found in released particles by cryo-et and cryo-em confirming that these particles are the mature form of pfv and that maturation occurs in the cell before virus egress. we also observed capsids accumulating in intracellular membrane tubes. although pfv capsid assembly was reported to take place at the pericentriolar region [ ] , we provide evidence that capsids accumulate in a membrane compartment whose origin is currently unknown. we suggest that they are immature unprocessed capsids, because of their slightly larger radius compared to the capsid at the plasma membrane (s fig). notably the intermediate shell of density present in mature capsids [ ] is absent and a closer interaction of the nterminal domain of gag with the core capsid could explain the larger capsid radius. because they lack glycoprotein they are most likely not related to budding into intracellular vacuoles as observed before [ ] . however, the nature of these potentially capsid-like structures has to be confirmed by further experiments as it cannot be ruled out that they are only indirectly related or completely unrelated to pfv. for instance, virus-related small vesicles have been recently reported during tick-borne encephalitis virus (tebv) infection [ ] . in contrast, these tebv related vesicles were variable in size and not arranged regularly as in the case of pfv infection. the glycoprotein plays a central role in entry and virus budding [ ] . cryo-et combined with sub-tomogram averaging is a well tailored method to study glycoprotein in situ (embedded in the viral membrane). several such structures have been determined to medium resolution for different enveloped viruses (up to Å for hiv, siv env [ ] ). here we used a different strategy and acquired d images on a direct electron detector by cryo-em. we took advantage of the high density of glycoprotein at the surface of pfv as well as its symmetrical organization [ ] and advanced automatic particle picking to reconstruct in situ pfv glycoprotein to~ Å resolution. we propose that pfv env is a class i glycoprotein due to the two predicted coiled coil regions in the gp tm fusion protein subunit and consistent with this prediction, we identify three rods of density centrally located in the structure which we interpret as a coiled coil region, a hall mark of class i fusion glycoproteins [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . furthermore, the maps show clear density for three tmhs derived from gp tm and three from gp lp . the central gp tm tmhs are in close proximity to interact with each other and can be therefore considered to exert the role of a trimerization domain. because of the importance of the transmembrane region for membrane fusion [ ] , it will be interesting to study pfv env gp tm induced fusion due to the presence of the gp lp transmembrane region. current models of membrane fusion suggest that the trimeric tm region has to come apart to rearrange the fusion protein to catalyze membrane fusion [ , ] . the extra gp lp tmhs may prevent the dissociation of the gp tm tmhs and the tm "complex" may therefore move as a block during fusion. in line with the hypothesis that gp lp affects fusion, mutations within its cytoplasmic region strongly enhance the fusogenic activity of such mutant glycoproteins [ ] . an unusual feature of pfv env is the physical interaction of glycoproteins with each other on the surface of the viral membrane to form intertwined hexagonal networks [ ] . the interaction might be mediated by the extracellular part of gp lp and/or parts of gp tm , which could act as a spacer between glycoproteins. while this topology is not systematic, it seems to be the preferred one. clustering of the glycoproteins on the surface has been reported for a number of other viruses having class i spike-like glycoproteins such as aslv env [ ] , influenza hemagglutinin [ ] , and class iii hsv gb [ ] . in all these examples, the glycoproteins rather form patches or clusters arranged without symmetry or regular pattern. it has been suggested that the concentration of glycoprotein on the virus surface may confer an advantage for the virus to more efficiently recognize and attach to cellular receptors. bunyaviruses, having class ii glycoproteins [ ] , are known to have local or global symmetrical arrangement of glycoprotein on the viral membrane [ ] [ ] [ ] : trimeric, tetrameric and icosahedral organization have been reported but none of these resembles pfv's. clusters of env glycoproteins have been shown to be the preferred site of host cell interaction and thus entry for hiv- [ ] and it is therefore likely that pfv env arrangement in hexameric clusters confers also an advantage for host cell entry. the cellular receptor(s) essential for pfv env-mediated membrane fusion is/ are currently unknown but must be ubiquitously expressed molecule(s) due to the large range of permissive cells. therefore the hexagonal arrangement of env could enhance low affinity receptor interactions by increased avidity. in summary our structural analysis of isolated pfv and of pfv infected cells provides new medium resolution insights into the structural proteins gag and env, two important players in the pfv life cycle and will thus help to further develop pfv as a gene therapy transfer vector. for production of replication-deficient, wild type pfv vector supernatant a component pfv vector system, consisting of the expression-optimized packaging constructs pcopg (pfv gag), pcope (pfv env), pcopp (pol), and the enhanced green fluorescent protein (egfp)expressing pfv transfer vector puc md was used, which has been described previously [ , , ] . mutant pfv vector particles deficient in nucleic acid incorporation (inab) were generated using the pfv gag packaging construct pcopg gr r/a whereas particles deficient in pfv env fusion (ifuse) were generated using the pfv env packaging construct pcope instead of the respective wild type packaging constructs [ , ] . the cmv-driven proviral expression vector pczhsrv (wt), described previously [ ] , was used for production of replication-competent pfv supernatants. the human kidney cell line t [ ] was cultivated in dulbecco's modified eagle's medium (dmem) supplemented with % heat-inactivated fetal calf serum and antibiotics. cell culture supernatants containing recombinant viral particles were generated by transfection of t cells with the corresponding plasmids using polyethyleneimine (pei) as described previously [ , ] . for subsequent western blot analysis the supernatant generated by transient transfection was harvested, passed through a . -μm filter and centrifuged at °c and , rpm for h in a sw ti rotor (beckman) through a % sucrose cushion. the particulate material was resuspended in phosphate-buffered saline (pbs). for cryo-em analysis, viral particles were produced in serum-free medium and a further concentration step using amicon ultra . ml k concentrators was included following the first concentration by ultracentrifugation through % sucrose similar as described recently [ ] . cells from a single transfected -mm cell culture dish were lysed in detergent-containing buffer and the lysates were subsequently centrifuged through a qiashredder column (qia-gen). protein samples from cellular lysates or purified particulate material were separated by sds-page on a % polyacrylamide gel and analyzed by immunoblotting as described previously [ ] . hybridoma supernatants specific for pfv env lp (env lp, clone p b -b ), pfv env su (env su, clone p e ) or pfv gag (gag, clone sgg- ) were employed [ , , ] . after incubation with a horseradish peroxidase (hrp)-conjugated secondary antibody, the blots were developed with immobilon western hrp substrate. the chemiluminescence signal was digitally recorded using a las- (fujifilm) imager and quantified using imagegauge (fujifilm). wt pfv, inab and ifuse mutants were all prepared following the same procedure except that wt pfv particles were first inactivated for at least h in % paraformaldehyde before being processed. μl of sample containing nm gold beads was applied to : quantifoil holey carbon grid (quantifoil micro tools gmbh, germany) and the grid was plunge frozen in liquid ethane with a vitrobot mark ii (fei, the netherlands). for cryo-et, the frozen grid was transferred to a fei f feg cryo electron microscope. tilt series were recorded at kv with fei tomography at a nominal magnification of , on a k by k eagle ccd camera from - to + °in °steps for a total dose of~ e -/Å . the defocus was set between - and - μm. tilt series were aligned using gold particles as fiducials, binned two times (final sampling of . Å/ pixel) and tomograms were calculated with imod [ ] . for visualization, the tomograms were denoised by anisotropic diffusion in imod. for cryo-em, samples were observed with a fei polara at kv. images were recorded on a k summit direct detector (gatan inc., usa) in super resolution counting mode. for the inab sample, movies were recorded at a nominal magnification of , ( . Å/pixel at the camera level) for a total exposure of s and ms per frame resulting in frames movies with a total dose of~ e -/Å . for ifuse, the magnification was , ( . Å/pixel at the camera level), the total exposure was s with frames of . ms resulting in frames movies with a total dose of~ e -/Å . and movies were manually recorded with digital micrograph (gatan inc., usa) for inab and ifuse pfv respectively. data interpretation and processing were done with imod and peet [ , ] . the protocol described below was applied for subtomogram averaging of glycoproteins of wt pfv, inab and ifuse mutants. the center and radius (defined as the distance from the center to the outer margin of the viral membrane) of each virus was defined in dmod. initial centers for the glycoprotein sub volumes were defined on each virus surface on a regular grid (defined by the spacing between the centers of the neighbor subvolumes and the distance between the subvolume and virus centers) with seedspikes in peet. two of the three euler angles were estimated based on the spike orientation on the virus surface with spikeinit in peet. sub tomogram averaging was done in peet. the initial reference was an average of all the sub volumes. in the first iterations, no symmetry was enforced but a cylindrical mask was applied to eliminate contributions from the neighbor spikes. the resulting asymmetric reconstruction clearly shows -fold symmetry (which was also seen in raw tomograms). c was then enforced in the subsequent steps. at that stage, the subtomogram positions were checked manually in dmod and sub volumes that converged at positions where there was no glycoprotein or at the same position as other subvolumes were discarded. a final d reconstruction of voxels was calculated with this new set of "clean" subvolumes using a soft edged mask around the reference. to obtain reconstruction of the hexagonal network, few more iterations were done without any masking and by increasing the reconstructed volume to voxels. the final d models include , and subvolumes for wt, inab and ifuse pfv respectively. the final resolutions calculated by fourier shell correlation between two half reconstructions were . , . and . nm at fsc = . for respectively wt, inab and ifuse pfv. glycoprotein d reconstructions from d images acquired by cryo-em images were first motion corrected with unblur [ ] . from the d reconstruction of a glycoprotein hexagonal network obtained by subtomogram averaging (see above), we extracted one hexagon of six trimers and centered it on its -fold axis. the resulting reconstruction was used as a template for automatic particle picking using the fast projection matching (fpm) method developed in the lab [ ] . to speed up calculation, the raw micrographs were binned by a factor of ( . Å/pixel) and ( . Å/pixel) for inab and ifuse samples respectively. masks were designed manually around each viral particle to further restrict the area where the automatic picking of glycoproteins was performed. for this initial automatic picking by template matching, the resolution was limited to nm. for each micrograph, only particles having cross correlation with the reference higher that the mean correlation of all the particles were kept. the output coordinate files from fpm were converted to boxer [ ] one (.box) and imported in relion [ ] where all subsequent steps were done with two-times binned images (final sampling of . and . Å/pixel for inab and ifuse respectively). ctf estimation, particle extraction (in boxes of and pixels) for inab and ifuse respectively) and preprocessing were done in relion. the data sets were first cleaned by d classification and only classes showing clear glycoprotein network were kept. a first d autorefine was then done using the model obtained by subtomogram averaging as an initial reference (low pass filtered to Å) and imposing c symmetry. the obtained d model was used as input for a d classification with classes and c symmetry. the particles belonging to the classes showing the best resolved features were used to do another d autorefine. the resulting d reconstruction showed much improved features than the subtomogram d model. therefore, we did another round of automatic picking with fpm using the latter model calculated with relion as a reference and extending the resolution limit from to nm. the new coordinate files were imported again in relion and the same procedure described above was repeated ( d classification, dautorefine, d classification and d autorefine) leading to new improved maps. we noticed that masking away the viral membrane by keeping only the densities corresponding to the extracelllular domains of the glycoprotein improved the alignment and quality of the d reconstruction. following the last d autorefine, we therefore decided to do a focused d classification within a mask containing only the extracellular domains of the spikes and their tm regions ( classes total without alignment). this leads to one class having much sharper details. we used the particles from this class to do a last d autorefine. the final reconstructions of hexagonal assemblies include out of and out of particles for respectively inab and ifuse and have resolutions of . and . Å at fsc = . . each hexagonal assembly consists of six identical trimers and each one of them has an additional -fold symmetry that was not yet applied. the center of one trimer was defined in dmod and volumes of , voxels for inab and ifuse dataset respectively were extracted from the two half unfiltered maps calculated by relion in the last dautorefine run. the two volumes were then aligned such as their fold axis lies parallel to the z axis with e align d.py from eman [ ] . the two aligned volumes were then -fold symmetrized before being post processed in relion with a mask to remove the contribution from neighbor spikes. this leads to an improvement of the density maps to . and . Å for inab and ifuse dataset respectively at fsc = . . intact capsids from the ifuse mutant were picked manually from the same cryo-em micrographs (binned times to . Å/pixel) used for the glycoprotein reconstruction with boxer [ ] . particles were extracted in boxes of pixels with relion and d classification in relion was performed which resulted in the isolation of a subset of intact and regular particles. these particles were assembled in a stack, low pass filtered to Å and further d classified in classes by multi variate statistical analysis in imagic [ ] . the d reconstructions of single trimer were sharpened with bfactors of - Å and - Å for inab and ifuse dataset respectively in relion. density maps visualization, segmentation and figure preparation were done with chimera [ ] . pfv env amino acid sequence was submitted to the genesilico metaserver (https://genesilico.pl/meta [ ] ) to predict the number and position of transmembrane helices. no structural homologues was found by the metaserver. coiled coil prediction in pfv gp was done either with coils [ ] or multicoils [ ] . human fibrosarcoma cells ht were cultivated in dulbecco's modified eagle's medium (dmem) supplemented with % heat-inactivated fetal calf serum and antibiotics. after to h, cells were infected at a moi of and harvested to hours post infection. the cells were fixed and the virus inactivated with % paraformaldehyde. the cells were gently detached, pelleted at g and most of the supernatant removed to obtain a thick cell slurry. the cell suspension was loaded in μm deep gold carrier and cryo immobilized by high pressure freezing with a hpm (leica). the carriers were then transferred in an afs device (leica) maintained at - °c. the temperature was slowly raised to - °c before starting the cryo-substitution protocol (adapted for hawes et al. [ ] ). the freeze substitution cocktail ( . % urac, % etoh, % h in acetone) was added for h at - °c. the temperature was then raised at °c/h to - °c. samples were washed with ethanol ( h) and then infiltrated with increasing concentrations ( , , %; h each) of lowicryl (hm ) in ethanol followed by three incubation in % hm ( x h + h). uv polymerization was initiated for h at - °c, the temperature was then raised to °c ( °c/hour) and polymerization continued for h. sections ( to nm thick) were obtained with a uc (leica) ultramicrotome and post-stained with % urac and % lead citrate. for electron tomography, nm gold beads were added to both sides of the sections. the grids were loaded in a f (fei, the netherlands) feg electron microscope. tilt series were recorded at kv with fei tomography at a nominal magnification of , on a k by k eagle ccd camera from - to °in °steps. the defocus was set around - μm. tilt series were aligned using gold particles as fiducials, binned two times (final sampling of . Å/pixel) and tomograms were calculated with imod [ ] . segmentation was done in dmod as well. appearance of pfv spike d reconstruction with filtering to lower resolution. a-c side views (right column: slab through the center of the structure) of the unsharpened pfv ifuse spike filtered to (a), (b) and Å (c). as the resolution is lowered the overall shape and the large space (indicated with a white star) of the map are unaltered. the large space observed in the map is therefore not likely fully occupied by protein density which would fade into the background at low resolution and get resolved at higher resolution. this artifact has been described by bartesaghi et al. [ ] (tif) s table. summary of data acquisition and analysis for cryo-em and subtomogram averaging (tif) s movie. ht cells infected with pfv. the tomogram depicts an area where capsid-like objects are contained within membrane-delimited tubes (relates to s fig). (avi) foamy virus budding and release human infection with foamy viruses replication in a superficial epithelial cell niche explains the lack of pathogenicity of primate foamy virus infections foamy virus biology and its application for vector development molecular characterization of proteolytic processing of the gag proteins of human spumavirus expression and maturation of human foamy virus gag precursor polypeptides prototype foamy virus protease activity is essential for intraparticle reverse transcription initiation but not absolutely required for uncoating upon host cell entry novel functions of prototype foamy virus gag glycine-arginine-rich boxes in reverse transcription and particle morphogenesis a particleassociated glycoprotein signal peptide essential for virus maturation and infectivity a unique spumavirus gag n-terminal domain with functional properties of orthoretroviral matrix and capsid characterization of the r t point mutant of a putative cleavage site in human foamy virus env an evolutionarily conserved positively charged amino acid in the putative membrane-spanning domain of the foamy virus envelope protein controls fusion activity prototype foamy virus envelope glycoprotein leader peptide processing is mediated by a furin-like cellular protease, but cleavage is not essential for viral infectivity the intact retroviral env glycoprotein of human foamy virus is a trimer foamy virus capsid assembly occurs at a pericentriolar region through a cytoplasmic targeting/retention signal in gag specific interaction of a novel foamy virus env leader protein with the n-terminal gag domain identification of domains in gag important for prototypic foamy virus egress how to get out: ssrna enveloped viruses and membrane fission divergent pathways lead to escrt-iii-catalyzed membrane fission the cooperative function of arginine residues in the prototype foamy virus gag c-terminus mediates viral and cellular rna encapsidation foamy virus particle formation do lipid rafts mediate virus assembly and pseudotyping? foamy virus envelope glycoprotein is sufficient for particle budding and release ubiquitination of the prototype foamy virus envelope glycoprotein leader peptide regulates subviral particle release multicoil: a program for predicting two-and three-stranded coiled coils predicting coiled coils from protein sequences virus membrane fusion analysis of human t-cell leukemia virus type particles by using cryo-electron tomography structural organization of authentic, mature hiv- virions and cores the mechanism of hiv- core assembly: insights from three-dimensional reconstructions of authentic virions rsv capsid polymorphism correlates with polymerization efficiency and envelope glycoprotein content: implications that nucleation controls morphogenesis conserved and variable features of gag structure and arrangement in immature retrovirus particles structure and assembly of immature hiv structure of the immature hiv- capsid in intact virus particles at . Å resolution the structure of immature virus-like rous sarcoma virus gag particles reveals a structural role for the p domain in assembly hiv- assembly, budding, and maturation. cold spring harb perspect med visualization of a -helix bundle in the hepatitis b virus capsid by cryo-electron microscopy morphogenesis of hepatitis b virus and its subviral envelope particles electron tomography analysis of tick-borne encephalitis virus infection in human neurons molecular architecture of native hiv- gp trimers structure of the haemagglutinin membrane glycoprotein of influenza virus at a resolution structure of the parainfluenza virus f protein in its metastable, prefusion conformation structure of the ebola virus glycoprotein bound to an antibody from a human survivor prefusion structure of trimeric hiv- envelope glycoprotein determined by cryo-electron microscopy crystal structure of a soluble cleaved hiv- envelope trimer cryo-em structure of a fully glycosylated soluble cleaved hiv- envelope trimer structure and immune recognition of trimeric pre-fusion hiv- env cryo-electron microscopy structure of a coronavirus spike glycoprotein trimer gpi-anchored influenza hemagglutinin induces hemifusion to both red blood cell and planar bilayer membranes viral membrane fusion virus membrane fusion visualization of the two-step fusion process of the retrovirus avian sarcoma/leukosis virus by cryo-electron tomography structure and accessibility of ha trimers on intact h n pandemic influenza virus to stem region-specific neutralizing antibodies the structure of herpesvirus fusion glycoprotein b-bilayer complex reveals the protein-membrane and lateral protein-protein interaction crystal structure of glycoprotein c from rift valley fever virus orthobunyavirus ultrastructure and the curious tripodal glycoprotein spike electron cryotomography of tula hantavirus suggests a unique assembly paradigm for enveloped viruses insights into bunyavirus architecture from electron cryotomography of uukuniemi virus super-resolution microscopy reveals specific recruitment of hiv- envelope proteins to viral assembly sites dependent on the envelope cterminal tail analysis of prototype foamy virus particle-host cell interaction with autofluorescent retroviral particles improved primate foamy virus vectors and packaging constructs differential ph-dependent cellular uptake pathways among foamy viruses elucidated using dual-colored fluorescent particles human foamy virus reverse transcription that occurs late in the viral replication cycle analysis of mutation in human cells by using an epstein-barr virus shuttle system computer visualization of three-dimensional image data using imod the molecular architecture of axonemes revealed by cryoelectron tomography clustering and variance maps for cryo-electron tomography using wedge-masked differences measuring the optimal exposure for single particle cryo-em using a . Å reconstruction of rotavirus vp fast projection matching for cryo-electron microscopy image reconstruction eman: semiautomated software for high-resolution single-particle reconstructions relion: implementation of a bayesian approach to cryo-em structure determination a new generation of the imagic image processing system ucsf chimera-a visualization system for exploratory research and analysis genesilico protein structure prediction meta-server rapid freeze-substitution preserves membranes in high-pressure frozen tissue culture cells conceived and designed the experiments: ge dl gs ww. performed the experiments: ge na pr ns. analyzed the data: ge lfe dl gs ww. wrote the paper: ge dl gs ww. designed the software used for particle picking in cryo-em micrographs: lfe. key: cord- -b v c c authors: de lang, anna; baas, tracey; teal, thomas; leijten, lonneke m; rain, brandon; osterhaus, albert d; haagmans, bart l; katze, michael g title: functional genomics highlights differential induction of antiviral pathways in the lungs of sars-cov–infected macaques date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: b v c c the pathogenesis of severe acute respiratory syndrome coronavirus (sars-cov) is likely mediated by disproportional immune responses and the ability of the virus to circumvent innate immunity. using functional genomics, we analyzed early host responses to sars-cov infection in the lungs of adolescent cynomolgus macaques (macaca fascicularis) that show lung pathology similar to that observed in human adults with sars. analysis of gene signatures revealed induction of a strong innate immune response characterized by the stimulation of various cytokine and chemokine genes, including interleukin (il)- , il- , and ip- , which corresponds to the host response seen in acute respiratory distress syndrome. as opposed to many in vitro experiments, sars-cov induced a wide range of type i interferons (ifns) and nuclear translocation of phosphorylated signal transducer and activator of transcription in the lungs of macaques. using immunohistochemistry, we revealed that these antiviral signaling pathways were differentially regulated in distinctive subsets of cells. our studies emphasize that the induction of early ifn signaling may be critical to confer protection against sars-cov infection and highlight the strength of combining functional genomics with immunohistochemistry to further unravel the pathogenesis of sars. infection with sars-cov causes lower respiratory tract disease with clinical symptoms that include fever, malaise, and lymphopenia [ ] . approximately %- % of sars patients require management in intensive care units, and the overall fatality rate has approached %. interestingly, children seem to be relatively resistant to sars, but the reason for this restriction is not known [ ] [ ] [ ] . the clinical course of sars follows three phases [ , ] . in the first phase, there is active viral replication and patients experience systemic symptoms. in the second phase, virus levels start to decrease while antibodies, which are effective in controlling infection, increase. however, pneumonia and immunopathological injury also develop in this phase. ultimately, in the third phase, fatal cases of sars progress to severe pneumonia and acute respiratory distress syndrome (ards), characterized by the presence of diffuse alveolar damage (dad) [ , ] . it has been hypothesized that the pathological changes are caused by a disproportional immune response, illustrated by elevated levels of inflammatory cytokines and chemokines, such as cxcl (ip- ), ccl (mcp- ), interleukin (il)- , il- , il- , il- b, and interferon (ifn)-c [ ] [ ] [ ] [ ] [ ] [ ] . these in vivo data have been confirmed with in vitro experiments, demonstrating that sars-cov infection induces a range of cytokines and chemokines in diverse cell types [ ] [ ] [ ] [ ] [ ] [ ] . in contrast, production of type i ifns seems to be inhibited or delayed by sars-cov in vitro [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . moreover, no ifn-a or ifn-b has been detected in the sera of sars patients or in lungs of sars-cov-infected mice [ ] [ ] [ ] . recent in vitro studies demonstrated that type i ifn inhibition or delay may be orchestrated by sars-cov proteins orf b, orf , and n [ ] . the inhibition of ifn production would benefit sars-cov replication, since pretreatment of cells with ifn before sars-cov infection efficiently prevents replication in these cells [ , [ ] [ ] [ ] [ ] . furthermore, prophylactic treatment of macaques with pegylated ifn-a reduces sars-cov replication in the lungs [ ] . although ifn production was absent in clinical samples, gene and protein expression profiles in these patients were likely impacted by clinical treatments and concurrent preexisting disease. in addition, most if not all virus-host response information is from clinical blood/sera samples that were taken relatively late during infection-little is known about what happens early during infection. animal studies are of great value to decipher the host's initial innate immune response, without confounding clinical treatment (steroid and mechanical ventilation) or underlying co-morbidity. in order to elucidate early host responses during the acute phase of sars-cov infection, we infected cynomolgus macaques with sars-cov and used macaque-specific microarrays and real-time (rt)-pcr techniques to study host gene expression profiles. adolescent cynomolgus macaques infected with sars-cov develop dad similar to sars patients, but clear most of the virus in the lungs by day [ ] . because sars-cov replicates predominantly in the lower respiratory tract of macaques, the virus infects a range of cells, including type and type pneumocytes, that are different from those analyzed in vitro. the ability to simultaneously examine virus replication and host response gene expression profiles in the lungs of these animals during the acute phase of sars offers the opportunity to further unravel the pathogenesis of sars. six cynomolgus macaques were inoculated with sars-cov strain hku- and lung tissues were collected at day (n ¼ , a and b) or day (n ¼ , a- d). no lesions or clinical symptoms were detected on day after sars-cov infection, whereas on day , three out of four monkeys were lethargic, with one of these animals showing mildly labored breathing. pathological changes at day post infection included dad, characterized by flooding of the alveoli with edema fluid, infiltration of neutrophils, damage to the alveolar and bronchial epithelia, and occasional type pneumocyte hyperplasia, as described earlier [ ] . four mock-infected animals were included in the study to serve as a reference for host response without viral challenge and to examine outbred inter-animal variation. our previous experience with a/ texas/ / influenza virus demonstrated that viral mrna was detected in representative samples of the lung rather than throughout the whole lung [ ] . based on this experience, the level of infection in separate lung samples was evaluated using rt-pcr. sars-cov mrna was detected in all animals, and pieces out of the total of lung pieces from infected animals contained high levels of virus, while the three remaining pieces of lung contained very low levels of virus (; - logs lower, figure a) . no viral rna could be detected in the samples from the mock-infected animals. for gene expression experiments, lung samples from sars-cov-infected animals were compared to a reference lung sample from mockinfected animals. the three samples with lower virus levels ( a-low, a-low, and d-low) were analyzed individually so as not to dilute the gene expression of pooled pulmonary samples with higher sars-cov levels and also to potentially further define pulmonary infection. samples from animals with high viral mrna levels showed greater gene expression changes (; , genes day , ; genes day ) compared to samples from animals with low levels of viral mrna (; genes), indicating a response of lung tissue to the virus ( figure b) . additionally, the two day animals showed higher numbers of differentially expressed genes than the day animals. in contrast, gene expression analysis of the separate mock samples revealed limited differentially expressed genes. in order to examine how gene expression would be influenced by presence of virus, timing after inoculation, and individual animal variation, global expression profiling was performed. hierarchical clustering methods were used to order rows (genes) and columns (samples) to identify groups of genes or samples with similar expression patterns [ , ] . these data were plotted as a heat map in which each matrix entry represents a gene expression value (figure a ). red corresponds to higher gene expression than that of the controls; green corresponds to lower gene expression. this analysis yielded , genes with day samples on one side of the heat map and day samples on the other side of the heat map, indicating an influence of timing after inoculation. there are two major roots to the hierarchical dendrogram, with the larger root composed of all the day samples and the three day samples with the highest virus levels. the smaller root is composed of the remaining day samples with the lowest sars-cov levels. although transcriptional profiling shows some variation when comparing samples from the same animal, the underlying gene expression is similar with a reduction in fold change in the ''low'' samples. these comparisons suggest that both individual animal variation and the ''asynchronous'' nature of the infection in the animals' lungs are factors involved in determining transcription of cellular genes. to validate that the host response from infected animals comprises a stronger transcriptional profile than individual variation from mock-infected animals, differential gene expression patterns in the separate mock samples were investigated, but only genes were differentially expressed ( figure b ). these results suggest that underlying basal levels of gene transcription do not confound expression levels after infection. even in a basal state, some low-level lung-to-lung variations were identified within the same animal but not enough to disrupt segregation of lung pieces based on mock-infected animals. severe acute respiratory syndrome coronavirus (sars-cov) infection causes a progressive atypical pneumonia. in typical cases, largely confined to adult and elderly individuals, acute respiratory distress syndrome develops, and admission to an intensive care unit is required. although these complications can be fatal, most sars patients recover, suggesting that protective immune responses are operational. in this study, we simultaneously examined virus replication and host-response gene expression profiles in macaque lungs during the acute phase of sars to gain more insight into the early events that take place after sars-cov infection. we show that a strong host response is induced in the lungs of sars-cov-infected macaques, illustrated by the induction of several pathogenic cytokines and chemokines. interestingly, antiviral pathways are activated as well, demonstrated by the presence of phosphorylated signal transducer and activator of transcription (stat ) transcription factors throughout the lung, but not in sars-cov-infected cells. a subset of cells was shown to produce interferon-b, a cytokine involved in the resistance to many viral infections and able to activate stat . activation of this antiviral pathway upon sars-cov infection may be an important escape route of the host to withstand the devastating effects of sars-cov. different time points after inoculation, a venn diagram was generated with each set (circle) holding to the parameters of an absolute fold change . and p , . in at least two animals ( figure a ). the day set contained , genes and the day set contained genes. when examining host responses that were similar throughout the course of the infection, the intersection of the day and day sets indicates that genes show shared responses. the heat map of these genes is shown in figure b . if more stringent criteria were used to find common responses in all six animals, using the , genes from the day set and the genes that are differentially expressed in all day animals, a subset of genes was identified. this subset included ifnstimulated genes (isgs), like ifits, mx , gbp , and g p , and also various chemokines and cytokines, such as cxcl (ip- ), ccl (mcp- ), il- , and il- ( figure s ). these same cytokines and chemokines have been reported to be upregulated in human sars cases [ ] [ ] [ ] [ ] . this set also included cathepsin l (ctsl), which has been shown to be required for sars-cov entry into a cell [ ] . even though only genes were commonly regulated in all animals, indicated with blue bars in figure b , the heat map highlights that the other genes show similar expression trends. both sets of commonresponse genes showed similar functionality: cellular growth and proliferation, cell death, cellular movement, immune response, and cell-to-cell signaling. next, we analyzed genes that were differentially expressed exclusively on either day or day in order to find signature gene expression patterns for each day. genes identified as unique responses at day ( genes) and at day ( genes) in the venn diagram showed unique functionality ( figure c ). the gene expression profile at day shows a prominent innate host response to viral infection; top functional categories on day are the immune response, the hematological system, and the immune and lymphatic system. genes like ifn-c, ccl (mip- -b), csf , il a, and tnf are included in these categories. at day , a smaller panel of unique differentially expressed genes that play a role in cell cycle, cellular assembly, and dna repair were identified like ccnb , ccne , cdca , cenpa, chaf a, and prc . in order to investigate genes that are most strongly regulated after sars-cov infection, genes included in the venn diagram ( figure a ) that also held to an absolute fold change . were queried ( figure s ). from this set, genes that were involved in the immune response and lung repair processes were used to generate a heat map ( figure ) . a number of genes that have been reported to be up-regulated in sars patient sera, such as ccl (mcp- ), cxcl (ip- ), il- , and il- , were strongly (; -fold) induced in all animals. many cell cycle and matrix genes indicative of tissue repair processes were also highly differentially expressed at day (e.g., anln, areg, cdc , cdkn , cks , fosl , and kif c). likewise, tissue factor pathway inhibitor (tfpi ), an anticoagulant, was strongly up-regulated during infection in all animals (averaging ; -fold), as well as plscr , ser-pine (pai ), and thbs , all genes involved in procoagulation and platelet activation, were induced. concomitant expression of tfpi with these pro-coagulation genes might function as an inhibitory response to restrain the activation of the coagulation pathway during acute inflammation. surprisingly, expression of diverse ifn-a genes and expression of ifn-b was up-regulated ; to -fold in the day samples. furthermore, ifn-c, a type ii ifn, was efficiently transcribed on day after sars-cov infection (; -fold). other genes associated with the induction of ifns like ddx (rig-i), irf- , and signal transducer and activator of transcription (stat ), were also highly induced (; fold). up-regulation of type i ifns in these sars-cov infected macaques is remarkable, since sars-cov inhibits ifn production in many in vitro studies. we did not detect induced ifn-b mrna expression using ma cells or caco cells and the sars-cov-hku virus (unpublished data). not only ifns, but also several ifn-responsive genes (e.g., g p , gbp / , ifi/ifits, mx / , isg , and oas / /l) were highly transcribed, showing a persistent activation of the innate immune response. furthermore, suppressor of cytokine signaling (socs ) is induced at the onset of infection, presumably to establish negative feedback to attenuate cytokine signaling. of note, ifit (isg /ifi ), often used to gauge ifn induction, was up-regulated an average of ; fold. to further explore some of the pathogenic and antiviral pathways that are induced after sars-cov infection, we investigated the transcription of various cytokines, chemokines, ifns, isgs, and transcription factors involved in the jak/stat pathway. as can be seen in figure a , a wide range of chemokines and cytokines are differentially expressed after sars-cov infection in macaque lungs, especially on day after infection. besides previously mentioned chemokines, we detected monocyte chemotactic protein genes like ccl (mcp- ) and ccl (mcp- ), but also ccl (eotaxin), a chemotactic protein for eosinophils. in the samples with low sars-cov mrna levels, the induction of chemokines is less evident, suggesting that the presence of these molecules is restricted to areas in the lung where virus is present. furthermore, sars-cov-infected macaques showed a stronger induction of ifns ( unique genes) and isgs ( unique genes) on day than day and when virus was present at high levels. note that besides ifn-a, ifn-b, and ifn-c, the ifn-ks (il- , il- a, il- b), which are type i ifns, were induced in samples with high sars-cov levels. in the absence of viral rna, no ifns, but interestingly, a number of isgs ( unique genes) were detected, suggesting paracrine stimulation ( figure b ). differential expression of a selection of strongly upregulated genes, cxcl (ip- ), il- , il- , and ifn-b, was confirmed using rt-pcr ( figure ). in accordance with the microarray data, the rt-pcr data showed that cxcl (ip- ), il- , il- , and ifn-b were all expressed at levels that were approximately times higher in the sars-cov-infected animals at day than in the uninfected control animals and were still elevated on day after infection. as can be seen in figure , the induction of ifn-b was strongly correlated to the presence of virus (r spearman ¼ . , p , . ). for cxcl (ip- ), il- , and il- the correlation is less evident, which is not surprising since these cytokines can be induced by other factors than the virus itself. in order to visualize the host response in the lungs of sars-cov-infected macaques, ifn-b production and translocation of phosphorylated stat was studied using immunohistochemistry. in the lungs of the sars-cov-infected macaques, a modest number of cells stained positive for ifnb at day post infection, whereas no ifn-b-positive cells could be detected in mock-infected macaques ( figure a - c). notably, most of the cells that stained positive for ifn-b were located very close to blood vessels, but not in the alveoli where most sars-cov antigen-positive cells (mainly type pneumocytes at day post infection) are located. to examine whether the ifns that are produced in the lungs of these sars-cov-infected macaques are biologically active and able to induce stat phosphorylation and translocation, lung sections of the infected macaques were stained with antibodies against phosphorylated stat . as shown in figure d and e, no phosphorylated stat could be detected in the lungs of pbs-infected macaques, while in the lungs of sars-cov-infected macaques, cells with phosphorylated stat in their nucleus were abundantly present. subsequently, the same pieces of lung from sars- . genes were included if they met the criteria of a -fold change or more (p . ). a two-of-nine strategy allowed samples to cluster together if profile similarities existed based on timing of inoculation (n ¼ samples for day ). (b) the number after pbs refers to the animal (i.e., pbs ), while the number after the dash refers to the lung piece (i.e., pbs - ). thirty-eight genes are displayed with an absolute fold change . and p , . in at least two animal samples. up-regulated genes are indicated in bold underline. only one gene, hla-dqa , was down-regulated . . no up-regulated genes met these criteria in mock-infected animals. separate mock samples (i.e., pbs - ) were compared to the total mock pool. doi: . /journal.ppat. .g cov-infected macaques at day were double stained for phosphorlylated stat and sars-cov (figure f) . notably, phosphorylated stat was not detected in the nucleus of sars-cov-infected cells (type pneumocytes), while cells directly adjacent to these sars-cov-infected cells stained for phosphorylated stat in many, but not all, foci containing sars-cov-positive cells. thus, type i ifns are produced in the lungs of sars-cov-infected macaques, and are able to activate the jak/stat pathway. however, translocation of stat does not occur in sars-cov-infected pneumocytes. although recent studies indicate that the sars-cov orf protein is able to inhibit nuclear translocation of stat in vitro, this was not demonstrated in experiments using infectious sars-cov [ ] . in order to assess whether sars-cov inhibits phosphorylation and translocation of stat , ma cells were infected with sars-cov for h and then either fixed directly or treated with type i ifn. cells infected with sars-cov, but not treated with ifn, stained positive for sars-cov (unpublished data), but lacked staining for phosphorylated stat , indicating that sars-cov or other soluble mediators are not able to induce stat phosphorylation ( figure ). after treatment of the ma cells with ifn, phosphorylated stat could be detected in the nucleus of most cells, but not in the nucleus of sars-cov-infected cells (figure ). this demonstrates that sars-cov inhibits the translocation of phosphorylated stat to the nucleus, confirming our in vivo data. besides inhibiting translocation of phosphorylated stat , sars-cov also seems to reduce stat phosphorylation, as the majority of sars-covinfected cells contained low levels of phosphorylated stat in their cytoplasm. pathogenic viruses escape the antiviral action of the ifn system by inhibiting both ifn production and signaling pathways. here, we report that even though production and signaling of type i ifns is inhibited by sars-cov in vitro as well as in sars-cov-infected cells in vivo, high levels of type i ifns are induced in the lungs of sars-cov-infected macaques. these ifns are able to activate stat , followed by the transcription of numerous isgs. using immunohistochemistry, we revealed that these antiviral signaling pathways were differentially regulated in distinctive subsets of cells. our results emphasize the strength of combining functional genomics with immunohistochemistry to further unravel the pathogenesis of sars-cov infection in cynomolgus macaques. to our knowledge, this study represents the first functional genomics investigation of sars-cov infection in cynomolgus macaques. all experimental animals showed signs of infection because viral mrna could be detected in random samples from the lung, indicating that the virus had spread throughout the whole lung at the time of necropsy. furthermore, pathological examination of sars-cov-infected macaques at day post infection revealed multifocal dad [ ] . unlike % of humans with sars, which are mainly restricted to the elderly, adult macaques used in this study do not succumb to sars-cov infection. however, the sars-cov-induced pathology in these macaques likely resembles the pathological changes seen in the majority of human sars patients that recover from the disease. although none of the current animal models has fully reproduced all features of sars, the most important aspects of this disease are observed in experimentally infected macaques, providing valuable insights into the initial innate immune response after infection without confounding clinical treatment or underlying co-morbidity. using macaque-specific microarrays, we were able to observe that with early infection, high levels of viral mrna corresponded to a strong cellular host response. this strong host response is dominated by genes involved in the immune response and includes a wide range of genes corresponding with what is seen in human ards. during the acute phase of human ards, activated neutrophils and macrophages enter the alveoli and produce a number of cytokines and chemokines such as il- , il- , and cxcl (ip- ) [ ] , as were found in the lungs of our sars-cov-infected macaques. researchers have postulated that these genes also predict adverse sars patient outcome [ ] . during the chronic phase of human ards, type pneumocytes start to proliferate and differentiate in order to repair the damaged lung. at day , [ , ] . we also detected a strong presence of genes involved in the coagulation pathway, including tfpi , serpine , and timp . the idea of a pro-coagulation profile mimics the clinical-pathological observations of sars patients that showed unusually disseminated small vessel thromboses in the lungs [ , ] . additionally, cathepsin l was up-regulated in all sars-cov-infected macaques. induction of this gene after sars-cov infection is quite interesting because cathepsin l is an endosomal protease that is necessary for sars-cov to infect a cell [ ] . remarkably, sars-cov infection in macaques leads to a strong transcription of ifns. not only ifn-a, ifn-b, and ifnk (all type i ifns), but also ifn-c, a type ii ifn, were all highly up-regulated, especially on day after infection. the expression of ifn-b, which strongly correlated to the amount of virus present, continued throughout day and was confirmed using immunohistochemistry; ifn-b-positive cells could be detected in the lungs of the sars-cov-infected macaques. the induction of ifn-b in these sars-covinfected macaques is surprising, because several reports have shown that sars-cov inhibits or delays type i ifn production in a number of cell types [ ] [ ] [ ] [ ] [ ] , ] . for example, sars-cov blocks a step in the activation of irf- , a transcription factor that is required for ifn-b induction [ ] . in addition, the sars-cov proteins orf b, orf , and nucleocapsid have been shown to function as ifn antagonists, as has the sars-cov nsp gene that prevents the production of sendai virus-induced ifn-b in cells [ , ] . interestingly, it was recently shown that plasmacytoid dendritic cells (pdcs) are able to produce ifn-a and ifn-b after sars-cov infection, while conventional dcs did not produce these type i ifns [ ] . pdcs are known for their ability to produce very high amounts of ifn-a and ifn-b and are considered firstline sentinels in immune surveillance in the lung [ ] [ ] [ ] [ ] [ ] . we speculate that the ifn-b-producing cells detected in the lungs of sars-cov-infected macaques are pdcs. future studies may address the nature of these ifn-producing cells once technical difficulties in detecting pdcs in macaque tissues have been tackled. these studies may also shed light on whether decreasing numbers of pdcs observed in clinical blood samples from human sars patients are caused by sequestering of pdcs by the lungs, destruction of pdcs by sars-cov, or destruction or suppression of pdcs by steroid treatment [ ] . when ifns are produced, they bind to their receptors on the cell membrane, after which stat , a key member of the jak/stat pathway, is phosphorylated and subsequently translocated to the nucleus, followed by the production of a wide range of ifn-stimulated genes. in vitro, sars-cov inhibited translocation of stat to the nucleus, and phosphorylation of stat was strongly reduced. however, the inhibition of stat phosphorylation was not absolute because cells with low levels of phosphorylated stat in their cytoplasm were also detected. in accordance with our data, kopecky-bromberg et al. recently showed that the sars-cov protein orf is able to inhibit stat translocation [ ] . this strategy is not unique to sars. other viruses have been shown to be able to block signaling of ifns by affecting phosphorylation and/or translocation of the stat proteins. for example, measles virus v protein inhibits translocation of stat , but does not affect phosphorylation, whereas measles virus p protein blocks both of these processes [ ] . other paramyxoviruses, like rinderpest virus, nipah virus, hendra virus, and mumps virus, as well as flaviviruses like west nile virus and japanese encephalitis virus, are able to block activation of stat and stat [ ] [ ] [ ] [ ] . inhibition of stat phosphorylation is not always complete. for example, sendai virus suppresses tyrosine phosphorylation of stat during the early stages of infection, but this block becomes leaky after a couple of hours with phosphorylated stat accumulating in the cytoplasm [ ] . in contrast to these in vitro data, we observed phosphorylated stat in the nuclei of numerous cells in the lungs of sars-cov-infected macaques, indicating that these cells had been activated by the ifns produced in the lung. however, phosphorylated stat was not detected in sars-covinfected cells. the observations made in this study indicate that sars-cov-infected macaques produce ifns in response to virus infection and are further capable of activating the stat pathway in cells surrounding the sars-cov-infected cells. the importance of ifns in controlling sars-cov infection has been suggested in several animal studies. mice clear sars-cov in the absence of nk cells, t cells, or b cells, suggesting that innate immune responses are sufficient to limit sars-cov infection in these animals [ ] . indeed, stat knock out mice, which are resistant to the effects of ifns, to some extent show a worsening of pulmonary disease and an increase in viral replication in the lungs compared to normal mice after infection with sars-cov [ ] . although ifn treatment was not conducted in sars-cov infection mouse studies, prophylactic treatment of macaques with pegylated ifn-a protects type pneumocytes from infection with sars-cov [ ] . in addition, potent antiviral activity is observed in vitro when cells are treated with ifns before they are infected with sars-cov [ , , ] . although we cannot determine the effect of neutralizing ifn-b in sars-covinfected animals, based on the experiments utilizing recombinant ifns in these animals, we postulate that type i ifns are partly responsible for the relatively mild clinical symptoms that are seen in sars-cov-infected macaques. in addition, a recent study again demonstrated the importance of ifns in viral infections, as macaques infected with the highly pathogenic and fatal influenza virus showed limited induction of type i ifns (only ifna reached fold changes . ) and delayed induction of isgs, while macaques infected with the low-pathogenic k influenza virus showed a strong induction of these antiviral molecules early during infection [ ] . notably, ifn-b was not up-regulated (absolute fold change , ) in any of the influenza virusinfected animals, even in those animals that recovered, unlike sars-cov-infected macaques that showed a very strong presence of ifn-b. in conclusion, our study demonstrates that cynomolgus macaques can be infected with sars-cov, as indicated by presence of viral mrna at different locations throughout the lung at day and day , with gross pathology becoming noticeable at day . furthermore, we show that infection of cynomolgus macaques with sars-cov leads to a strong immune response, including the induction of various cytokines and chemokines, resembling the host response seen in human sars patients. strikingly, despite the fact that sars-cov infection blocks the production of ifns in vitro, type i ifns are strongly induced in the lungs of sars-cov- infected macaques. the production of ifn early during infection leads to widespread activation of stat and the production of isgs. this suggests that, although sars-cov blocks ifn signaling in infected cells, locally produced ifns are capable of activating non-infected cells and possibly can prevent infection of these cells. thus, sars-cov infection in macaques leads to the differential activation of both pathogenic and antiviral signaling pathways in vivo, and the outcome may be determined by the relative contribution of these signaling pathways. were infected intratracheally with tcid sars-cov (hku- ) as described earlier [ ] . virus stocks were generated in vero e cells that were defective in ifn production. two animals were euthanized on day after infection and four animals were euthanized on day . in addition, four animals were mock (pbs) infected and euthanized on day , serving as a negative control group. one lung from each monkey was fixed in % formalin for histopathology and immunohistochemistry while the other was used for real-time pcr and microarrays. lung samples were randomly excised from three different lung areas (cranial, medial, caudal) and stored in rnalater (ambion, http://www.ambion.com/). sixteen pieces of lung were taken from the sars-cov-infected animals, two to three pieces of lung per animal. twelve pieces of lung were taken from the mock-infected animals, three pieces of lung per animal. individual lung samples in rnalater were transferred to trizol reagent (invitrogen, http:// www.invitrogen.com/), homogenized using polytron pt tissue grinders (kinematica, http://www.kinematica.ch), and then processed to extract rna. all experiments were executed under a biosafety level , and approval for animal experiments was obtained from the institutional animal welfare committee. oligonucleotide microarray analysis. infected macaque lung samples were co-hybridized with a reference mock-infected macaque lung sample on macaque oligonucleotide arrays containing viral probes, corresponding to viruses, and , rhesus probes, corresponding to ; , rhesus genes. the reference mock-infected sample was created by pooling equal mass quantities of total rna extracted from the individual lung pieces from mock-infected animals. an agilent bioanalyzer was used to check the purity of the total rna prior to crna probe production with the agilent low rna input fluorescent linear amplification kit (agilent technologies, http://www.agilent.com/). arrays were scanned with an agilent dna microarray scanner, and image analysis was performed using agilent feature extractor software (agilent technologies). each microarray experiment was done with two technical replicates using dye reversal [ ] . all data were entered into a custom-designed database (expression array manager) and analyzed with resolver . (rosetta biosoftware, http://www.rosettabio.com/) and spotfire deci-sionsite for functional genomics (spotfire, http://www.spotfire.com/). in our data analysis, genes were selected to be included for transcriptional profile based on two criteria: a greater than . % probability of being differentially expressed (p . ) and an expression level change of -fold or greater. ingenuity pathway analysis (ingenuity systems, http://www.ingenuity.com/) was used to functionally annotate genes according to biological processes and canonical pathways. in accordance with proposed miame standards, primary data are available in the public domain through expression array manager at http://expression.microslu.washington.edu/ expression/index.html [ ] . quantitative real-time rt-pcr. rt-pcr was performed to detect sars-cov mrna and to validate cellular gene expression changes as detected with microarrays. each reaction was run in triplicate using taqman x pcr universal master mix (applied biosystems, http:// www.appliedbiosystems.com/) with primers and probe specific for the sars-cov nucleoprotein gene [ ] , or for macaque cellular genes (sequences shown in table ). differences in gene expression are represented as the fold change in gene expression relative to a calibrator and normalized to a reference, using the Àddct method [ ] . gapdh (glyceraldehydes- -phosphate dehydrogenase) or s rrna were used as endogenous controls to normalize quantification of the target gene. the samples from the mock-infected macaques were used as a calibrator. immunohistochemistry. formalin-fixed, paraffin-embedded lung samples from sars-cov-infected and mock-infected macaques were stained for sars-cov, phosphorylated stat , and ifn-b using mouse-anti-sars-nucleocapsid (clone ncap , mouse igg b; imgenex, http://www.imgenex.com/), mouse-anti-phospho-stat (clone st p- a , mouse igg a-j; zymed laboratories, http://www. invitrogen.com/), and rabbit-anti -ifn-b (chemicon, http://www. chemicon.com/), respectively. after deparaffinization, antigen retrieval was performed using a citrate buffer for the sars-cov and stat staining. no antigen retrieval was performed when staining for ifn-b. goat-anti-mouse igg a hrp, goat-anti-mouse igg b ap (southern biotech, http://www.southernbiotech.com/), and anti-rabbit igg-hrp (dako, http://www.dako.com/) were used as secondary antibodies. signals were developed with fast red and dab (sigma, http://www.sigmaaldrich.com/) and counterstained with mayer's hematoxylin. in vitro sars-cov and stat staining. ma cells (african green monkey foetal kidney cells, ecacc) were cultured in eagle's minimal essential medium (emem; cambrex, http://www.cambrex. com/) supplemented with mm glutamine, % non-essential amino acids and % foetal bovine serum. cells were seeded in -well plates and infected with sars-cov (moi . ), and h after infection, selected wells were treated with universal type i ifn ( , u/ml, sigma) for min at c. subsequently, cells were fixed with % neutral-buffered formalin and treated with % ethanol. sars-cov-infected cells were visualized using purified human igg from a convalescent sars patient (csl), followed by staining with an antibody to human igg, linked to alexa fluor (invitrogen). phosphorylated stat was visualized using mouse-anti-phospho-stat (zymed), followed by staining with a fitc-linked antibody to mouse igg. lung pathology of fatal severe acute respiratory syndrome severe acute respiratory syndrome coronavirus pathogenesis, disease and vaccines: an update clinical presentations and outcome of severe acute respiratory syndrome in children clinical picture, diagnosis, treatment and outcome of severe acute respiratory syndrome (sars) in children clinical progression and viral load in a community outbreak of coronavirusassociated sars pneumonia: a prospective study the severe acute respiratory syndrome newly discovered coronavirus as the primary cause of severe acute respiratory syndrome an interferongamma-related cytokine storm in sars patients expression profile of immune response genes in patients with severe acute respiratory syndrome analysis of serum cytokines in patients with severe acute respiratory syndrome plasma inflammatory cytokines and chemokines in severe acute respiratory syndrome characterization of cytokine/chemokine profiles of severe acute respiratory syndrome a probable role for ifn-gamma in the development of a lung immunopathology in sars comparative host gene transcription by microarray analysis early after infection of the huh cell line by severe acute respiratory syndrome coronavirus and human coronavirus e cytokine responses in severe acute respiratory syndrome coronavirus-infected macrophages in vitro: possible relevance to pathogenesis chemokine upregulation in sars-coronavirus-infected, monocyte-derived human dendritic cells a human in vitro model system for investigating genome-wide host responses to sars coronavirus infection inhibition of cytokine gene expression and induction of chemokine genes in non-lymphatic cells infected with sars coronavirus modeling the early events of severe acute respiratory syndrome coronavirus infection in vitro severe acute respiratory syndrome coronavirus fails to activate cytokinemediated innate immune responses in cultured human monocyte-derived dendritic cells inhibition of beta interferon induction by severe acute respiratory syndrome coronavirus suggests a two-step model for activation of interferon regulatory factor interaction of severe acute respiratory syndrome-associated coronavirus with dendritic cells mechanisms of host defense following severe acute respiratory syndrome-coronavirus (sars-cov) pulmonary infection of mice severe acute respiratory syndrome coronavirus infection of mice transgenic for the human angiotensin-converting enzyme virus receptor lethal infection of k -hace mice infected with severe acute respiratory syndrome coronavirus severe acute respiratory syndrome coronavirus open reading frame (orf) b, orf , and nucleocapsid proteins function as interferon antagonists treatment of sars with human interferons potent inhibition of sars-associated coronavirus (scov) infection and replication by type i interferons (ifn-alpha/beta) but not by type ii interferon (ifn-gamma) ribavirin and interferon-beta synergistically inhibit sars-associated coronavirus replication in animal and human cell lines interferon-beta and interferon-gamma synergistically inhibit the replication of severe acute respiratory syndrome-associated coronavirus (sars-cov) pegylated interferon-alpha protects type pneumocytes against sars coronavirus infection in macaques integrated molecular signature of disease: analysis of influenza virus-infected macaques through functional genomics and proteomics hybrid hierarchical clustering with applications to microarray data analyzing microarray data using cluster analysis inhibitors of cathepsin l prevent severe acute respiratory syndrome coronavirus entry the acute respiratory distress syndrome early enhanced expression of interferon-inducible protein- (cxcl- ) and other chemokines predicts adverse outcome in severe acute respiratory syndrome the nucleocapsid protein of severe acute respiratory syndrome-coronavirus inhibits the activity of cyclincyclin-dependent kinase complex and blocks s phase progression in mammalian cells lung pathology of severe acute respiratory syndrome (sars): a study of autopsy cases from singapore severe acute respiratory syndrome coronavirus nsp protein suppresses host gene expression by promoting host mrna degradation control of coronavirus infection through plasmacytoid dendritic-cellderived type i interferon the nature of the principal type interferon-producing cells in human blood different roles for human lung dendritic cell subsets in pulmonary immune defense mechanisms plasmacytoid monocytes migrate to inflamed lymph nodes and produce large amounts of type i interferon plasmacytoid dendritic cells in immunity characterization of myeloid and plasmacytoid dendritic cells in human lung longitudinal alteration of circulating dendritic cell subsets and its correlation with steroid treatment in patients with severe acute respiratory syndrome tyrosine in the measles virus phosphoprotein is required to block stat phosphorylation inhibition of interferon signaling by the new york strain and kunjin subtype of west nile virus involves blockage of stat and stat activation by nonstructural proteins mumps virus v protein antagonizes interferon without the complete degradation of stat blocking of interferoninduced jak-stat signaling by japanese encephalitis virus ns through a protein tyrosine phosphatase-mediated mechanism rinderpest virus blocks type i and type ii interferon action: role of structural and nonstructural proteins sendai virus c protein impairs both phosphorylation and dephosphorylation processes of stat resolution of primary severe acute respiratory syndrome-associated coronavirus infection requires stat aberrant innate immune response in lethal infection of macaques with the influenza virus statistical design and the analysis of gene expression microarray data minimum information about a microarray experiment (miame)-toward standards for microarray data analysis of relative gene expression data using real-time quantitative pcr and the (-delta delta c(t)) method we thank s. smits for her assistance with sars-cov infections and rna isolations. author contributions. adl, tb, ado, blh, and mgk conceived and designed the experiments. adl, tb, tt, lml, and br performed the experiments. adl, tb, and blh analyzed the data. adl, tb, ado, blh, and mgk wrote the paper.funding. this work was supported by the us national institutes of health, r grant hl - a , and by the european union, grant sp- -ct- - .competing interests. the authors have declared that no competing interests exist. key: cord- -sufwsu authors: bär, séverine; rommelaere, jean; nüesch, jürg p. f. title: vesicular transport of progeny parvovirus particles through er and golgi regulates maturation and cytolysis date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: sufwsu progeny particles of non-enveloped lytic parvoviruses were previously shown to be actively transported to the cell periphery through vesicles in a gelsolin-dependent manner. this process involves rearrangement and destruction of actin filaments, while microtubules become protected throughout the infection. here the focus is on the intracellular egress pathway, as well as its impact on the properties and release of progeny virions. by colocalization with cellular marker proteins and specific modulation of the pathways through over-expression of variant effector genes transduced by recombinant adeno-associated virus vectors, we show that progeny pv particles become engulfed into copii-vesicles in the endoplasmic reticulum (er) and are transported through the golgi to the plasma membrane. besides known factors like sar , sec , rab , the erm family proteins, radixin and moesin play (an) essential role(s) in the formation/loading and targeting of virus-containing copii-vesicles. these proteins also contribute to the transport through er and golgi of the well described analogue of cellular proteins, the secreted gaussia luciferase in absence of virus infection. it is therefore likely that radixin and moesin also serve for a more general function in cellular exocytosis. finally, parvovirus egress via er and golgi appears to be necessary for virions to gain full infectivity through post-assembly modifications (e.g. phosphorylation). while not being absolutely required for cytolysis and progeny virus release, vesicular transport of parvoviruses through er and golgi significantly accelerates these processes pointing to a regulatory role of this transport pathway. egress of enveloped viruses is typically associated with the cell secretory pathway guiding the particles and/or their precursors through cellular organelles, in particular the endoplasmic reticulum (er) and the golgi cisternae [ ] [ ] [ ] [ ] [ ] [ ] [ ] . in this regard, enveloped viruses usurp the cellular secretory machinery in order to achieve the efficient transport of progeny viruses to the plasma membrane and the maturation of precursor particles into infectious virions [ ] [ ] [ ] [ ] [ ] [ ] [ ] . in contrast, non-enveloped viruses are thought to be released as mature virions through a cytolysis burst at the end of infection [ ] [ ] [ ] . however, there is evidence on non-enveloped virus egress through active transport besides the major lytic pathway. a few non-enveloped lytic viruses were shown to be actively transported to the cell periphery and released prior to cell lysis. for instance sv was detected in intra-cytoplasmic smooth membrane vesicles and described as being released from the cell before cytopathic effects are seen [ ] . similarly, cocksackie b virus was found to be transferred from cell to cell through microvesicles [ ] . we also recently reported that in the case of non-enveloped lytic parvoviruses that progeny virions are actively transported from the nucleus to the plasma membrane (pm) through vesicles in a gelsolin-dependent manner [ ] . the cellular secretory pathway has been characterized in great detail and a number of proteins involved in the specific recognition of cargos, the formation and loading of vesicles, and the guided transport through the cytoplasm have been identified [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . once in the er, transport of cargos is mediated by coat protein complex ii (copii) vesicles. engulfment of cargos into copii vesicles at er exit sites is triggered by the activation of the small gtpase sar , a process which in turn induces the recruitment of two heterodimeric complexes, sec -sec and sec -sec , and their assembly into a protein coat. in this complex, sec -sec is responsible for the selection and binding of the cargo. sec can either bind directly to the transported protein if it features a transmembrane domain, or interact with a transmembrane receptor specific to the cargo in the case of a secreted protein. in both cases, sec -sec bound to the cargo forms a ternary complex which concentrates the cargo and bends membranes. the budding vesicle is then enveloped by the sec -sec cage and released from the er by fission. during the last step of this budding, cellular factors responsible for directing the vesicle to the next compartment and fusing the vesicular membrane with the target membrane, associate with the departing vesicle [ , ] . targeting of cargo-vesicles to distinct organelles/compartments through the cytoplasm is achieved by distinct small gtpases, rab-proteins, which are associated with the surface of moving vesicles. for instance, rab targets vesicles from the er to the golgi apparatus, while rab is responsible for transport within the golgi cisternae and for retrograde transport from the golgi apparatus to er. transport from the transgolginetwork (tgn) to the plasma membrane (pm) can take different routes and is accordingly controlled by a variety of rab proteins. among these, rab is responsible for guiding the vesicles along the direct route from the tgn to the pm, while rab shuttles tgnvesicles through recycling endosomes to the pm [ ] . a number of other rab proteins are also involved in vesicle targeting to the endocytosis and secretion pathways, and new routes are constantly being discovered. when the vesicles reach the periphery of the cell, the release of the cargo is generally achieved by fusion of the vesicular membrane with the pm [ , ] . parvoviruses (pv) are small icosahedral non-enveloped particles with a . -kb linear single-stranded dna genome. during productive infection, pvs induce dramatic morphological and physiological changes in their host cells, culminating in cell death and lysis [ , , ] . this is mainly attributed to the large nonstructural viral protein ns , a multifunctional protein involved in particle production and spread (reviewed in [ ] ). ns was shown to modulate cellular pathways by physical interactions with distinct cell components [ , ] and/or induction of post-translational modifications of cellular target proteins [ , , , ] . these targets might be modified either directly by ns /ckiia, a recently described complex formed by ns with the catalytic domain of cellular ckii [ ] , or indirectly through activation/modulation of the pdk /pkc/pkb signaling cascade [ ] . the latter activation is mediated by the virus-induced association of the erm family protein radixin with pkcg, forming a complex that controls the activity and substrate specificity of the pdk . the cellular proteins targeted by the ns /ckiia complex include the erm-family protein radixin [ ] . erm proteins are known mediators of the interplay between filamentous actin and membrane structures [ ] . in particular, radixin (rdx) has been implicated in the parvovirus mvm infectious cycle, controlling progeny particles production and spreading as measured through the formation of lysis plaques [ ] . gelsolin, an actin-severing protein was found to be another target for the ns /ckiia complex. gelsolin shown to contribute to the egress of pv progeny virions [ ] through its function in the formation and/or the pvloading of cellular vesicles, which are then targeted through the cytoplasm to the pm [ ] . ns /ckiia-mediated phosphorylation seems to play a crucial role in this process. the present investigation aims to characterize vesicular egress of non-enveloped lytic parvoviruses in more detail and to determine the impact of this process on the pv life-cycle. using fluorescence microscopy, biochemical fractionation and selective inhibition of proteins involved in vesicular transport (e.g. rabproteins), we show that pv progeny particles become engulfed into vesicles in the er and are actively transported through the golgi apparatus to the pm. the erm family proteins moesin and radixin play an important role in the formation/loading and targeting of progeny virus-containing vesicles. finally, egress through the golgi apparatus appears to play an essential role in post-assembly modification, infectivity of progeny virions, and the stimulation of virus-induced cytolysis. previous investigations have shown that progeny parvoviral particles become associated to vesicles in a gelsolin-dependent manner and are actively transported from the nuclear periphery to the plasma membrane [ ] . to further characterize this pathway, we analyzed potential association of progeny particles in mvm infected a cells with cellular factors known to be involved in generation/loading of perinuclear vesicles. an obvious candidate for viral cargo shuttling out of the cell is the secretion pathway, which starts in the endoplasmic reticulum (er), with sar , sec / and sec / -dependent formation of copii vesicles. we therefore investigated the role of these factors in the association of progeny pv virions with vesicles and the further transport to the cell periphery. in a first step, we determined whether out-going mvm virions colocalize with sec , sec and b-cop in infected a cells using laser scanning microscopy. previous reports pointed that progeny particles are associated with lamp , a protein present in the membrane of lysosomes and late endosomes/multivesicular bodies [ , ] . we therefore used colocalizations with lamp and mitochondria as positive and negative controls, respectively. the absence of any significant capsid fluorescence at h p.i. was taken as a reference for the selective detection of newly produced virions after onset of virus propagation and not in-coming virus during the infection process. furthermore, cell reinfection with progeny particles was prevented by addition of neutralizing antibodies to the medium after the initial infection. as illustrated in fig. a , progeny pv particles showed clear colocalization with the cellular vesicle forming complex, giving a first hint that mvm virions may associate with cellular vesicles assembled at the er membrane. to functionally challenge this finding, we knocked down vesicle formation through transfection of neutralizing sec antibodies and expression of dominant-negative sar . association of progeny particles with vesicles was determined by cell fractionation and monitoring single-stranded virion dna presence in the corresponding biochemical fractions [ ] . to confirm association of virus with membranes structures and to assess internalization into vesicles, cell extracts were treated, or not, with or triton x- (disruption of membranes, release of membrane associated proteins into cytoplasm) and proteinase k (elimination of all proteins that are unprotected by membrane structures) prior to previously, it was thought that non-enveloped lytic parvoviruses were released through a lytic burst of cells at the end of infection. however, recent work demonstrated that these small non-enveloped single-stranded dna viruses are actively transported through vesicles from the nucleus, the site of replication and assembly, to the cell periphery. the current investigation demonstrates that progeny particles become engulfed into copii-vesicles in the endoplasmic reticulum (er) and are transported through the golgi to the plasma membrane (pm). erm family proteins radixin and moesin appear to play an essential role in this cellular secretion pathway. while passing through er and golgi cisternae, pvs maturate through post-assembly modifications, which significantly increase the infectivity of progeny virions. finally, the vesicular transport of parvoviral particles was shown to regulate virus-induced cytolysis, thereby accelerating the further release and spread of progeny virions. as rodent pvs are currently viewed as oncolytic agents for cancer virotherapy, it is important to further investigate the mechanism of pv egress -not only to improve the spreading of these agents through the tumor mass, but also to optimize the induction of an anti-tumor immune response upon virus -induced cytolysis. mvmp ( pfu/cell), and single rounds of infection were monitored in presence of neutralizing antibody b after initial infections. cells were fixed with paraformaldehyde at h (input) or h (mvm h) p.i., and analyzed by confocal laser scanning microscopy after double-staining with antisera specific for the indicated cell proteins (red) and mvm capsids (green). lack of interference from input virions is revealed by the absence of detectable capsid signal at h p.i. (nomarsky picture). colocalization areas appear yellow in the merge panel and are expressed as percent of total cytosolic capsids after quantification by image j analysis of infected cells from three individual experiments. to better visualize colocalization, zoom-ins were performed on indicated areas. bars indicate scales (in mm). (b-d) a cells were infected with mvmp ( pfu/cell), nch cells with hgh -pv ( pfu/cell) and harvested/fixed at h p.i. when indicated, sar functioning was jeopardized by over-expression of the dominant-negative sar k m variant (dnsar ) through transduction by raav h prior to parvovirus infection. sec was inhibited by transfection of neutralizing antibodies (asec ) h prior to infection, apkb served as control igg. to measure potential signals from incoming capsids cells untreated cells were harvested at h p.i. (b) cellular extracts were fractionated by differential (density) centrifugation to separate organelles. nuc, purified nuclei; hmf, large organelles; cyt, cytosol. the lmf-fraction was fractionated by centrifugation through iodixanol gradients allowing cellular vesicles to be identified according to their density (fractions [ ] [ ] [ ] [ ] . the presence of progeny particles was determined by southern blotting (revealing their singlestranded dna). to determine the nature of pv virion-containing vesicles, cell lysates were treated with triton x- (triton) or proteinase k (prot k) prior to fractionation. to rule out contamination from infecting particles, a fractionation experiment was performed after cell transfection with mvm infectious clone in the presence of neutralizing antibodies (transf+b panel). the migration of lamp (lysosomes/late endosomes) was determined by western blotting. (c) cells were fixed with paraformaldehyde and stained for mvm capsids (green) and counterstained with lamin b (red). fractionation. a h p.i. time-point was chosen to discriminate egress from the infection process. transfection experiments in the presence of neutralizing antibodies were performed to discriminate egress from (re-)infections. as shown in fig. b and in agreement with the above hypothesis, viruses were found in the subcellular vesicle fractions, from which they were removed by exposing cell lysates to triton prior to fractionation. this vesicle association was not detected when target cells were either transfected with sec neutralizing antibodies or if the cells expressed dominant-negative sar mutants. this suggests that mvm progeny virions become engulfed into vesicles by components of the cellular secretory pathway in the perinuclear er. similar results were obtained with the related rodent parvovirus strain hgh -pv after infection of the human glioblastoma cell line nch , suggesting that vesicular egress of progeny virions is a general feature of rodent pvs. we next studied the effect of functional sec / and sar on pv egress. to this end, mvm-infected a cultures were analyzed for the proportion of cells supporting cytoplasmic transport of progeny virions, as determined by immunofluorescence microscopy and the amount of infectious particles shed into the medium supernatant. cells lacking functional sec or sar were unable to transport progeny virions away from the nucleus, resulting in a marked increase in the proportion of cells showing only nuclear virus (fig. c ). while total virus production was not much affected by sec and sar knockdowns, the observed retention of virions in the (peri)nuclear area correlated with a significant reduction of virus release in the culture medium as determined by measuring the amounts of virion dna (dot blots) and/or infectious virions (plaque assays) in the supernatant (fig. d ). we next characterized the pathway followed by vesicles carrying pv progeny particles. many enveloped viruses are transported through one or the other compartment of the cellular secretory pathway before mature virions are released by budding at the plasma membrane [ ] [ ] [ ] [ ] [ ] [ ] . to determine the mode of parvoviral egress, we first examined the presence of mvm particles within cell organelles, and the association/colocalization of the particles with proteins involved in the targeting of intracellular vesicles. the latter proteins include the small gtpases, rab that are located on the outside of vesicular membranes and are guiding the vesicles between subcellular compartments. colocalisation with mitochondria (mitotracker), er (calnexin, sec ) and golgi (gm ) as well as the small gtpases rab (er to golgi), rab (trans golgi network [tgn] to pm), and rab (recycling endosomes) were investigated by confocal laser scanning microscopy. rab which is involved in retrograde transport from golgi to er and is mainly detectable in the golgi complex was used as a second marker for this organelle. it was shown previously that pvs become associated with lamp -containing vesicles (lysosomes, late endosomes/ multivesicular bodies (mvb) [ ] . therefore, this marker served as a positive standard. (re-)infections were inhibited by addition of neutralizing antibodies after the initial infections. as shown in fig. s and summarized in fig. a , strong colocalizations were observed between mvm particles and the er resident protein calnexin, the golgi complex marker gm , and the small gtpases rab and rab , known to control vesicular transfer between these compartments. interestingly, significant co-staining was seen with rab (tgn-re-pm), while only background levels were obtained with mitotracker and rab (tgn to pm). the presence of mvm progeny particles in er and golgi was then confirmed by biochemical fractionation of cellular extracts, separating er membranes from the golgi complex by ultracentrifugation on a nycodenz step gradient (fig. b ). altogether these results strongly argue for the active transport of progeny pv particles through er and golgi. to functionally support these findings, secretion of intracellular proteins was inhibited by selective inactivation of distinct rab proteins through (over-)expression of the corresponding dominantnegative mutants lacking gtpase activity [ , , ] . to this end, a cells were transduced with raav virions expressing dominantnegative (and for controls functionally active) rab proteins prior to mvm infection. to ensure efficient expression, the dnrab mutant genes were placed under the control of the parvoviral p promoter, and cells were co-transduced with raav:p -dnrab and, in absence of the pv ns protein (i.e. in absence of pv infection), with a second recombinant aav virus expressing a transactivator protein specific for this promoter (raav:p -transactivator). this synthetic transactivator protein is comprised of two distinct ns domains, the n-terminal site-specific dnabinding domain (aa - ) and the c-terminal transactivator domain (aa - ) linked by gfp. dimerization of this polypeptide takes place through an n-terminal gst-tag. in contrast to the natural pv ns protein, this polypeptide expressed from the pv p -promoter proved to be non-toxic as assessed by measuring metabolic activity and cell lysis (fig. s a ). under these conditions, selective inhibition of the secretion pathway was first tested independently of mvm infection, using cells transfected with a plasmid expressing secreted gaussia luciferase (gluc). indeed, this enzyme is known to be transported by vesicles from er to golgi and further to the plasma membrane following the regular rab and rab dependent secretion pathway [ ] . as shown in fig. a , secretion of gluc was efficiently blocked as a result of the expression of dnsar (driver for the formation of transport vesicles at er exit sites), dnrab and dnrab but only marginal dnrab , demonstrating the functional impact and specificity of these variant proteins. we next tested the effects of rab , rab and rab inhibition on the vesicular egress of mvm through er (sec ) and golgi (rab ) by colocalization with corresponding marker proteins ( fig. b; fig. s ), and on the release of progeny particles in the medium by dna dot blot analyses ( fig. c ) and plaque assays (fig. d ). as expected, none of the dominant-negative mutants prevented mvm particles from entering the er compartment. in contrast, mvm transport from er to golgi, and virus secretion into the supernatant were strongly impaired upon expression of dnrab . moreover, a very limited reduction of secreted particles was observed with dnrab , and nothing at all upon inhibition of rab , suggesting that pv particles take a different route from the golgi apparatus to the plasma membrane as compared to gluc. due to their impact on mvm plaque morphology in a cells, the erm family proteins moesin (moe) and radixin (rdx) have been implicated in the spreading capacity of this parvovirus [ ] . this observation, together with evidence of moesin being involved in endocytosis [ ] , led us to determine whether erm proteins may play a role in the vesicular egress of progeny pv virions. in agreement with this possibility, we first showed that moe and rdx protein can be detected together with sec and virion dna in vesicular fractions of mvm-infected a cells (fig. a, top panels). to put moe and rdx contribution to the test, we (over)expressed mutants thereof previously shown to interfere with endogenous protein functioning [ ] . impact on viral egress was then evaluated measuring virion dna presence in vesicles (fig. a , bottom), viral capsid intracellular distribution (fig. b ) and colocalization with cellular compartment marker proteins (figs. c and s ), as well as progeny virion release into the medium (fig. d ). the dominant-negative moesin (moet a) and radixin (rdxdl[p]) mutants strongly interfered with the loading of progeny virions into cytoplasmic vesicles ( fig. a and c), resulting in a dramatic inhibition of virion release into the medium (fig. d) . accordingly, viral capsids were hardly detectable in the cytoplasm under these conditions (fig. b ). in the presence of the functionally active rdxy f mutant (consensus phosphorylation site for receptor tyrosine kinases [ ] ), mvm virions became associated with the vesicular fractions ( fig. a ) and were readily found in the cytoplasm of infected cells (fig. s ). virus neutralizing antibodies were added after the initial infection to prevent cell re-infections with progeny viruses. colocalization was quantified by image j analyzing infected cells from three individual experiments with . cells. lys/le, lysosomes/late endosomes (lamp ); mit, mitochondria (mitotracker); er, endoplasmic reticulum (calnexin [clnx]); tgn, trans golgi network; re, recycling endosomes; pm, plasma membrane. (b) cellular extracts were fractionated to separate golgi-versus er-membrane structures by nycodenz gradient centrifugation. progeny virions were detected by southern blotting measuring single-stranded virion dna, the presence of gm (golgi) and calnexin (er), respectively, by western blotting. to preclude interference from input viruses, a control was made by transfecting cells with mvm infectious dna clone. transfected cells were further incubated in the presence of neutralizing b antibodies to ensure that infection is limited to a single round and cell do not get reinfected with progeny viruses (transf+b panel). doi: . /journal.ppat. .g (fig. b) . however, when vesicular egress through er and golgi was monitored, a strong decrease was observed in the colocalisation with the golgi marker (rab , see fig. s ), indicating the use of an alternative route seemingly involving secretory vesicles (increased rab colocalization) and recycling endosomes (preserved rab colocalization). interestingly, in the presence of rdxy f, progeny virions were not released efficiently into the medium (fig. d) , suggesting that virion transport through er and golgi plays an important role in the efficient release and spreading of pvs. erm proteins are known mediators of the interplay between actin cytoskeleton and membrane structures. the involvement of rdx and moe in the formation/loading of pv-containing vesicles raised the question, whether these proteins are selectively recruited by pvs or are genuine components of the cellular secretory machinery. this was investigated by expressing the secreted protein gluc in mouse a and human nch cells, and by determining the impact of over-expressing dominant-negative erm variants on the proportion of enzyme secreted in culture supernatants in the absence of pv infections. as shown in fig. e , inhibition of endogenous rdx by rdxt a strongly reduced the amount of gluc in the medium from both cell cultures. a lesser but significant inhibition of gluc secretion was also caused by dnmoe (moet a). in contrast dnez (ezt a) failed to impair secretion. these data strongly argue for the essential role played by the erm family proteins rdx and moe in the secretion of cellular proteins. the fact that dn mutant forms of these proteins inhibited in similar way pv egress gave further support to the involvement of vesicular transport in this process. a productive pv infection typically culminates in the rupture of the cell plasma membrane, as apparent from the generation of lysis plaques [ ] and the release of intracellular polypeptides into medium supernatants [ ] . this final lytic event clearly contributes to virus release and spreading, questioning the role of the vesicular egress of pv particles taking place prior to cell death. the above-mentioned impairment of progeny particle release upon down-modulation of vesicular transport (fig. d, d , and d) made us wonder whether the latter transport may not only contribute to pre-lytic virion release but also control the lytic process which follows. to this end, time-course experiments were performed with mvm infected a cells under conditions where viral egress and release were modulated as shown in previous experiments. permeabilization of the plasma membrane was then measured by propidium-iodide incorporation using fluorescent microscopy and image j software quantification. mock-treated a cells served as negative controls. in addition, the relation of the lytic activity observed to the presence/absence of pv egress was tested by performing experiments with a recombinant mvm vector (rmvm). this vector produces similar amounts of ns as the wild type virus in a single-round infection, yet it fails to produce capsid proteins, and as a result, does not lead to virus egress [ ] . therefore, infection of a cells with rmvm was used to measure the mere lytic activity induced upon ns expression. the results are summarized in fig. . mvm-induced cytolysis was strongly reduced when (i) the virus lacked the capacity for progeny particle production and in consequence transport to the pm (rmvm), or (ii) vesicular transport was either inhibited (dnsar , rdxdl[p], dnrab ) or by-passing the golgi apparatus (rdxy f). inhibition of particle loading (dngln, moet a) resulted in an intermediate phenotype as apparent from the delayed and significantly reduced pi incorporation. this was also the case of cells expressing dnrab , correlating with a reduction of virus release into the medium (see fig. c and d), presumably due to inhibition of the golgi-tgn-re pathway. in contrast, no or little effects on cell permeabilization were seen with dnrab , which did not impair egress of pvs ( fig. c and d) . altogether these results suggest that vesicular transport of progeny particles through the golgi complex plays an important role in the induction of cytolysis and to ensure the further release of progeny particles at the end of infection. progeny mvm particles become modified during egress through er and golgi, resulting in a gain of infectivity vesicular trafficking through the golgi apparatus raises the intriguing possibility of assembled pv particles undergoing maturation and gaining full infectivity in this compartment. this prompted us to first determine whether the egress process was accompanied with post-assembly phosphorylations of mvm capsids. unlike ns whose phosphorylation is targeted at serine and threonine residues [ ] , mvm (as aav ) progeny particles purified from infected cells were also phosphorylated at tyrosines (fig. a&b) . after metabolic p-labeling, these phosphorylations resolved into a complex pattern of seven distinct tryptic phosphopeptides in two-dimensional electrophoresis/chromatography analysis (fig. c) . to determine the impact of vesicular transport on mvm capsid phosphorylation, effector protein variants known to inhibit viral egress (ckiiae a [ ] , moet a, and rdxy f [see above]) were tested for their effects on the capsid tryptic phosphopeptide profile. as shown in fig. c , the inhibition of vesicular egress or its re-routing away from the golgi compartment, led to remarkable changes in the capsid phosphorylation pattern, in particular the characteristic loss of three distinct phosphopeptides (a, c, and e). these data provide strong evidence of parvoviral capsid modifications during egress through er and golgi. we next determined the impact of vesicular egress on viral infectivity. virus stocks were produced both in parental a cells, and in stable a -transfectants, which have been previously shown to suppress vesicular egress of progeny pv particles due to the expression of variant cellular proteins (glny a, ckiiae a [ ] , rdxy f, moet a [see above]). virus stocks were purified on cscl-gradients to remove empty capsids, matched for their ssdna content (fig. a ) and tested for their infectivity in a cells by counting both ns -positive cells (fig. b ) and lysis plaques (fig. c ) in infected cultures. progeny particles that had transited (at least in part) through the golgi complex in parental a cells proved to be significantly more infectious (lower particle (dna) to infectivity ratio) than their counterparts that had bypassed this compartment (rdxy f, moet a, glny a, ckii-e a). for the same amounts of viral genome, virus produced in the latter four cells gave rise to to times less ns -positive cells and up to fold fewer plaques than virus produced in vesicular egressproficient cells (a ). together with the above finding that egress through er and golgi is accompanied by capsid modifications, these results suggest that the transport pathway to the plasma membrane represents an important step in the maturation of progeny virions. progeny virion shuttling from the nucleus, the site of parvovirus replication and assembly, to the plasma membrane is an important step in virus release and cell to cell spread. the present analysis shows that the release of non-enveloped pvs is not a mere consequence of the cellular lytic burst occurring at the very end of the viral life-cycle but involves a pre-lytic active vesicular transport of virions. this transport makes virions transit through er and golgi, and appears to have two major implications: the maturation of progeny particles into fully infectious virions, and the induction of cytolysis with the ensuing virus spread. for this transport process, pvs usurp cellular components allowing progeny virion engulfment into copii vesicles at the er and moving through the golgi to the plasma membrane under control of small rab gtpases. this vesicular trafficking is a general feature of rodent parvoviruses as it was demonstrated for both, the mouse virus mvm and rat virus h- pv in mouse and human cancer cells, respectively. besides known cellular components of vesicular transport (sar , sec / , sec / ), the erm family proteins radixin and, to a minor extent, moesin were found to be involved in the formation of pv-loaded copii-vesicles. given their known mediator function between actin and membrane structures [ ] , we hypothesize that erm proteins may control the engulfment of viral cargo into membrane structures. in keeping with this possibility, we recently reported that progeny pv particles associate with actin fragments, and that the actin-severing protein gelsolin controls pv loading [ ] . interestingly, the present results indicate that, radixin and (at least in human cells) moesin are essential players not only for pv egress but for the secretion of gluc as well. this speaks for the fact that erm family proteins are not simply involved in pv virion sensing, but play a general role in the copii vesicle-mediated secretion. in contrast, the actin-processing protein gelsolin, whose knock-down had no impact on gluc secretion, seems to be recruited specifically to promote (parvo)virus egress and is apparently not required for cellular secretion in general. during their traffic from the nucleus to the pm, pvs appear to transit through the golgi compartment, as evidenced by biochemical fractionations and co-staining data. this is unexpected, since in contrast with enveloped viruses, pvs do not acquire, during this transit, a lipid envelope harboring proteins mediating cell attachment and/or entry. however, passage through the er-golgi pathway still proved to be important for pv maturation in that capsids gained post-assembly modification such as phosphorylations. these capsid modifications did not occur under conditions inhibiting the vesicular secretory pathway. mvm viruses lacking the golgi-associated capsid modifications were less efficient at infecting naïve cells as compared with viruses produced in cells with an intact secretory pathway. this is not without precedent for non-enveloped lytic viruses. indeed, tyrosine phosphorylations at the capsid surface of the defective parvovirus aav also seem to control the infectivity of this agent [ ] . furthermore, other viruses become processed after assembly. for instance, assembled polioviruses present in autophagosomes, were shown to mature upon acidification of these vesicles prior to their fusion with lysosomes. this acidification induces the cleavage of poliovirus capsid proteins, an essential step for these virions to gain full infectivity [ ] . interestingly, mvm and h- pv virions were shown to colocalize with the late endosomal/lysosomal marker lamp (see also below). this leads us to speculate that the maturation of these viruses may involve the induction of additional post-assembly capsid modifications in these compartments besides the above-mentioned golgi-associated phosphorylations. these observations raise the question how parvoviruses get sorted into the intracellular cisternae system. a number of enveloped viruses (e.g. filo-, retro-rhabdo-, arena-, paramyxo viruses) transit through multi vesicular bodies (mvb) during their transport to the pm. a common feature of these viruses consists in the presence in their structural proteins, of (a) short recognition motif(s) facilitating their interaction with cellular factors that participate in mvb sorting. in the case of hiv- , the ptap-motif of gag interacts with tsg , thereby inducing the recruitment of the escrt complex which constitutes the sorting machinery for incorporation of ubiquitylated proteins into mvbs. thus, this interaction promotes entry of viral particles into mvbs during hiv-infection [ ] [ ] [ ] [ ] . other motifs known to mediate vesicular uptake include ypx(n)l and py (ppxy), which bind to the cellular factors aip /alix and nedd ubiquitin ligase, respectively [ , ] . it is noteworthy that ppxy motifs are present in the parvovirus vp protein. together with the strong and weak colocalization of pv capsids with the late endosome markers lamp [ , ] , and rab (present study), respectively, this feature raises the possibility of late endosomes/mvbs playing a role in the transport of pvs from the golgi to the pm. besides contributing to virus intracellular trafficking, vesicular transport of progeny pv particles through er and golgi apparently serves additional purposes. importantly, this vesicular transit was found to result in post-assembly modification (in particular phosphorylation) of capsids, and in a marked increase in virion infectivity. furthermore, vesicular egress proved to have an impact on the still poorly understood mechanism of pvinduced cytolysis. the present results indicate that progeny virus transport through er and golgi up-regulates this process. this is apparent from the delay in or inhibition of infected cells lysis upon alteration of the virus egress route. the mechanism underlying this dependence of cytolysis on vesicular pv transport remains a matter of speculation. it may be traced back to the co-transport of cellular or viral products involved in cytolysis, which become engulfed into virion-containing vesicles, are brought to the pm and cause its permeabilization. besides cellular or viral phospholipases, the viral ns protein is an intriguing candidate for this process, since it appears to exert lytic activities upon (prolonged) ectopic expression [ ] and is known to be present in cellular vesicles [ , ] . at late stages of infection, the vesicular co-transport of cytolytic factors might not be required anymore since the cytoskeletal collapse [ , ] would allow direct access of cellular and viral lytic factors to the pm. this would explain the delayed cell lysis and small plaque phenotype observed after pv infection of cells deficient in er-golgi vesicular transport. as a whole, the present study revealed not only the subcellular pathway followed by progeny pvs to exit infected cells, but also the unexpected role played by this vesicular egress in the interconnected process of cytolysis, virus maturation and spread. the latter processes are of particular importance in the context of the current development of pvs as oncolytic agents for anti-cancer therapeutic applications [ ] . it is noteworthy in this regard that tumor cell killing by parvoviruses is immunogenic [ ] , resulting in an immune bystander effect that takes over from the initial direct oncolytic effect of the viral treatment to complete eradication [ ] . it will be of great interest to determine whether the hereby reported vesicular egress of parvovirus particles also contributes to the display and/or release of tumor-associated antigens and/or damage/pathogen-associated molecular patterns that stimulate the immune component of pv oncosuppression. primary antibodies. sec , sec , calnexin (abcam ab ), gm , rab a (sta. cruz biotechnologies sc ), rab (sta. cruz biotechnologies sc ), rab a (sta. cruz biotechnologies sc ), rab a (abcam ab ), radixin (sta. cruz biotechnologies sc ), moesin (sta. cruz biotechnologies sc ) antibodies as well as antibodies against flag-(m ) and myc-tag (sigma: f , m ), lamin b (sta. cruz biotechnologies: m- sc ), lamp (abcam ab ), sar (abcam ab ), sec (sta. cruz biotechnologies sc ); sec (sta. cruz biotechnologies sc ) and pkb (sta. cruz biotechnologies sc ). rabbit antiserum recognizing the mvm ns (ans c ) [ ] , antiserum recognizing vp (avp ) [ ] , and monoclonal anti-capsid antibody b [ ] were described previously. phosphotyrosine specific mouse monoclonal antibodies ( ca ) were kindly provided by prof. dr. angel alonso (german cancer research center). secondary iggs. horseradish-peroxidase-conjugated (hrpconjugated) anti-rabbit and anti-mouse iggs (promega), hrpconjugated anti-goat iggs (sta. cruz), alexa fluor fluorescent-dyelabeled igg (dianova and invitrogen). others: protein g sepharose beads (pharmacia amersham), [ p]-labeled a-dctp (perkin elmer), [ p]-orthophosphate (mp biomedicals). protein kinases. flag-tagged ckiiae a (dominant-negative) [ , ] . erm-family proteins. fl-ezt a (dominant-negative), fl-rdxt a (dominant-negative), fl-rdxt e (constitutiveactive), fl-rdxdl[p] (dominant-negative), fl-rdxy f (active), fl-moet a (dominant-negative) [ ] . gelsolin. fl-gln y a and fl-d n [ ] . site-directed mutagenesis was performed by single or chimeric pcr, cloned into pcr . vectors (invitrogen) and verified by sequencing [ ] . effector genes. dnsar was obtained by the substitution of lysine at position by a methionine using following primers: sar k m-f: ggataatgccgggatgacaactttgctacac and sar k m-r: gtgtagcaaagttgtcatcccggcattatcc. the dnrab mutant corresponds to the substitution of serine by an asparagine [ ] using following primers: rab s n-f: gttggaaagaactgccttctcc and rab s n-r: ggagaaggcagttctttccaac. the dnrab mutant corresponds to the substitution of serine by an asparagine [ ] using following primers: rab t n-f: ggggtggggaagaactgtgtgctg and rab t n-r: cagcacacagttctcccccacccc. the dnrab mutant corresponds to the substitution of serine by an asparagine [ ] using following primers: rab s n-f: ggtgttggaaagaataatctcctg and rab s n-r: gagattattctttccaacacc. all four variants were cloned in order to express an nterminal myc tag using an n-terminal primer harboring the respective sequence in frame with the original start codon. (fig. s a) . production of expression constructs for generating stably transfected cell lines. mvm ns -inducible expression vectors were constructed from plasmid paav :pp -gfp, where myc/ flag-tagged protein variants were transferred from pcr . vectors, replacing the gfp reporter gene [ ] . raav :p -x and raav :(pa)p -x constructs. paav:p -gfp contains the gpf-gene under the control of the mvm flanked by multiple cloning sites. this allows easy replacement with candidate gene-sequences [ncoi,pmei,xbai,eco iii]-gfp-[ecorv,hindiii,xhoi,stui,-noti]. paav :(pa)p -gfp contains the same gfp cassette under the control of the ns -inducible (h -pv) p promoter. potential promoter activity through the left-end itr was blocked by insertion of the mvm poly(a) sequence at h -ns position nt . it was constructed as follows: ptrh -gfp [ ] was first cleaved with pmei/bst i, ligated. the pmei/not cleaved gfp cassette was then inserted into the stui/noti cleaved p -less ptrh -gfp. finally the pcr-generated mvm poly(a) sequence was inserted into the nhei-cleaved construct and the correct orientation was checked by a bamhi-digest due to the presence of a new bamhi-site at the left-end of the poly(a) sequence. effector genes, cut blunt-end (n-terminus) and noti (c-terminus) were then inserted into the eco iii-and noti-cleaved paav :p -x or paav :p -x vectors, respectively, to generate the corresponding paav-p /p -x constructs. reporter construct pcmv-gluc. the gaussia luciferase expressing construct under the control of the cmv promoter was generated by transferring the gluc gene from the pgluc-basic vector (new england biolabs) as a pcr-derived ecori-fragment into similarly cleaved pcr . (invitrogen). all cell lines were maintained as monolayers in dmem containing % fcs. stable transfectants were generated with pp -x and the selection plasmid psv neo or ptk-hyg at the molar ratio of : . colonies were pooled after growth under selection and frozen stocks prepared. experiments were performed in the absence of drugs [ ] . mvm was propagated in a cells. human-glioma propagation-competent hgh -pv strain was obtained after serial passaging in nch cells and propagated in nb k cells [ ] . to produce virus stocks under conditions lacking vesicular egress through er and golgi, a derivative cell lines a :p -fl-glnd n [ ] , a :p -ckii-e a [ , ] , a :p -moet a, and a :p -rdxy f [ ] , respectively were infected with , pfu/cell mvmp and harvested upon detection of severe cpe. all virus stocks were purified after freezing and thawing over cscl density gradient [ ] and quantified by their amount of single-stranded dna by southern blotting and standard plaque assays. pv-infection was limited to a single round by addition of either . u/ml neuraminidase (sigma) to remove sialic acid from the cell receptor / dilution of neutralizing antibody b as from h after the initial infection (supplement s )o recombinant adeno-associated viruses (raavs) were generated in t cells in absence of ad by co-transfection with paav-p -x and pdg, purified by iodixanol step gradients and the genomic titers determined by dot blot hybridization [ ] . transduction efficiencies were determined by immunofluorescence microscopy measuring the proportion of trans-gene expressing cells. toxicity was assessed by measuring metabolic activity through mitotracker incorporation, and cell lysis through pi-staining (supplement fig. s a, b) . determination of infectious titers by standard plaque assays [ , ] to determine the presence of infectious particles monolayer cultures (a for mvmp, nb k for hgh -pv) were seeded at a concentration of cells per mm dish, infected h later and covered with a bacto-agar overlay. after incubation for days, cultures were stained for h by addition of neutral-red containing bacto-agar. stained cells were fixed on the plates with formaldehyde after removing the agar overlay. detection and quantification of viral parvoviral dna by southern blot or dot blot hybridization accumulation of parvoviral dna species (monomer/dimer replication intermediates and single-stranded virion dna) were determined by southern blotting [ ] , total viral dnas by dot blot hybridization. at the indicated times p.i., medium was removed and kept separately. adherent cells were washed, harvested in dmem without serum by scraping from the dish, and collected by centrifugation. medium-and cell-associated virions were quantified, after repeated freezing and thawing, in standard plaque assays (see above) or after proteinase k digest by southern and/or dot blot hybridization: cell associated viral dnas were harvested in vte, digested with proteinase k and total dna was shared through syringe. to determine the amount of virion dna in purified virus stocks serial dilutions were performed, medium associated viral dnas were harvested and adjusted to ''vteconditions'' and digested by proteinase k. to detect different viral dna species, samples were analyzed by agarose gel electrophoresis and transferred onto nitrocellulose membranes. total viral dna was transferred to nitrocellulose using a dot-blot apparatus. viral dna was detected by hybridization with a p-labeled probe corresponding to nts - of the ns -encoding region of mvm dna and when indicated quantified after autoradiography with gelquant.net software. western blot analyses [ ] cellular extracts were produced in co-ip buffer and cleared by centrifugation. protein extracts were then fractionated by discontinuous sds-page and blotted onto nitrocellulose membranes. blocking was performed in % dry milk/pbs or for phosphospecific antibodies in % casein, mm tris ph . , mm nacl, mm edta, . % triton x- for h. staining with horseradish-peroxidase-conjugated secondary antibodies for h followed by chemiluminescence detection (amersham). immunofluorescence microscopy [ ] cells were grown on spot slides (roth). cultures were fixed with % paraformaldehyde and permeabilized with . % triton x- . specimens were pre-adsorbed with % fcs, incubated with primary antibodies, and stained with specific alexa fluor or/ and conjugated anti-species antibodies. dapi ( mg/ml) was added to the secondary antibody solutions. analyses were performed with a leica dmirbe confocal microscope ( lens, laser: red nm, green nm) and powerscan software or with an olympus fluoview fv confocal microscope ( lens, laser: green , red nm, far red: nm) presenting a single slice of a stack. quantitative analyses and mean colocalizations were calculated with imagej software. for pi staining, cells were incubated with propidium iodide ( mg/ml) for min at uc. pictures of living cells were taken on an inverted leicadfc fx microscope using transmitted and fluorescent light, pi positive and the total number of cells counted using imagej and the proportion of pi positive cells calculated. separation of nuclear, mitochondrial, and vesicular fractions from the soluble cytosol [ ] . nuclear components were obtained by pelleting at g and further purification through m sucrose. the -g supernatant was centrifuged at g to pellet large organelles like mitochondria in a ''heavy mitochondrial fraction'' (hmf), and the supernatant was centrifuged at g to pellet smaller organelles, including vesicles, in a ''light mitochondrial fraction'' (lmf). the final supernatant represented the soluble cytosolic fraction. the lmf-fraction was used to determine association of newly synthesized virions with vesicles. the lmf pellet suspended in hypotonic buffer was added to % iodixanol/ mm sucrose ( : v/v), and the components were separated according to their density by centrifugation for h at uc in a self-forming gradient in a vertical rotor at , g. fractions were collected from the top, volume-matched with the nuclear, hmf, and cytosolic fractions, and analyzed individually by southern and western blotting. separation of endoplasmic reticulum (er) and golgi membranes. separation of er and golgi proteins was essentially achieved as previously published [ ] . a cells were washed, scraped into ice-cold pbs and centrifuged at g for min at uc. the cell pellet was suspended in ml homogenization buffer ( mm tris ph . , mm nacl, mm kcl, mm edta) and passed multiple times consecutively through a syringe ( g and g ). nuclei were then separated by centrifugation at g for min and discarded. the remaining supernatant was adjusted to ml and centrifuged through a nycodenz step-gradient ( ml %, . ml %, . ml %, . ml %, . ml . %, . ml %, . ml . % (w/v nycodenz in homogenization buffer) at , g for min using a sw rotor (beckman instruments). nine fractions of . ml were collected from the top and analyzed by southern and western blotting respectively. metabolic labeling, purification, phosphopeptide analyses and phosphor amino acid analyses [ , ] a cell cultures were subjected to labeling medium for h ( . nci/cell of [ p]. labeled cells were harvested and the respective proteins isolated by immunoprecipitations and purified by sds-page. p-labeled proteins blotted on pdf-membranes were revealed by autoradiography, excised and digested trypsin. tryptic peptides were analyzed on thin-layer cellulose plates (merck) in two dimensions, first by electrophoresis in a ph . buffer, and then by chromatography in phosphochromatography buffer. phospho amino acid analyses were performed with excised p-labeled protein bands. individual labeled amino acids were obtained by hydrolization in m hcl at uc for h and phosphoserine, phosphothreonine, and phosphotyrosines were separated by thin-layer electrophoreses in two-dimension using ph . and ph . buffers, respectively. figure s a cells grown on spot slides were infected (mvm h) or not (a ) with mvmp ( pfu/cell) and further incubated under conditions neutralizing progeny particles released in the medium. cells were then fixed with paraformaldehyde h p.i., and analyzed by confocal laser scanning microscopy after double-staining using specific antisera for the indicated cell proteins (red) and mvm capsids (green). colocalization areas appear yellow in the merge and are quantified by image j analyzing infected cells from three individual experiments. scale bar mm. infection. a cells grown on spot-slides were transfected with control serum (apkb) or neutralizing anti-sec antibodies (asec ) at mg/ cells. twenty-four hours post-transfection, cells were infected with mvm, washed extensively after h to remove the inoculum, further incubated for h and fixed with paraformaldehyde. transfected antibodies were detected with alexa-conjugated secondary igg and counterstained with dapi to determine the proportion of transfected cells. the amount of infected cells was determined by means of ns expression. scale bar mm. (pptx) assembly and maturation of the flavivirus kunjin virus appear to occur in the rough endoplasmic reticulum and along the secretory pathway, respectively molecular determinants and dynamics of hepatitis c virus secretion herpesvirus assembly and egress characterization of the budding compartment of mouse hepatitis virus: evidence that transport from the rer to the golgi complex requires only one vesicular transport step viral and host control of cytomegalovirus maturation hepatitis b virus maturation is sensitive to functional inhibition of escrt-iii, vps , and gamma -adaptin roles for the recycling endosome, rab , and rab in hantavirus release from epithelial cells structural maturation of the transmissible gastroenteritis coronavirus polymorphism and structural maturation of bunyamwera virus in golgi and post-golgi compartments silencing the morphogenesis of rotavirus herpesviruses remodel host membranes for virus egress structure of the hemagglutinin precursor cleavage site, a determinant of influenza pathogenicity and the origin of the labile conformation proproteinprocessing endoproteases pc and furin both activate hemagglutinin of virulent avian influenza viruses intracellular processing of the gp hiv- envelope precursor. endoproteolytic cleavage occurs in a cis or medial compartment of the golgi complex replication of the parvovirus mvm. i. dependence of virus multiplication and plaque formation on cell growth the autonomously replicating parvoviruses of vertebrates modulation of minute virus of mice cytotoxic activities through site-directed mutagenesis within the ns coding region release of simian virus virions from epithelial cells is polarized and occurs without cell lysis coxsackievirus b transmission and possible new roles for extracellular vesicles vesicular egress of nonenveloped lytic parvoviruses depends on gelsolin functioning targeting rab gtpases to distinct membrane compartments role of rab gtpases in membrane traffic and cell physiology role of sec isoforms in selective export of membrane proteins from the endoplasmic reticulum the mechanisms of vesicle budding and fusion copii-cargo interactions direct protein sorting into er-derived transport vesicles protein sorting receptors in the early secretory pathway dynamin ii regulates hormone secretion in neuroendocrine cells bridging membrane and cytoskeleton dynamics in the secretory and endocytic pathways coordination of copii vesicle trafficking by sec the copii cage: unifying principles of vesicle coat assembly imaging constitutive exocytosis with total internal reflection fluorescence microscopy fusion of constitutive membrane traffic with the cell surface observed by evanescent wave microscopy selective alterations of the host cell architecture upon infection with parvovirus minute virus of mice molecular pathways: rodent parvoviruses-mechanisms of oncolysis and prospects for clinical cancer treatment regulation of non-structural protein functions by differential synthesis, modifications and trafficking ns interaction with ckii alpha: novel protein complex mediating parvovirus-induced cytotoxicity a viral adaptor protein modulating casein kinase ii activity induces cytopathic effects in permissive cells ezrin-radixin-moesin family proteins are involved in parvovirus replication and spreading parvovirus interference with intracellular signalling: mechanism of pkceta activation in mvm-infected a fibroblasts erm proteins: head-to-tail regulation of actinplasma membrane interaction cytosolic activation of cathepsins mediates parvovirus h- -induced killing of cisplatin and trail-resistant glioma cells a gdpbound of rab inhibits protein export from the endoplasmic reticulum and transport between golgi compartments mutant rab impairs docking and fusion of rhodopsin-bearing post-golgi membranes and causes cell death of transgenic xenopus rods rab is required for trans-golgi network-to-plasma membrane transport and a preferential target for gdp dissociation inhibitor a highly sensitive assay for monitoring the secretory pathway and er stress moesin regulates the trafficking of nascent clathrin-coated vesicles identification of the two major epidermal growth factor-induced tyrosine phosphorylation sites in the microvillar core protein ezrin cis requirements for the efficient production of recombinant dna vectors based on autonomous parvoviruses phosphorylation of the viral nonstructural protein ns during mvmp infection of a cells tyrosine-phosphorylation of aav vectors and its consequences on viral intracellular trafficking and transgene expression intracellular vesicle acidification promotes maturation of infectious poliovirus particles structure and functional interactions of the tsg uev domain hiv gag mimics the tsg -recruiting activity of the human hrs protein tsg and the vacuolar protein sorting pathway are essential for hiv- budding ubiquitin-dependent sorting into the multivesicular body pathway requires the function of a conserved endosomal protein sorting complex retrovirus budding retrovirus budding biosynthetic transport of a major lysosome-associated membrane glycoprotein , lamp- : a significant fraction of newly synthesized lamp- is delivered to lysosomes by way of early endosomes through its nonstructural protein ns , parvovirus h- induces apoptosis via accumulation of reactive oxygen species phase i/iia study of intratumoral/intracerebral or intravenous/intracerebral administration of parvovirus h- (parvoryx) in patients with progressive primary or recurrent glioblastoma multiforme: parvoryx protocol parvoviruses-tools to fine-tune anticancer immune responses immune cells participate in the oncosuppressive activity of parvovirus h- pv and are activated as a result of their abortive infection with this agent the ns proteins of parvovirus minute virus of mice are required for efficient nuclear egress of progeny virions in mouse cells novel pkceta is required to activate replicative functions of the major nonstructural protein ns of minute virus of mice augmented transgene expression in transformed cells using a parvoviral hybrid vector intranasal vaccination with recombinant adeno-associated virus type against human papillomavirus type l cabp , a calcium binding protein of the thioredoxin family, is a resident kdel protein of the er and not of the intermediate compartment biochemical activities of minute virus of mice nonstructural protein ns are modulated in vitro by the phosphorylation state of the polypeptide special thanks are due to claudia plotzky for excellent technical assistance. we are also grateful to michèle vogel and drs nathalie salome, rainer pepperkok, britta brügger and angel alonso for providing us with antisera as well as helpful comments. confocal microscopy on the olympus microscope was performed at the light microscopy facility of the dkfz (inf , d- heidelberg). key: cord- -tsvzg ax authors: fensterl, volker; wetzel, jaime l.; ramachandran, srividya; ogino, tomoaki; stohlman, stephen a.; bergmann, cornelia c.; diamond, michael s.; virgin, herbert w.; sen, ganes c. title: interferon-induced ifit /isg protects mice from lethal vsv neuropathogenesis date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: tsvzg ax interferon protects mice from vesicular stomatitis virus (vsv) infection and pathogenesis; however, it is not known which of the numerous interferon-stimulated genes (isg) mediate the antiviral effect. a prominent family of isgs is the interferon-induced with tetratricopeptide repeats (ifit) genes comprising three members in mice, ifit /isg , ifit /isg and ifit /isg . intranasal infection with a low dose of vsv is not lethal to wild-type mice and all three ifit genes are induced in the central nervous system of the infected mice. we tested their potential contributions to the observed protection of wild-type mice from vsv pathogenesis, by taking advantage of the newly generated knockout mice lacking either ifit or ifit . we observed that in ifit knockout (ifit (−/−)) mice, intranasal vsv infection was uniformly lethal and death was preceded by neurological signs, such as ataxia and hind limb paralysis. in contrast, wild-type and ifit (−/−) mice were highly protected and survived without developing such disease. however, when vsv was injected intracranially, virus replication and survival were not significantly different between wild-type and ifit (−/−) mice. when administered intranasally, vsv entered the central nervous system through the olfactory bulbs, where it replicated equivalently in wild-type and ifit (−/−) mice and induced interferon-β. however, as the infection spread to other regions of the brain, vsv titers rose several hundred folds higher in ifit (−/−) mice as compared to wild-type mice. this was not caused by a broadened cell tropism in the brains of ifit (−/−) mice, where vsv still replicated selectively in neurons. surprisingly, this advantage for vsv replication in the brains of ifit (−/−) mice was not observed in other organs, such as lung and liver. pathogenesis by another neurotropic rna virus, encephalomyocarditis virus, was not enhanced in the brains of ifit (−/−) mice. our study provides a clear demonstration of tissue-, virus- and isg-specific antiviral action of interferon. virus infection of mammals induces the synthesis of type i interferons (ifn), which, in turn, inhibit virus replication. the high susceptibility of type i ifn receptor knockout (ifnar / ) mice to infection by a variety of viruses [ ] [ ] [ ] provides strong evidence for the major role of the ifn system in protecting from viral pathogenesis. in these mice, although ifn is induced by virus infection, it cannot act on target cells. similarly, in genetically altered mice that are defective in ifn production due to the absence of specific pathogen-associated pattern recognition receptors, signaling proteins or specific transcription factors, viral pathogenesis is enhanced [ ] [ ] [ ] . although the critical importance of the ifn system in regulating viral pathogenesis is now well established, in many cases it is still unclear how ifn inhibits the replication and spread of a specific virus in vivo. in this context, activation of different components of the immune system plays a major role in controlling viral diseases that are relatively slow to develop [ ] [ ] [ ] . in contrast, in acute infection by viruses that cause severe pathogenesis and death within a few days after infection, protection is primarily provided by the intrinsic antiviral actions of ifn-induced proteins encoded by the hundreds of ifn-stimulated genes (isgs) [ ] [ ] [ ] , several of which often contribute to the overall effect of ifn against a given virus. our knowledge of the antiviral and the biochemical properties of individual isg products is mostly limited to a few intensively studied examples such as pkr, oas/rnase l or mx [ ] . however, recent systematic investigation of the antiviral functions of the entire family of isgs has started producing exciting new information [ ] . in the above context, we have been investigating the biochemical and biological functions of the members of the ifit family of isgs, which are very strongly induced by ifn. there are three members of this family of genes in mice: ifit /isg , ifit / isg and ifit /isg ; all of the encoded proteins contain multiple tetratricopeptide repeats (tpr), which mediate proteinprotein and protein-rna interactions [ ] . in vitro, p and p , the products of ifit and ifit , respectively, bind to the translation initiation factor eif and inhibit protein synthesis [ ] . the third member, p , the product of ifit , does not share this property [ ] . recently, it has been reported that ifit proteins form a multiprotein complex that can bind to the triphosphorylated end of rnas, an rna-species produced during the replication of some, but not all, viruses [ ] . in vivo, these genes are strongly induced in brains of mice infected with west nile virus (wnv) or lymphocytic choriomeningitis virus (lcmv); surprisingly, different ifit genes are differentially induced in different regions of the brain, suggesting non-redundant functions [ ] . to further explore the antiviral properties of the ifit proteins, we generated ifit knockout (ifit / ) mice and challenged them with different viruses. we observed that ifit / mice were particularly susceptible to a wnv mutant that is defective in its mrna cap -o methylation; the mutant virus killed ifit / mice but not the wild-type (wt) mice [ ] . here, we report on the antiviral properties of the newly generated ifit / mice; these mice, but not ifit / mice, were highly susceptible to neuropathogenesis after intranasal infection with vesicular stomatitis virus (vsv), a negative sense, singlestranded rna rhabdovirus. vsv replication is highly sensitive to the inhibitory action of ifn and is routinely used to assay the antiviral activity of ifn in vitro [ ] . as expected, ifnar / mice are highly susceptible to vsv pathogenesis and the same is true for mice that specifically lack expression of ifnar on the cells of their central nervous system (cns) [ ] . in spite of these observations, little is known about how ifn inhibits vsv replication in vivo. our new results indicate that in the brain, but not in other organs, ifit is a major mediator of ifn's protective effect against vsv. in contrast, ifit could not protect mice from neuropathogenesis caused by encephalomyocarditis virus (emcv), a picornavirus. thus, we have uncovered a virusspecific, tissue-specific and isg-specific antiviral effect of the ifn system. generation of ifit /isg and ifit /isg knockout mice ifit gene knockout (ifit / ) mice were generated by deleting the entire protein-encoding region of the gene, which was achieved by flanking exons and with frt recombinase sites in c bl/ embryonic stem cells and excising the flanked region with flp recombinase ( figure a ). ifit / mice were bred to homozygosity ( figure b) , and deficiency for induced expression of ifit protein was confirmed in lysates of ifn-b-treated primary murine embryonic fibroblasts (mef) ( figure c ). mice deficient for ifit (ifit / ) were derived from c bl/ embryonic stem cells lacking the entire ifit coding region ( figure a ). genotypic homozygosity of the ifit / mice and deficiency for ifit protein induction were confirmed ( figure b and c ). both knockout mouse lines were healthy and fertile. moreover, deletion of one gene within the ifit locus did not alter the pattern of induction of other adjacent gene family members, as compared to wild-type (wt) mice ( figure c ). to determine the impact of ifit on the outcome of viral infections in vivo, we compared susceptibilities of ifit / and wt mice to vsv infection, using ifnar / mice as positive controls of enhanced susceptibility. virus was administered at a low dose [ plaque forming units (pfu)], intranasally, reflecting a natural route of infection for vsv [ ] . as seen previously, % of ifnar / mice rapidly succumbed to vsv infection within days ( figure a , and [ ] ), after suffering symptoms of lethargy. on the other hand, % of wt mice survived, the remaining % succumbed to vsv, and this occurred later, at - days post infection (d.p.i.). in contrast, % of ifit / mice died by d.p.i. (figure a ), with most succumbing by d.p.i.; thus, we observed uniform and more rapidly occurring death of ifit / compared to wt mice after vsv infection. within h before death, both wt and ifit / mice developed neurological signs including ataxia, hind limb paralysis, and hyper-excitability. ifit +/ mice displayed an intermediate survival curve, demonstrating a gene dosage effect ( figure b ). next, the role of a related gene, ifit , in vsv pathogenesis was evaluated by infecting ifit / mice. unlike the results observed with ifit / mice, no statistically significant increase in mortality was observed in ifit / mice ( figure b , % death for wt versus % for ifit / , respectively; p. . ). consistent with this, survival kinetics of ifit / and wt mice were similar. increasing the virus dose by , -fold (to pfu) did not appreciably change the survival curves of wt, ifit / , or ifit / mice ( figure c ). these results demonstrate functional differences between the two closely related proteins encoded by ifit and ifit . the virus-specificity of the antiviral action of ifit was evaluated by infecting ifit / mice with emcv, an unrelated neurovirulent positive-strand rna virus of the picornavirus family ( figure d ). ifnar / mice were highly susceptible to emcv infection with all mice succumbing within d.p.i.; in contrast, wt mice died with a slower kinetics and at a rate of only %. notably, ifit / mice behaved similarly to the wt mice, without enhanced or accelerated mortality ( figure d ). the same conclusion was true for a lower dose of emcv ( figure s ). the survival pattern of emcv-infected ifit / mice also was similar to that of the wt mice ( figure d ). mice of all genotypes either succumbed after developing neurological symptoms, mainly hind limb paralysis, or survived without symptoms. these results demonstrate that the antiviral action of ifit is both virus-and ifitspecific. the uniform penetrance of neuropathogenesis and lethality of vsv-infected ifit / mice, even at a low virus dose, prompted us to examine viral spread along its route from the nasal cavity into the cns ( figure a ). after intranasal administration, vsv infects in mammals, the first line of defense against virus infection is the interferon system. viruses induce synthesis of interferon in the infected cells and its secretion to circulation. interferon acts upon the as yet uninfected cells and protects them from oncoming infection by inducing the synthesis of hundreds of new proteins, many of which interfere with virus replication. vesicular stomatitis virus (vsv), a virus similar to rabies virus, is very sensitive to interferon but it is not known which interferon-induced protein inhibits its replication. here, we have identified a single interferon-induced protein as the protector of mice from death by vsv infection. knocking out the gene encoding this protein, ifit , made mice very vulnerable to neuropathogenesis caused by vsv infection; a related protein, ifit , did not share this property. moreover, ifit failed to protect mice from another neurotropic virus, encephalomyocarditis virus, nor was it necessary for protecting organs other than brain from infection by vsv. our observation that a single ifn-induced protein protects a specific organ from infection by a specific virus revealed an unexpected degree of specificity of the antiviral action of ifn. shared wt mice (n = number of animals used). c, survival of ifit / , ifit / and wt mice after intranasal infection with a higher dose of vsv ( pfu). d, survival of ifit / , ifit / , ifnar / and wt mice after infection with pfu of emcv. in a-d, data are cumulative from at least two independent experiments (exceptions: figure b , ifit +/ mice and figure d , ifit / mice infected in a single experiment). statistical significance of survival differences relative to wt mice is indicated by p-values; n.s., not significant; i.n., intranasal. doi: . /journal.ppat. .g the nasal epithelia including olfactory sensor neurons, which project to the outer layer of the olfactory bulbs (ob) [ ] . this represents the entry step into the cns, which we examined by immunostaining of ob sections. in wt mice, vsv p protein was detected exclusively within the glomeruli of the ob at d.p.i. ( figure b , upper right panel and [ ] ), whereas in ifnar / mice, vsv antigen had spread into deeper layers of the ob ( figure b , lower left panel). in ifit / mice ob, viral antigen was restricted to the glomeruli, as seen in wt mice ( figure b , lower right panel). this similar pattern of viral antigen expression between wt and ifit / mice was reflected in the equivalent levels of viral rna in ob at d.p.i. ( figure c ). in contrast, , times more vsv rna was present in ob of ifnar / mice ( figure c , right panel, p, . ). a comparison of the infectious viral burden between wt and ifit / mice in the ob confirmed these findings: at d.p.i., , pfu/g of vsv was present in both wt and ifit / mice ( figure d , p = . ). however, later in the course of infection, by day , viral ob titers in ifit / mice were not significantly changed, whereas in wt mice average titers of infectious vsv as well as viral rna levels had decreased by , -fold ( figure c and d, both p, . ). these results suggest that vsv initially enters and replicates with similar efficiency in both wt and ifit / ob before spreading into the rest of the brain. the efficiency of vsv replication in the brain, excluding the ob, was examined by quantifying infectious vsv as well as viral rna. early after infection, at d.p.i., virus titers in brains were low (, to pfu/g) and roughly equivalent in wt and ifit / mice ( figure a , p. . ). similarly, viral rna levels at the same figure . ifit does not inhibit vsv entry and replication in olfactory bulbs. a, schematic entry route of vsv into the central nervous system of wt mice after intranasal infection, and vsv spread within brain, as reported in the literature. ob, olfactory bulbs; cx, cortex; mb, midbrain; cb, cerebellum; bs, brain stem; sc, spinal cord. b, vsv p protein in ob of vsv-infected wt, ifit / and ifnar / mice at d.p.i., detected by immunohistofluorescence. c, vsv rna levels in ob of uninfected or vsv-infected wt, ifit / and ifnar / mice at , or d.p.i., plotted as mean+sd on log scale; nd, none detected. d, infectious vsv titers in wt and ifit / ob at and d.p.i.; plotted as pfu/g with mean on log scale; dashed line depicts threshold of detection. in c and d, n = - mice per infected group accumulated from three independent experiments; in b, n = mice from two independent experiments. all infections were pfu of vsv administered intranasally. asterisks indicate statistical significance: ** p = . , * p, . ; n.s.: not significant. doi: . /journal.ppat. .g time were low and comparable between wt and ifit / ( figure b , p. . ). however, at the same time, levels of vsv rna ( -fold, p, . ) were much higher in the brains of ifnar / mice ( figure b , right panel). later in the course of infection ( d.p.i.), brains of wt mice accumulated only , -fold more infectious vsv, with occasional clearance of the virus. in contrast, we detected markedly higher vsv titers in the brains of ifit / mice (, -fold higher compared to wt mice, p = . ), reaching , pfu/g ( figure a ); the high virus load likely caused the pronounced lethality. differences in viral rna levels in brains of wt and ifit / mice at d.p.i. correlated well with levels of infectious vsv ( figure b ). to determine whether ifit selectively restricts replication of vsv in particular regions of the brain, we measured viral rna levels in cortex, midbrain, cerebellum and brain stem at d.p.i. in wt mice, vsv rna was present prominently in the cortex, midbrain and brainstem, but not in the cerebellum ( figure c ), which is consistent with published results [ ] . however, in ifit / mice, viral rna was -fold or more (p, . ) abundant in all regions of the brain examined, including the cerebellum. the increase of vsv replication in ifit / brains was not due to a broadened cell tropism of the virus; immunostaining for viral p protein showed exclusive localization to neurons and not other cell types, such as astrocytes ( figure d ). from the above observations, we conclude that after intranasal infection by vsv, ifit protects mice from neuropathogenesis by suppressing replication or spread of the virus in brain neurons. ifit and ifit are induced in vsv-infected regions of ob and brain the protective effect of type i ifn signaling and in particular, ifit , against vsv neuropathogenesis prompted us to confirm its expression in ob and brain of wt mice, and whether it was induced in a type i ifn-dependent manner. in wt ob, ifit , ifit , and ifn-b mrna was induced strongly by d.p.i., and ifit and ifit rna remained abundant until day d.p.i. (figure a ). the induction of these genes was dependent on type i ifn receptor in ob as well as in brain ( figure b and e, and data not shown). ifit suppresses vsv replication in the brain after intranasal infection. a, infectious vsv titers in wt and ifit / brains at and days after intranasal infection, plotted as pfu/g with mean on log scale; dashed line depicts threshold of detection. b, vsv rna levels in brains of uninfected or vsv-infected wt, ifit / and ifnar / mice at or d.p.i., plotted as mean+sd on log scale. c, vsv rna levels in different regions of the brains of uninfected or vsv-infected wt and ifit / mice at d.p.i., plotted as mean+sd on log scale. d, vsv p protein in midbrain neurons of ifit / mice at d.p.i.; detection by immunohistofluorescence-labeling of vsv-p (red) and neuron (neun) or astrocyte (gfap) markers (green); in a and b: n = - mice per infected group accumulated from three independent experiments; in c: n = mice per infected group; in d: n = mice per infected group; all infections in a-d were intranasal with pfu of vsv. nd, none detected. brains in a and b were separated from obs assayed in figure d and c, respectively. asterisks indicate statistical significance: *** p# . ; n.s.: not significant. doi: . /journal.ppat. .g furthermore, expression of ifit mrna in wt ob coincided with the presence of detectable levels of the encoded ifit protein ( = p ) at d.p.i. and d.p.i., as seen by immunohistochemistry ( figure c , and data not shown). ifit protein staining was observed in vsv-infected cells within ob glomeruli as well as in surrounding and distant viral antigen-free cells, consistent with a remote ifn-dependent induction of ifit expression ( figure c , arrowheads in magnified images of right panel). ifit and ifn-b mrnas were induced as strongly in ob of ifit / as in wt mice, which correlated well with similar abundance of vsv rna in wt and ifit / ob (figure a compared to figure c ). in brains, at d.p.i., in contrast to ob, induction of ifit and ifn-b mrnas was considerably stronger in ifit / mice compared to wt mice ( figure d , -fold and -fold, respectively, both p, . ). the enhanced gene induction in vsv-infected ifit / mice was not restricted to specific regions of the brain ( figure s ). enhanced cellular gene expression also was observed for several virusinduced cytokine and chemokine genes, as measured by quantitative rt-pcr ( figure s a ). gene expression profiling of brain tissue at day d.p.i., using microarray analysis, revealed that many other genes, including isgs, were also more strongly induced (table s ). these results demonstrated that enhanced virus replication in the brains of ifit / mice led to enhanced type i ifn, other cytokines and isg induction, which nevertheless failed to restrict vsv replication in the absence of ifit . wt mice are as susceptible as ifit / mice to intracranial vsv infection our results from intranasal vsv infection indicated that ifit induction in the brain was mediated by type i ifn that was, in all likelihood, produced by infected cells in the ob ( figure a ). virus replication and resultant ifn induction at d.p.i. were similar in the obs of wt and ifit / mice (figs. c, d and a); presumably, the newly produced ifn diffused into the rest of the brain and induced local ifit expression in the wt mouse brains, prior to the arrival of the infectious virus. if this were the case, one would anticipate that direct infection of the brain, without prior action of ifn produced in infected ob, would minimize the difference between the phenotypes of wt and ifit / mice. to test this idea, we injected a very low dose ( pfu) of vsv intracranially. as hypothesized, wt and ifit / mice were now equally susceptible; almost all mice died by d.p.i. even at this low dose ( figure a ) and there were equally high virus titers and viral rna levels in the brains of mice of both genotypes ( figure b and c). concomitant with virus replication, there was similar induction of ifit and ifn-b ( figure c ) and other cytokines and chemokines ( figure s b ). these results indicate that in the absence of prior induction of ifit by ifn, brain neurons are highly susceptible to vsv infection. unlike the brain, other organs of ifit / mice are not more susceptible to intranasal vsv infection ifnar / mice succumbed within two days after vsv infection without accumulating very high vsv rna levels in the brain ( figure b ). these mice did not develop cns-related signs of disease, but showed severe lethargy before death, suggesting that death was due to efficient replication of the virus in peripheral organs, due to the absence of an otherwise effective type i ifn-mediated antiviral protection of the same organs in wt mice. to test this, we assessed the kinetics of vsv accumulation in brains, livers and lungs of wt, ifnar / and ifit / mice (figure ) . at d.p.i., vsv titers were very high in the liver of ifnar / mice, reaching pfu/g ( figure a ). in contrast, no or little infectious virus was detected in the liver of wt mice at or d.p.i., indicating efficient ifn-dependent suppression of vsv replication; intriguingly, this was also observed in ifit / mice, demonstrating that ifit did not mediate the anti-vsv effects of type i ifn in the liver. in lungs, which directly received a part of the virus inoculum from intranasal inhalation of vsv, the virus also replicated efficiently in ifnar / mice, reaching pfu/g before death ( figure b ). in comparison, lungs of wt and ifit / mice exhibited much lower levels of vsv at and d.p.i. ( , to , -fold lower for wt and ifit / compared to ifnar / mice, all p, . ). by days and d.p.i., the virus was cleared from the lungs of a subset of wt and ifit / mice. in contrast, in brains from the same animals, to -fold higher average titers (p, . ) of vsv accumulated in ifit / compared to wt mice at all time points between and d.p.i. ( figure c ). as expected, in wt mice, both ifit and ifit were induced not only in brains ( figure d ), but also in livers ( figure d ) and lungs ( figure e ); ifn-b was also induced in lungs, but not livers. ifit , ifit and ifn-b mrnas were also induced in the brains of emcv-infected wt mice ( figure s c ). these findings demonstrate an unexpected brain-restricted and virus-restricted function of ifit in the context of the type i ifn-mediated antiviral response to vsv infection. they also indicate that in ifit / mice, other isgs, which presumably protect the peripheral organs of vsv-infected wt mice, are either not induced in neurons or insufficient to protect them. ifns are defined by their antiviral activities. they inhibit the replication of many, if not all, viruses mostly by direct inhibition of replication in the infected cells but also by promoting the ability of immune cells to recognize and eliminate the virus-infected cells [ ] . the direct effects are mediated by isgs, which number in the hundreds, and different isgs are thought to have more potent antiviral activities toward different families of viruses [ ] . however, in most cases, it is not known which isg inhibits the replication of a given virus; the rare exception is the mx-mediated inhibition of influenza viruses, the underlying effect which allowed for the discovery of ifns [ ] . the task of connecting a specific ifn-induced protein to a specific antiviral action is compounded by the fact that often several ifn-induced proteins act in concert to inhibit the same virus at different stages of its life cycle. moreover, a specific ifn-induced protein may be more relevant for inhibiting a virus in one specific cell-type than another. recent systematic investigation of the specific antiviral effects of different isgs has started providing significant insight into this problem [ ] . such findings are complemented by the analyses of the spectra of the antiviral effects of a specific isg or a family of isgs [ ] . we have undertaken an investigation of the ifit family of mouse isgs. the corresponding human proteins are known to have antiviral activities against human papillomavirus (hpv) and hepatitis c virus (hcv), neither of which replicate in mouse cells. the anti-hpv activity of human ifit ( = p ) has been attributed to its ability to bind hpv e protein and to inhibit its helicase activity, which is essential for hpv dna replication [ , ] . the antiviral effect on hcv, on the other hand, is manifested at the level of inhibiting viral protein synthesis as a consequence of the ability of ifit to bind the translation initiation factor eif and inhibit its various actions in translation initiation [ ] . it has been reported recently that the ifit protein can form a complex and bind to rnas with triphosphorylated ends, presumably providing another means to inhibit specific viruses that produce such rnas [ ] . the ifit genes are clustered at a single locus in both human and mouse. in the latter species, two alleles of ifit genes are flanked on two sides by one allele of ifit and one allele of ifit [ ] . to identify their physiological functions, we have separately deleted the entire coding regions of ifit or ifit genes. the ifit / mice exhibited an interesting phenotype in allowing the replication of and resultant pathogenesis by a wnv mutant, which failed to replicate in wt mice [ ] . because this mutant is defective in -o methylation of the cap structure of viral mrnas, its rescue in the ifit / mouse indicates that this antiviral protein recognizes the ends of mrnas, a conclusion that is consistent with the observation that, in vitro, it can bind to rnas having specific structures at the ends [ ] . it remains to be seen whether the proposed property of ifit proteins to recognize ends of rna is connected in any way to their ability to inhibit the functions of eif [ ] , which participates in several steps of translation initiation taking place at or near the ends of mrnas. replication of vsv is highly sensitive to the antiviral activity of ifns, and vsv is widely used to determine the specific activities of ifn preparations quantitatively [ ] . in spite of this strong connection, it is unclear how ifn inhibits vsv replication. an early report indicated that viral primary transcription is inhibited by ifn, but it is not known which ifn-induced protein mediates this inhibition [ ] . the observed sensitivity of vsv replication in vitro is reflected in vivo. ifnar / mice are extremely susceptible to vsv infection; they rapidly die within days after infection and the virus replicates to very high titers in many organs of the infected mice. the extreme sensitivity of ifnar / mice to vsv infection suggests that type i ifn provides the majority, if not all, of the protective innate immune defense. eventually, protection may be facilitated by immune cell-mediated antiviral actions, but this is a slow process that does not appear to function before - days post-infection [ , ] . thus, it is likely that one or more isgs directly inhibit vsv replication in vivo. in this context, it has been reported that mice lacking pkr, a well-studied isg, display higher susceptibility to vsv pathogenesis [ ] . however, detailed investigation of the underlying mechanism revealed that pkr did not execute ifn's antiviral action; rather, it was required for efficient induction of ifn-a/b in the infected mice [ ] . in vivo vsv-infection induces ifn synthesis in many cell types, using either the cytoplasmic rig-i pathway or the endosomal tlr pathway [ , ] ; however, it is unclear how pkr aids this process. our results show that ifit / mice are highly susceptible to intranasal vsv infection and the effect is gene dosage-dependent: ifit +/ mice had an intermediate susceptibility phenotype. infected ifit / mice displayed symptoms of severe neuropathogenesis late after vsv infection accompanied by efficient replication of the virus in many regions of the brain. however, virus replication was restricted to neurons and did not spread to other types of cells in the brain, such as astrocytes. our results are consistent with the hypothesis that prior, ifn-induced, ifit expression in the brain restricts vsv replication. supporting genetic evidence for the requirement of ifn action is provided by the high susceptibility of the ifnar / mice, which possess the functional ifit gene but ifit is not induced by vsv infection because these mice cannot respond to type i ifn. additional evidence comes from a previous study using brain-specific ifnar / mice, which displayed a pattern of susceptibility to intranasal vsv infection similar to that of our ifit / mice [ ] . in our experimental system, the source of the ifn production was figure c ). ifit was also induced at this time in the rest of the brain, without any induction of ifn mrna ( figure d ) suggesting that the source of ifn was the ob. in accord with the well-established concept of ifn action, pre-induction of ifit in neurons, before the onset of infection, was essential for the antiviral effect. in comparison, induction of ifn and ifit that was concomitant with vsv infection failed to have an appreciable antiviral effect, as manifested by robust virus replication at directly infected sites, such as the obs of wt mice infected intranasally ( figure d ) or the brain of wt mice infected intracranially ( figure b ). high mortality of the infected mice correlated with high virus titers in the brains of intranasally infected ifit / mice or intracranially infected wt and ifit / mice. in the intranasally infected ifit / mice, death was not preceded by widespread apoptosis in the brain ( figure s ). however, as expected with high viral loads, ifn and other cytokines and chemokines were strongly induced ( figures d, s and s a) ; consequently, many isgs, except ifit , were also induced (table s ) . pre-induced ifit prevents efficient vsv replication in the brain, most probably by blocking one or more essential step of the viral life cycle including viral entry, uncoating, primary transcription, viral protein synthesis, rna replication, virion assembly or egress. it also might block trans-synaptic spread of the virus, although unlike another rhabdovirus, rabies virus, vsv is not known to depend on transit from neuron to neuron. in this context, it is important to note the observations made by iannacone et al. [ ] using a footpad vsv infection model. they concluded that type i ifn, produced by infected macrophages and plasmacytoid dendritic cells in infected mice, blocked infection of peripheral neurons resulting in lowered infection of the cns and prevention of neuropathogenesis. it is worth noting that in our studies, the absence of ifit did not affect ifn induction by vsv (figures a and c ). further investigation of the biochemical mechanism behind the observed in vivo effect of ifit / is hampered by the absence of a suitable cell culture model of the phenomenon. for example, ifit was not required for mediating the anti-vsv effect of ifn in mouse embryonic fibroblasts ( figure s ), in primary fetal neurons or in ifit -ablated neuroblastoma cells (data not shown), results that are not surprising given the strong tissuespecificity of ifit action observed in vivo (figure ) . specific rnabinding properties of ifit proteins have been recently reported [ ] . following this lead, we examined the rna-binding properties of recombinant murine ifit and ifit using vsv leader rna as the probe in an electrophoretic mobility shift assay: ifit bound rna with a -ppp end but not with a -oh end; however, ifit bound neither ( figure s ). to obtain meaningful leads, future investigation of this kind may require using brain extracts from infected mice to detect protein-viral rna complexes that may contain ifit along with adult neuronspecific proteins. our results revealed several layers of specificity of ifn action, some of which were not anticipated. first, compared to ifit / mice, ifit / mice were much less susceptible to intranasal vsv infection; this was true for both low and high doses of virus. this finding was surprising in view of a recent report on vsv susceptibility of ifit / mice [ ] and the observation that ifit , but not ifit , could bind vsv leader rna in vitro ( figure s ) . the above results demonstrate that different ifit proteins have nonredundant functions in vivo. the second layer of specificity was directed toward the nature of the infecting virus. although both vsv and emcv caused neuroinvasive disease, induced ifn-b, ifit and ifit in the brain and type i ifn action was required for protection against both viruses, ifit was critical only for protection against vsv; the absence of either ifit or ifit did not exacerbate susceptibility to emcv. the third layer of specificity was revealed by the organ-specific action of ifit . in the complete absence of type i ifn action in the ifnar / mice, intranasally infected vsv replicated vigorously not only in brains, but also in livers and lungs ( figure a-c) . in contrast, in ifit / mice, efficient vsv replication was restricted to the brain suggesting that ifit does not act as an anti-vsv isg in the liver or the lung because its absence did not impact virus titers, even though ifit was induced in these organs of infected wt mice ( figure d and e) . the efficient vsv replication in livers and lungs of ifnar / mice, but not wt and ifit / mice, indicates that other isgs must have anti-vsv effects in those organs. further investigation is needed to determine the basis of neuronal specificity of ifit action and the identities of other isgs that inhibit vsv replication in other organs. all animal experiments were performed in strict accordance with all provisions of the animal welfare act, the guide for the care and use of laboratory animals, and the phs policy on humane care and use of laboratory animals. the protocol was approved by the cleveland clinic institutional animal care and use committee (iacuc), phs assurance number a - . all experimental manipulations or intranasal instillations of mice were performed under anesthesia induced by pentobarbital sodium or isofluorane, respectively, and all efforts were made to minimize suffering. all mice used were of c bl/ background and of both sexes; ifit / mice were custom-generated by taconic farms, inc. by flanking exons and of ifit , encompassing the complete protein-encoding region, with frt sites in c bl/ embryonic stem (es) cells, and deleting the flanked region by transfection of flp recombinase. es cell clones were injected into bl/ blastocysts, and heterozygous offspring mice were crossed to homozygosity. ifit / mice were generated from c bl/ es cells lacking the whole coding region of ifit ( ); es cells were obtained from the nih knockout mouse project (komp, allele ifit tm (komp)vlcg ). the same es cell line was independently used to generate mice in another study [ ] . ifnar / mice (lacking ifnar ) were a gift of murali-krishna kaja (emory university, atlanta, ga). congenic wild-type mice were obtained from taconic farms. vesicular stomatitis virus (vsv) indiana was a gift from amiya k. banerjee, lerner research institute, cleveland, ohio. for intranasal infections, between and pfu of vsv in ml of endotoxin-free pbs were inhaled by isofluorane-anesthetized - week-old mice, with pbs-only as control. for intracranial infections, pfu of vsv in ml of endotoxin-free pbs were injected into the brains of - week-old mice, with pbsonly as control. thereafter, mice were monitored daily (twice daily after i.c. injection) for weight loss and symptoms of disease. encephalomyocarditis virus (emcv) k strain was a gift from robert h. silverman, lerner research institute, cleveland, ohio. for intraperitoneal infections, between and pfu of emcv in ml of pbs were injected into the peritoneal cavity of mice. mice were monitored daily for weight loss and symptoms of disease. mice were anesthetized with pentobarbital ( mg/kg) and blood was removed from organs by cardiac perfusion with ml of pbs, followed by perfusion with ml of % paraformaldehyde/pbs for fixation. brains were placed in % paraformaldehyde overnight for complete fixation, submerged in % sucrose/ pbs overnight for cryoprotection, and frozen in o.c.t. compound (sakura finetek usa, torrance, ca, usa). mm sagittal sections were cut at uc in a leica cm cryostat, mounted on coated slides (superfrost plus, fisherbrand, fisher scientific); membranes were permeabilized by . % triton x- /pbs treatment for min. for immunohistochemistry, the envision+ dab kit (dako, carpinteria, ca) was used with antimouse ifit /p [ ] or anti-vsv-p protein (a gift from amiya k. banerjee, lerner research institute, cleveland, ohio) as primary antibodies. for immunohistofluorescence, anti-vsv-p or anti-neun (chemicon intl./millipore, billerica, ma) or anti-gfap (sigma-aldrich, st. louis, mo) were used; labeled brain sections were stained with alexafluor- secondary antibody (invitrogen/molecular probes, carlsbad, ca). for detection of apoptotic cells in brain sections, the deadend fluorometric tunel system (promega) was used according to manufacturer's instructions. all objects were then mounted with vectashield (with dapi, vector labs, burlingame, ca), and examined with a leica drm fluorescence microscope. mice were anesthetized with pentobarbital ( mg/kg) and blood was removed from organs after cardiac perfusion with ml of pbs. brains were separated into olfactory bulbs and the remainder of the brain, snap-frozen in liquid nitrogen (as well as livers and lungs) and rna was extracted using trizol reagent (invitrogen). dnase i treatment (dnafree, applied biosystems/ ambion) and reverse transcription with random hexamers (improm-ii, promega) were performed according to manufacturer's instructions. . ng of rna was used in well-format realtime pcrs in a roche lightcycler ii using applied biosystem's sybr green pcr core reagents. pcr primers for murine isg /ifit , isg /ifit , isg /ifit and s rrna have been published previously [ ] ; primers targeting murine ifnb [ -cttctccgtcatctccataggg- [ ] , with the alternative reverse primer: -cacagccctctccatcaact- ], vsv n rna [ ] or emcv d polymerase genomic region [ ] were described previously. primers for ccl , il b, il , tnf, il b and nos have been described previously [ , ] . average expression levels, relative to s rrna and normalized by use of calibrator samples, were graphed with prism . software. for analysis of different regions of the brain, brains without ob of perfused mice were separated into cortex, cerebellum, brain stem and remaining ''midbrain'', and tissue was submerged into rnalater stabilizing reagent (qiagen) overnight and frozen. rna was then extracted via trizol and further processed and assayed by realtime rt-pcr as described above. for microarray analysis, trizol-extracted and dnase i-treated rna was additionally purified using spin columns (rneasy mini kit, qiagen) before subjection to mrna expression microarray analysis via illumina mouse ref- v beadchip and genomestudio software v . (illumina, inc.); rna hybridization to chips was performed by the lerner research institute genomics core at the cleveland clinic. microarray raw data were deposited in the ncbi gene expression omnibus (geo), accession number gse . for quantification of infectious vsv in organs, mice were anesthetized with pentobarbital ( mg/kg) and blood was removed from organs by cardiac perfusion with ml of pbs. organs were snap-frozen in liquid nitrogen, weighed, pestle/tubehomogenized (kimble/kontes) in ml of pbs per brain or peripheral organ or . ml per pair of olfactory bulbs, and virus was titered in -fold serial dilutions on vero cells by plaque assay. results are expressed as plaque-forming units (pfu) per gram of tissue. for quantification of infectious vsv yields in mef, cells ( /+ifn-b pretreatment as indicated) were infected with vsv inoculum for h, and after another h, cells were freeze/ thawed, and cleared supernatants of lysates were assayed for vsv by plaque assay on vero cells. primary murine embryonic fibroblasts (mefs) were stimulated with u/ml murine ifn-b (pbl, inc., piscataway, nj) for h and lysed in lysis buffer [ mm tris ph . , mm nacl, . % triton x- , mm sodium orthovanadate, mm sodium fluoride, mm sodium pyrophosphate, mm bglycerophosphate and complete edta-free protease inhibitor (roche, indianapolis, in)]. mg of whole cell extract were separated via % sds-page, transferred to pvdf membranes, blocked with % dry milk in tris-buffered saline/ . % tween- overnight and labeled with anti-ifit /p , anti-ifit /p or anti-ifit /p polyclonal rabbit sera [ , ] . single-stranded vsv leader rna (nucleotides - ) was t polymerase-transcribed in presence of [a- p]-ctp, yielding radiolabeled -triphosphorylated (ppp-) rna, followed by alkaline phosphatase treatment for generation of -hydroxyl (ho-) rna. ppp-rna or ho-rna were added to bacterially expressed and purified xhis-tagged ifit or ifit protein in reaction buffer ( mm tris ph . , mm nacl, mm edta, mm dtt, . % triton x- , % glycerol) and incubated for min on ice. reaction products were separated by % native polyacrylamide gel electrophoresis followed by exposure to film. statistical significance of mouse survival differences was calculated by mantel-cox log rank test. to assess significance of differences in gene expressions or virus titers, the two-tailed mann-whitney test was used. all calculations were performed using graphpad prism . software. previously published transcript sequences in the ncbi entrez nucleotide database: ifit , nm_ ; ifit , nm_ ; ifit , nm_ ; ifnb , nm_ ; ifnar , nm_ . figure s survival of wt and ifit / mice after infection with low emcv dose ( pfu). statistical significance of survival differences is indicated by p-value; n.s., not significant. (pdf) figure s enhanced isg and ifn-b induction in intranasally vsv-infected ifit / brain regions. ifn-b-, and ifit / / mrna levels in different regions of brains of uninfected or vsv-infected wt and ifit / mice at d.p.i., plotted as mean+sd. n = mice per infected group; nd, not done. infections were intranasal with pfu of vsv. (pdf) figure s gene induction in brains after vsv or emcv infections. a, mrna levels of select genes in brains (without obs) of uninfected or intranasally vsv-infected wt and ifit / mice at d.p.i., plotted as mean+sd; n = mice per infected group; infection was intranasal with pfu of vsv. b, mrna levels of select genes in brains (without obs) of uninfected or intracranially vsv-infected wt and ifit / mice at h post injection, plotted as mean+sd; n = mice per infected group; infection was intracranial injection with pfu of vsv. c, ifit , ifit , ifn-b and emcv rna levels in brains days after emcv infection ( pfu, n = mice per infected group). (pdf) figure s region-selective induction of apoptosis in brains of intranasally vsv-infected ifit / mice. ifit / mice were i.n. infected with pfu of vsv; at d.p.i., adjacent sections of fixed brains were labeled to detect apoptotic cells (tunel) or vsv p protein (immunohistofluorescence), n = mice; only few regions such as striatum show positive tunel; infected wt brains and uninfected control brains of either genotype did not show appreciable signals, hence data not shown). (pdf) figure s vsv yields from infected wt and ifit / mef. immortalized mef were treated for h with u/ml ifn-b and infected with vsv at moi . hours after infection, virus yields were determined by plaque assay. results are plotted as mean+sd on log scale, representing one of two independent experiments. (pdf) figure s murine ifit protein does not bind ppp-rna. single-stranded radiolabeled vsv leader rnas (nt - ) with either -triphosphorylated or free -hydroxyl-ends (ppp-rna or ho-rna) were in vitro incubated with purified murine ifit ( = p ) or ifit ( = p ) proteins; formation of protein/rna complex was detected by electrophoretic mobility shift assay. (pdf) table s enhanced gene expression in brains incl. obs of intranasally vsv-infected ifit / versus wt mice at d.p.i. wt or ifit / mice were intranasally vsv-infected with pfu, and at or d.p.i., brain (incl. ob) rna expression profiles were obtained by microarray. genes are ranked by their ''fold expression level in ifit / over wt at d.p.i.''. only genes with at least -fold higher expression level in ifit / are included. note: the ifit /isg probe of the illumina mouse ref- chip is defective and therefore the gene is not included in this list. (pdf) local type i ifn receptor signaling protects against virus spread within the central nervous system functional role of type i and type ii interferons in antiviral defense a null mutation in the gene encoding a type i interferon receptor component eliminates antiproliferative and antiviral responses to interferons alpha and beta and alters macrophage responses differential roles of mda and rig-i helicases in the recognition of rna viruses distinct and essential roles of transcription factors irf- and irf- in response to viruses for ifn-alpha/beta gene induction essential role of ips- in innate immune responses against rna viruses modulation of host innate and adaptive immune defenses by cytomegalovirus: timing is everything dcs and nk cells: critical effectors in the immune response to hiv- pathogenesis of murine coronavirus in the central nervous system targeted disruption of the mouse stat gene results in compromised innate immunity to viral disease targeted disruption of the stat gene in mice reveals unexpected physiologic specificity in the jak-stat signaling pathway identification of genes differentially regulated by interferon alpha, beta, or gamma using oligonucleotide arrays interferon-inducible antiviral effectors a diverse range of gene products are effectors of the type i interferon antiviral response the isg /ifit gene family induction and mode of action of the viral stressinducible murine proteins, p and p novel characteristics of the function and induction of murine p family proteins ifit is an antiviral protein that recognizes -triphosphate rna coordinated regulation and widespread cellular expression of interferonstimulated genes (isg) isg- , isg- , and isg- in the central nervous system after infection with distinct viruses -o methylation of the viral mrna cap evades host restriction by ifit family members convenient assay for interferons vesicular stomatitis the earliest events in vesicular stomatitis virus infection of the murine olfactory neuroepithelium and entry of the central nervous system distribution of vesicular stomatitis virus proteins in the brains of balb/c mice following intranasal inoculation: an immunohistochemical analysis interferons alpha and beta as immune regulators-a new look human mxa protein: an interferon-induced dynamin-like gtpase with broad antiviral activity ifn-stimulated gene functions as a critical antiviral molecule against influenza, herpes, and sindbis viruses the inhibitory action of p on select functions of e mediates interferon's effect on human papillomavirus dna replication interferon-inducible protein, p , inhibits hpv dna replication by binding to the viral protein e alpha interferon induces distinct translational control programs to suppress hepatitis c virus rna replication inhibition of vesicular stomatitis viral mrna synthesis by interferons host immune response to vesicular stomatitis virus infection of the central nervous system in c bl/ mice vesicular stomatitis virus infection of the central nervous system activates both innate and acquired immunity essential role for the dsrna-dependent protein kinase pkr in innate immunity to viral infection protein kinase r contributes to immunity against specific viruses by regulating interferon mrna integrity autophagydependent viral recognition by plasmacytoid dendritic cells subcapsular sinus macrophages prevent cns invasion on peripheral infection with a neurotropic virus tissue-specific and inducer-specific differential induction of isg and isg in mice cell-specific irf- responses protect against west nile virus infection by interferondependent and -independent mechanisms replication and propagation of attenuated vesicular stomatitis virus vectors in vivo: vector spread correlates with induction of immune responses and persistence of genomic rna rapid diagnosis of encephalomyocarditis virus infections in pigs using a reverse transcription-polymerase chain reaction interleukin- (il- ), but not il- , deficiency ameliorates viral encephalitis without affecting viral control factors supporting intrathecal humoral responses following viral encephalomyelitis we thank michifumi yamashita and niranjan butchi for sharing technical expertise. key: cord- -andje wu authors: dorobantu, cristina m.; albulescu, lucian; harak, christian; feng, qian; van kampen, mirjam; strating, jeroen r. p. m.; gorbalenya, alexander e.; lohmann, volker; van der schaar, hilde m.; van kuppeveld, frank j. m. title: modulation of the host lipid landscape to promote rna virus replication: the picornavirus encephalomyocarditis virus converges on the pathway used by hepatitis c virus date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: andje wu cardioviruses, including encephalomyocarditis virus (emcv) and the human saffold virus, are small non-enveloped viruses belonging to the picornaviridae, a large family of positive-sense rna [(+)rna] viruses. all (+)rna viruses remodel intracellular membranes into unique structures for viral genome replication. accumulating evidence suggests that picornaviruses from different genera use different strategies to generate viral replication organelles (ros). for instance, enteroviruses (e.g. poliovirus, coxsackievirus, rhinovirus) rely on the golgi-localized phosphatidylinositol -kinase iii beta (pi kb), while cardioviruses replicate independently of the kinase. by which mechanisms cardioviruses develop their ros is currently unknown. here we show that cardioviruses manipulate another pi k, namely the er-localized phosphatidylinositol -kinase iii alpha (pi ka), to generate pi p-enriched ros. by sirna-mediated knockdown and pharmacological inhibition, we demonstrate that pi ka is an essential host factor for emcv genome replication. we reveal that the emcv nonstructural protein a interacts with and is responsible for pi ka recruitment to viral ros. the ensuing phosphatidylinositol -phosphate (pi p) proved important for the recruitment of oxysterol-binding protein (osbp), which delivers cholesterol to emcv ros in a pi p-dependent manner. pi p lipids and cholesterol are shown to be required for the global organization of the ros and for viral genome replication. consistently, inhibition of osbp expression or function efficiently blocked emcv rna replication. in conclusion, we describe for the first time a cellular pathway involved in the biogenesis of cardiovirus ros. remarkably, the same pathway was reported to promote formation of the replication sites of hepatitis c virus, a member of the flaviviridae family, but not other picornaviruses or flaviviruses. thus, our results highlight the convergent recruitment by distantly related (+)rna viruses of a host lipid-modifying pathway underlying formation of viral replication sites. picornaviridae is a large family of positive-sense rna viruses [(+)rna viruses] comprising many clinically relevant human and animal pathogens. members of the genus enterovirus include important human viruses like poliovirus (pv), the causative agents of poliomyelitis, coxsackieviruses (cv), causing meningitis and myocarditis, and rhinoviruses (rv), responsible for the common cold and exacerbations of asthma and chronic obstructive pulmonary disease. perhaps the best-known non-human picornavirus is foot-and-mouth-disease virus (fmdv, genus aphtovirus), which can cause devastating outbreaks in cattle leading to severe economic loss. closely related to the apthovirus genus is the genus cardiovirus, composed of three species: theilovirus (tv), encephalomyocarditis virus (emcv) and the more recently discovered boone cardiovirus. the species theilovirus includes, among others, theiler's murine encephalomyocarditis virus (tmev) and saffold virus (safv), a human cardiovirus. while tmev is known to cause enteric infections and sometimes more severe encephalitis or chronic infection of the central nervous system [ ] , as yet, safv has not been firmly associated with a clinical disease [ ] . emcv can infect a wide range of animals, of which rodents are considered the natural reservoir. of all domesticated animals, pigs are most prone to emcv infection, which can lead to fatal myocarditis [ ] , reproductive failure in sows or sudden death of piglets [ ] [ ] [ ] . like other (+)rna viruses-such as hepatitis c virus (hcv), dengue virus (denv), chikungunya virus (chikv) and coronavirus (cov)-picornaviruses replicate their genomic rna on specialized, virus-modified intracellular membranes. these remodeled membranes termed replication organelles (ros) arise from the concerted actions of both viral nonstructural virus-host interactions, indicating that diverse rna viruses might have a limited choice of pathways in the remodeling of host membrane network for virus replication. unlike enteroviruses, cardioviruses do not require pi kb for replication [ ] . to investigate whether other pi ks might be involved in cardiovirus replication, we depleted each of the four distinct cellular pi ks by sirna-mediated gene knockdown using a set of sirna sequences (ambion) which we previously tested for efficiency and toxicity [ ] , and monitored the subsequent effects on replication of emcv. we observed inhibitory effects on emcv replication when silencing pi ka, but not upon silencing of the other pi ks (fig a) . to confirm the importance of pi ka for emcv replication, we performed another series of knockdown experiments using another set of sirna sequences (qiagen). depletion of pi ka, but not pi kb, significantly reduced emcv infection, measured by end-point titration of progeny virus production ( fig b) . we next wondered which step in the virus life cycle is dependent on pi ka. to omit the step of virus attachment and cell entry, emcv rna was in vitro transcribed and subsequently transfected in cells depleted of pi ka by sirnas. virus replication was strongly inhibited upon pi ka silencing, as measured by end-point titration of progeny virions ( fig c) . this indicated that pi ka is involved in a post-entry step in the virus life cycle. to elucidate whether emcv requires pi ka for viral genome amplification, we infected cells with a renilla luciferase-encoding emcv (rluc-emcv) and quantified the luciferase activity as a measure of viral rna replication. emcv rna replication was severely impaired in cells lacking pi ka, but not pi kb (fig d) . we excluded that inhibition of emcv replication by pi ka silencing was due to cytotoxic effects by a cell viability assay ( fig e) and verified the knockdown efficiency by western blot analysis ( fig f) . altogether, these results showed that pi ka plays a key role in emcv genome rna replication. next, we investigated whether emcv required the enzymatic activity of pi ka using al- , a pi k inhibitor that also blocks pi kb, but at -fold higher concentration [ ] . cells were infected with emcv or rluc-emcv at moi . and treated with increasing concentrations of al- for h. coxsackievirus b (cvb ), as well as all other enteroviruses, has been previously shown to hijack the golgi-localized pi kb for replication [ , ] and was included as a control. as measured by end-point titration and analysis of the luciferase activity ( fig g and h ), emcv replication was efficiently inhibited by al- in a dose-dependent manner with complete inhibition detected at μm, while cvb replication was hampered only at μm ( fig g) , which is in line with the -fold preference of al- for pi ka over pi kb. dipyridamole, a well-established inhibitor of emcv rna replication, was included here as positive control. importantly, al- inhibited emcv replication also when infection was performed at high moi (s a fig, moi ). to corroborate that pi ka activity is required for the step of viral genome replication, we performed a time-of-addition experiment in which al- was added to the cells at different time points after infection with rluc-emcv. similar to dipyridamole, al- strongly inhibited replication when added up to h after infection (s b fig), indicating that not entry but rather a step during genome replication was blocked by al- . next, we tested whether other members of the cardiovirus genus also depended on pi ka for replication. similar to emcv, replication of the human cardiovirus saffold virus (safv ) (species theilovirus) was also sensitive to al- treatment (fig i) . the cell viability assay demonstrated that al- treatment only exerted slight cytotoxic effects at the highest concentration tested (fig j) . these results indicated that different cardiovirus species required the enzymatic activity of pi ka for genome replication. soon after infection, the cytoplasm of emcv-infected cells accumulates an impressive amount of vesicular membranous structures [ , ] . as yet, there is little information available regarding which viral proteins and host factors are associated with these new virus-induced organelles [ , ] . we set out to investigate whether pi ka was present at emcv ros. despite repeated efforts, we were unable to detect the endogenous kinase by immunofluorescence staining in any of the cell lines tested. as an alternative, we chose to analyze possible changes in the subcellular distribution of ectopically expressed pi ka upon emcv infection. in mock-infected cells (fig a, upper panel) , gfp-pi ka was distributed diffusely throughout the entire cytoplasm, as previously reported by others [ , ] . in infected cells visualized by dsrna staining, we instead observed a clear difference in the localization of the kinase, which was redistributed to discrete cytoplasmic punctae in a perinuclear region (fig a, lower panel) . we next aimed to elucidate whether these pi ka punctae coincided with the viral ros. the small picornaviral protein a and its precursor ab are membrane-associated and play key roles in viral rna replication and recruitment of essential host factors [ , [ ] [ ] [ ] [ ] . hence, we considered ab as a suitable marker for emcv ros and compared the staining of pi ka to that of ab in infected cells. we observed a striking overlap of gfp-pi ka with ab-positive structures ( fig b, lower panels) and could confirm this phenotype when analyzing the localization of ectopically expressed pi ka bearing an ha-tag (s fig). by contrast, the signal for gfp-pi kb, which was mainly localized at the golgi in non-infected cells (fig c, top panel) , failed to overlap with ab ( fig c, lower panels) . interestingly, although in close proximity to dsrna signals ( fig a, lower panel) , pi ka did not clearly overlap with dsrna (fig a, insets) . taken together, these data demonstrated that pi ka is selectively recruited to emcv ros. interestingly, we noticed a loss of the typical golgi localization of pi kb in emcv-infected cells ( fig c, lower panel) , suggesting that golgi integrity might be affected upon emcv infection. prompted by this and our finding that emcv utilizes the er-localized pi ka for replication, we set out to elucidate whether other er or golgi components are present at emcv ros. in order to be able to use more antibody combinations in immunofluorescence, we constructed a recombinant emcv bearing an ha-tag in the nonstructural protein c. the tag was introduced after the second amino acid, leaving the b- c cleavage site intact (s a fig), and did not impair virus replication (s b fig). first, we checked whether c-ha and ab are present on the same membranes by immunofluorescence microscopy. indeed, c and ab signals greatly overlapped (s c fig), supporting the idea that these proteins occupy the same membranes of the ros. using this tagged emcv, we noticed that the golgi structure was indeed altered in infected cells, from h p.i. onwards, as revealed by the dispersed pattern of both cisand trans-golgi markers gm ( fig a) and tgn , respectively ( fig b) . however, neither tgn nor gm were present at c-ha-positive structures, suggesting that emcv ros are not golgi-derived. ergic , a marker of the er-golgi intermediate compartment also , cells were infected with emcv (a-c) or rluc-emcv (d) at an moi of . or transfected with full-length infectious emcv in vitro-transcribed rna (c). after h, cells were freeze-thawed to release intracellular virus particles and the total virus titers were determined by endpoint titration (a-c). alternatively, cells were lysed and renilla luciferase activity was determined as a measure of viral rna replication (d). in parallel, a cell viability assay was performed to evaluate the cytotoxicity of the sirna treatment (e). (f) western blot analysis showing efficient knockdown of pi ka and pi kb. actin was used as loading control. (g-i) al- treatment inhibits cardiovirus replication. hela r cells were infected with virus at an moi of . (g and h) or (i), followed by al- treatment for h, after which cells were lysed and virus replication was measured by endpoint titration (g and i) or by determining the renilla luciferase activity (h). cytotoxicity of al- was determined in a cell viability assay run in parallel (j). bars represent mean values of triplicates ± standard error of the means (sem). means were statistically compared using unpaired t tests. *p < . , **p< . ; ***p< . . appeared scattered throughout the cytoplasm in infected cells, but without overlapping c-ha ( fig c) . we next compared the localization of ab with sec (copii-coatomer complex component), an er exit site (eres) marker, and the er marker calreticulin. while in noninfected cells, sec displayed mainly a typical perinuclear localization, in emcv-infected cells it appeared dispersed, but without significantly colocalizing with ab ( fig d, mander's colocalization coefficient m = . ± . , fraction of sec overlapping ab). we observed a greater degree of overlap between ab and calreticulin ( fig e, m = . ± . , fraction of calreticulin overlapping ab). images acquired with higher magnification revealed that most of ab was in close contact with er tubules (fig f) . taken together, these data suggested that emcv possibly replicates on er-derived membranes. based on the extensive overlap between pi ka and ab and the drastic change in pi ka pattern in infected cells, we hypothesized that pi ka might be recruited to replication sites by interacting (directly or indirectly) with one or more of the viral nonstructural proteins. to investigate this, we used the stable cell line huh -lunet/t that allows ectopic protein expression under the control of a t promoter and has been previously optimized and validated as a reliable and reproducible cellular system to study pi ka-protein interactions by radioactive co-ip assays [ , ] . myc-tagged emcv nonstructural proteins a, b, c, a, ab, c and d were individually co-expressed together with ha-pi ka in huh -lunet/t cells, radioactively labeled, and affinity purified from cell lysates using anti-myc specific antibodies. autoradiography analysis showed that ha-pi ka was specifically co-purified by a and ab, but not by the other viral proteins (fig a) . to confirm this interaction by co-immunoprecipitation (co-ip) coupled with western blot analysis, myc-tagged emcv a was co-expressed with ha-pi ka and subjected to affinity purification using either monoclonal or polyclonal antimyc antibodies. as shown in fig b, ha-pi ka only interacted with emcv a, but not with cvb a, which interacts with pi kb [ , , ] and was included here as a negative control. these data implied that emcv nonstructural protein a is responsible for pi ka recruitment to ros. interestingly, a diffuse band just below kda appears to co-purify with emcv c when ha-pi ka is co-expressed ( fig a, indicated by à ). we reasoned this could be indicative of a temporal regulation of the pi ka activity during infection via c-dependent degradation. to explore this possibility, we performed western blot analysis of endogenous pi ka during the time course of infection, but did not detect any bands indicative of degradation, neither in huh -lunet/t or hela r cells (s fig). to test if a alone can recruit pi ka, we examined by immunofluorescence the subcellular localization of ha-pi ka when co-expressed with a, ab or b, which we considered as a negative control. when expressed alone, ha-pi ka localized throughout the cell in a diffuse pattern (fig c, top panel) , as previously described [ ] . emcv a-and ab-myc were both localized throughout the cytoplasm and at discrete punctate structures, of which a subset was also positive for pi ka ( fig c) . b-myc was also distributed in punctae throughout the cytoplasm, but failed to recruit pi ka (fig c) . collectively, these results indicated that emcv a is the viral protein responsible for engaging pi ka in replication. while pi kb produces pi p at golgi membranes, pi ka is responsible for the synthesis of the pi p pool at the plasma membrane, where it dynamically localizes [ , [ ] [ ] [ ] . our finding that pi ka activity was critical for emcv rna replication prompted us to investigate whether pi p metabolism is altered during virus replication. given that emcv replicates on the picornavirus emcv converges on the host lipid pathway used by hcv intracellular membranes, we monitored potential changes in the subcellular distribution of both plasma membrane (pm) and intracellular (ic) pools of pi p in huh lunet/t cells following emcv infection. the two pools of pi p can be selectively visualized using two different immunocytochemistry protocols previously established by hammond et al [ ] . while the plasma membrane pool of pi p appeared unaffected in emcv-infected cells (fig a) , the intracellular pi p distribution changed from a perinuclear, golgi-like pattern in mock-infected cells to dispersed throughout the cytoplasm in emcv-infected cells ( fig a) . we observed similar pi p phenotypes in hela cells (s fig), indicating that the observed effects were not cell line-specific. notably, quantitative analysis of the fluorescent pi p signals revealed a marked increase in the level of intracellular pi p in infected cells ( fig b) . to rule out a possible involvement of pi kb in establishing the elevated pi p levels observed in emcv infected cells, we treated cells with the pi kb inhibitor bf (compound ) [ ] . for simultaneous detection of pi p and viral ros by immunofluorescence, we infected cells with emcv- c-ha. short treatment with bf severely depleted the golgi pi p pool in non-infected cells (fig c) , thus reflecting an effective inhibition of pi kb activity. however, the pi p phenotype remained unaltered in infected cells (fig c) , demonstrating that the emcv-induced accumulation of intracellular pi p was not mediated by pi kb. most pi p localized in the vicinity of c-ha, with at least a small subset of pi p overlapping with c-ha ( fig c) . these data together with the finding that emcv requires pi ka activity suggested that pi ka-derived pi p lipids play a central role in emcv genome replication. various cellular proteins carrying a pi p-binding domain called the pleckstrin-homology domain (ph), such as the ceramide-transfer protein (cert), four-phosphate-adaptor protein (fapp ), or the oxysterol-binding protein (osbp) can sense and specifically bind pi p lipids [ , [ ] [ ] [ ] . recently, we and others showed that enteroviruses generate pi p-enriched membranes to recruit osbp, which in turn exchanges pi p for cholesterol at ros [ , , ] . moreover, we showed that emcv is sensitive to itraconazole, which we identified to be an osbp inhibitor [ ] , and that cholesterol shuttling is important for emcv replication [ ] . we therefore reasoned that in emcv-infected cells one purpose of pi p lipids might be to recruit osbp to replication membranes to support viral rna replication. to test if osbp is required for emcv replication, we efficiently reduced osbp expression in hela cells by sirna gene silencing ( fig a) and evaluated the subsequent effects on emcv replication by end-point titration analysis. replication of emcv was significantly reduced in cells in which osbp was depleted compared to control-treated cells (fig a) , indicating that osbp is required for efficient replication. we further used osw- , an osbp ligand that interferes with normal osbp functioning [ ] , to pharmacologically inhibit osbp and analyze whether its lipid transfer function is linked to emcv infection. using luciferase-encoding emcv, we observed a complete inhibition of genome rna replication after h of treatment with osw- at nanomolar concentrations, with no cytotoxicity present (fig b) . a similar inhibition by osw- was observed when infection was performed at high moi (s fig, moi ) . furthermore, by performing osw- time-of-addition experiments, we excluded the possibility that osbp was involved in early steps in the virus life cycle (fig c) . similar results were obtained when using -hydroxycholesterol ( -hc, fig c) , another established osbp ligand [ , ] . next, we wondered whether endogenous osbp was present at emcv ros and if so, whether this localization was dependent on the pi p pool generated by pi ka. to this end, cells were infected with emcv for . h and then treated with dmso or al- for min to acutely deplete pi p, prior to immunofluorescence analysis. while in non-infected cells osbp huh -lunet/t cells were mockinfected or infected with gfp-emcv at an moi of . at h p.i., cells were fixed and stained with antibodies against pi p using specific protocols for detection of plasma membrane and intracellular pi p pools, as described previously [ ] . nuclei were stained with dapi (blue). (b) quantification of pi p levels by immunofluorescence analysis. huh -lunet/t cells were mock-infected or infected with emcv at an moi of . at h p.i., cells were fixed and stained for intracellular pi p and viral dsrna. the intensities of fluorescent pi p signals from whole-cell z-stacks were quantified using imagej. shown are mean values ± sem of cells per condition. means of mock and infected cells were statistically analyzed using the mann-whitney test. ***p< . . (c) emcv ros contain pi p. huh -lunet/t cells were mock-infected or infected with emcv- c-ha at an moi of . at . h p.i., cells were treated with dmso or μm bf (pi kb inhibitor) for min, then fixed and stained for intracellular pi p and ha using specific antibodies. nuclei were stained with dapi (blue). the crop panels at the bottom depict an enlargement of the boxed areas. scale bars represent μm. the picornavirus emcv converges on the host lipid pathway used by hcv localized throughout the cytoplasm and at the golgi, osbp was mainly found at ros in infected cells, where it largely colocalized with ab ( fig d, pearson's correlation coefficient = . ). since other golgi proteins were not present at the ros (fig a and b) , these results suggested that osbp is specifically recruited by emcv. following inhibition of pi ka by short treatment with al- , we observed a strong and significant reduction of osbp and ab colocalization (fig d, pearson's coefficient = . ). importantly, the subcellular localization of osbp in non-infected cells was not affected by al- treatment (fig d) , demonstrating that the presence of osbp at emcv replication structures is conditioned by pi ka-produced pi p. given the colocalization of osbp with ab, we sought to verify whether emcv a was responsible for osbp recruitment. to this end, myc-tagged emcv a-, ab-or b-myc were ectopically expressed in huh -lunet/t cells and recruitment of endogenous osbp was analyzed by immunofluorescence analysis. in cells expressing a and ab, osbp was redistributed in punctate structures throughout the cytoplasm, with some of these punctae colocalizing with a/ ab (fig e) . by contrast, osbp remained localized at the golgi and did not localize at c-positive punctae (fig e) . these data indicated that during emcv infection, osbp is recruited to ros via a. to test whether osbp is involved in transferring cholesterol to ros in a pi p-dependent manner, cells were infected with emcv for h, treated with al- or osw- for h to block pi ka activity or osbp function respectively, and subjected to immunofluorescence analysis. in non-infected cells, cholesterol mainly localizes at endosomes in the perinuclear area and at the plasma membrane, as visualized by filipin staining [ ] . in infected cells treated with dmso, we detected cholesterol primarily colocalizing with ab-positive structures (pearson's coefficient = . ), while in drug-treated cells this colocalization was markedly reduced (fig , pearson's coefficient = . for al- and . for osw- ). this result confirmed that emcv ros acquire cholesterol through the actions of both pi ka and osbp. (+)rna viruses display immense genetic diversity, yet they all rely on remodeled membranes for viral genome replication. a diverse array of cellular organelles can be remodeled into viral replication structures. for instance, picornaviruses from the genera enterovirus and kobuvirus are thought to replicate at modified golgi membranes [ , , ] , while the flavivirus hcv replicates on a membranous web originated from the er [ ] . to do so, viruses rewire host pathways involved in lipid synthesis and transport to generate replication membranes with unique lipid signatures [ ] [ ] [ ] [ ] . how picornaviruses from the genus cardiovirus build their ros is currently unknown. here, we identified pi ka and osbp as essential host factors for genome replication of the cardiovirus emcv. our data suggest that emcv ros may be derived from the er and that pi ka was recruited to ros by interacting with the viral protein a(b). pi ka recruitment led to a significant increase of intracellular pi p levels in infected cells, which proved important for the downstream recruitment of osbp. finally, data are presented suggesting that the osbp-mediated exchange of pi p and cholesterol at ro-mcss is critical for emcv genome replication and the global organization of ros. membrane alterations in the cytoplasm of cardiovirus-infected cells were already observed decades ago by electron microscopy [ , , ] . as also described for enteroviruses, cardiovirus-induced membranes consist of perinuclear clusters of heterogeneous single-and doublemembrane vesicles (dmvs). while recent studies greatly contributed to our understanding of the origin and biogenesis of enterovirus ros [ , , , , , ] , for cardioviruses these details have remained scarce. in a report by zhang et al it was proposed that emcv subverts the autophagy pathway to promote virus replication and ro formation [ ] . the authors observed induction of autophagy and accumulation of cytoplasmic double-membrane vesicles (dmvs), a hallmark of autophagosomes, upon emcv infection. however, inhibition of autophagy had stronger effects on extracellular than intracellular virus yields, which pointed towards a role of autophagy in virus release. indeed, recent studies using enteroviruses and flaviviruses support the hypothesis that autophagy-derived membranes rather serve as means of non-lytic virus release and spread than as a membrane source for the viral ros [ ] [ ] [ ] [ ] . as opposed to enteroviruses, members of the cardiovirus genus are insensitive to gbf depletion by sirna [ ] or treatment with bfa [ , , ] , a compound that targets gbf and subsequently blocks activation of arf , a key regulator of membrane trafficking in the secretory the picornavirus emcv converges on the host lipid pathway used by hcv pathway. furthermore, cardiovirus replication does not require the golgi-localized pi kb, which is essential for enterovirus replication [ ] . collectively, these data suggested that cardioviruses do not rely on golgi components for replication. in line with these previous findings, we here present data suggesting that emcv may derive its ros from er membranes. confocal microscopy analysis revealed that emcv nonstructural proteins partially overlapped with the er marker calreticulin, but not with markers of eres, ergic, cis-or trans-golgi network, which appeared dispersed in infected cells, even at the earliest stages of infection. furthermore, pi ka, which normally resides at the er network, was redistributed in emcv-infected cells to discrete cytoplasmic structures that also contained the viral protein ab. interestingly, the majority of pi ka-positive punctae were detected in close proximity to viral dsrna, but did not completely overlap with dsrna, suggesting a spatial segregation of dsrna from the viral replication membranes, previously also shown for coronaviruses [ , ] . using a pharmacological inhibitor of pi ka, we prove that emcv and safv, which belong to distinct cardiovirus species, both require the lipid kinase activity for replication. in agreement with this result, we observed elevated pi p levels at intracellular membranes in infected cells, suggesting an important role of pi p lipids in cardiovirus replication. in non-infected cells, osbp plays a critical role in lipid homeostasis by exchanging cholesterol for pi p at the interface of er and golgi membranes, to which it localizes under normal conditions [ ] . in this process, pi p lipids also serve as a membrane anchor for osbp. we hypothesized that in cardiovirus infection, pi p may serve to recruit osbp and cholesterol to viral replication sites. indeed, osbp was present at emcv ros, where it colocalized with the viral protein ab. this colocalization was markedly reduced upon al- treatment, demonstrating that osbp is recruited through pi ka-produced pi p. osbp is an essential cardiovirus host factor, since both genetic depletion by sirna treatment and pharmacological inhibition by osw- and -hc blocked viral genome replication. cholesterol was redistributed to emcv ros upon infection, and treatment with al- or osw- resulted in a significantly reduced colocalization of cholesterol with ab, arguing that accumulation of cholesterol at ros is mediated by both pi ka and osbp. these data are in agreement with our recent findings that cholesterol shuttling is important for cardiovirus genome replication [ ] and that cardioviruses are sensitive to itraconazole, which we recently discovered to be an. inhibitor of osbp [ ] . our results indicate that pi p and cholesterol are vital for the global organization of emcv ros. however, as these lipids fulfill multiple functions in various cellular processes [ , [ ] [ ] [ ] , other roles in virus replication should be envisaged. a potential task of pi p in virus replication may be linked to the pi( , )p synthesis pathway, since pi p is the major precursor of pi( , ) p lipids, which were recently attributed an important role in hcv replication [ ] . cholesterol homeostasis was recently shown to play an important role in efficient pv polyprotein processing [ ] . whether cholesterol also ensures a proper microenvironment that supports cardiovirus polyprotein processing remains to be determined. interestingly and in apparent parallel with the distantly related enteroviruses, exploitation of the pi k-osbp pathway by hcv correlates with the induction of membranes of positive curvature [ ] . by contrast, the flavivirus denv, although closely related to hcv, does not require pi k or osbp [ ] and generates membranes of negative curvature [ ] . hence, the interplay between pi p and cholesterol may dictate the positive curvature of the membranes at which diverse rna viruses replicate their genomes. through co-ip assays, we identified pi ka as a novel interaction partner of emcv proteins a and its precursor ab. emcv a is a small protein ( amino acids) of unknown structure, containing a predicted hydrophobic domain in the c-terminus half. expression of a alone was sufficient for pi ka recruitment in intact cells, arguing that in infection, pi ka is recruited to ros by this viral protein. enteroviruses and kobuviruses recruit pi kb to ros also via their a protein [ , , , ] , raising the possibility that diverse picornaviruses might use an evolutionary conserved and a-mediated mechanism to generate pi p-enriched membranes. however, the a proteins of entero-, kobu-and cardioviruses do not share any apparent sequence similarity, their name simply reflecting the occupancy of the same locus ( a) in the respective viral genomes. with the exception of their catalytic domain, also the pi ka and pi kb isoforms do not share any sequence similarity [ ] . furthermore, unlike a of most enteroviruses, cardiovirus a does not interact with gbf nor blocks protein transport in the secretory pathway when expressed alone [ ] , highlighting the functional diversification associated with these small viral proteins. emcv and hcv converged to recruit a common lipid-modifying pathway in building replication sites several lines of evidence suggest that the picornavirus emcv and the distantly related flavivirus hcv have evolved to exploit common host components in assisting virus rna replication. first, hcv genome replication occurs at the "membranous web" (mw), a network of single and dmvs that, like the emcv ro, also mainly originates from the er [ ] . second, both emcv and hcv express a viral protein dedicated to recruitment of pi ka, in order to induce a pi p-rich environment at the replication sites [ , ] . third, in hcv infection, pi p lipids were also shown to be important for the recruitment of osbp, which mediates cholesterol transfer to the mw [ ] . fourth, inhibition of either pi ka or osbp induced clear alterations in the global distribution of emcv ros, which appeared more "clustered" upon treatment with al- or osw- . a similar clustering effect was also observed for replication structures of the hcv mw upon pi ka or osbp inhibition [ , ] , whereas no obvious disruption of the enterovirus ros was observed upon pi kb or osbp inhibition [ , , ] . together, these observations indicate that emcv and hcv replication structures share critical host components, and possibly also a similar architecture, although the latter still remains to be determined. based on at least two lines of emerging evidence in the context of phylogeny of flavi-and picornaviruses, emcv and hcv have likely converged on rather than retained their functional similarities upon divergence from the common ancestor (fig ) . first, the observed commonalities between emcv and hcv are not shared by other characterized viruses in their respective families, indicating that they are not a manifestation of the properties conserved among the two families. for instance, picornaviruses from different genera exhibit different host factor requirements. members of the cardiovirus genus hijack the er-localized pi ka (this study), whereas members of the enterovirus and kobuvirus genera depend on the golgi-localized pi kb [ , , ] . in contrast, equine rhinitis a virus (erav, member of aphthovirus genus, which is prototyped by fmdv) and hepatitis a virus (hepatovirus genus) seem to replicate independent of both pi kb and pi ka (s fig and [ , ] ). likewise, the flaviviruses denv and wnv, representing a sister genus to that of hcv, do not rely on either pi ka or pi kb [ , ] . while denv was also shown not to require osbp [ ] , for wnv this is not known yet. importantly, cardioviruses targeting pi ka occupy a lineage that is farther from the root compared to those of entero-and kobuviruses targeting pi kb (fig ) . this phylogenetic pattern is indicative of the relatively recent emergence of the emcv-specific target properties. second, emcv and hcv employ apparently unrelated proteins to mediate the interaction with pi ka, namely a and ns a (although hcv ns b may contribute as well [ , ] ). both proteins are membrane-bound, albeit through a hydrophobic domain located at either n-terminus (ns a) or in the c-terminus-half ( a), and each includes another region which is among the least conserved in the nonstructural proteins of the respective families [ , ] . our study contributes to the hypothesis that viruses may be confronted with powerful constraints that limit the diversity of host pathways recruited for efficient replication. thus, a common pathway is used by different rna viruses that either only moderately diverged (e.g. different species of same genus) or converged on a host target while diverging profoundly (different families-e.g. emcv and hcv). to date, only a small number of (+)rna viruses have been studied in terms of host lipid requirements. identification of the lipid pathways used by other viruses will hopefully provide a deeper insight on the constraints that viruses are confronted with during the endeavor to replicate their genome. buffalo green monkey (bgm) kidney cells, baby hamster kidney (bhk- ) and hela r cells were grown at °c and % co in dulbecco's modified eagle's medium (dmem, lonza) supplemented with % fetal bovine serum (fbs). huh lunet/t cells (provided by r. bartenschlager, department of molecular virology, university of heidelberg, heidelberg, germany) [ ] were grown in dmem (lonza) supplemented with % fbs and μg/ml blasticidin (paa) . bgm cells were purchased from ecacc and bhk- cells were purchased from atcc. hela r cells were obtained from g. belov (university of maryland and virginia-maryland regional college of veterinary medicine, us) [ ] . al- and osw- were kind gifts from j. neyts (rega institute for medical research, university of leuven, leuven, belgium) and m.d. shair (department of chemistry and chemical biology, harvard university, cambridge, usa) respectively. -hc was purchased from santa cruz. bf [ ] was provided by galapagos nv. filipin iii and dipyridamole were from sigma. [ ] , and the entire tree is rooted according to gibrat et al., [ ] . *in addition to ns a, ns b has also been suggested to be involved in the interaction with pi ka [ , ] the picornavirus emcv converges on the host lipid pathway used by hcv plasmids constructs ptm-ha-pi ka [ ] , pegfp-pi ka (provided by g. hammond, nichd, national institutes of health, bethesda, usa) [ , ] and p a(cvb )-myc [ ] were described previously. pgfp-pi kb was a kind gift from n. altan-bonnet (laboratory of host-pathogen dynamics, national institutes of health, bethesda, usa). to generate c-terminal myc-tagged emcv proteins, genes encoding emcv nonstructural proteins a, b, c, a, ab, c and d were amplified by pcr using the plasmid pm . [ ] and primers introducing restriction sites bamhi and hindiii. pm . contains the full-length infectious cdna sequence of emcv, strain mengovirus. the pcr products were then cloned into the p a (cvb )-myc backbone from which the cvb - a gene was removed using the same restriction enzymes. to allow ectopic expression of pi ka under a cmv promoter, the gene encoding ha-pi ka was amplified by pcr using ptm-ha-pi ka as template and introduced in the pegfp-n backbone using restriction enzymes sali and noti. emcv- c-ha infectious clone was generated by introducing the ha coding sequence (ypydvpdya) in-frame after codon in c of pm . using mutagenesis primers and the q site-directed mutagenesis kit (new england biolabs). emcv, emcv- c-ha and rluc-emcv, which contains the renilla luciferase gene upstream of the capsid coding region [ ] , were obtained by transfecting bhk- cells with rna transcripts derived from full length infectious clones pm . , pm . - c-ha and prluc-qg-m . , respectively, linearized with bamhi. gfp-emcv, which contains the egfp gene upstream the capsid region, was generated similar as rlucemcv [ ] . cvb (strain nancy) was obtained by transfecting bgm cells with rna transcripts of the full length infectious clone p cb /t [ ] linearized with sali. saffold virus (type ) was described previously [ ] . erav (nm / ) was kindly provided by david rowlands and toby tuthill (university of leeds, united kingdom). virus infections were performed by incubating subconfluent cell monolayers for min at °c with virus, after which the virus-containing medium was removed and fresh (compound-containing) medium was added to the cells (t = ). in the time-of-addition experiments, medium without compound was added at t = and replaced by medium with compound at the indicated time points. at the given time points post infection, cells were either fixed for immunolabeling, freeze-thawed to determine virus titers or, in the case of rluc-emcv, lysed to determine replication by measuring the intracellular renilla luciferase activity using the renilla luciferase assay system (promega). virus titers were determined by endpoint titration according to the method of reed and muench and expressed as % tissue culture infective doses (tcid ). hela r or huh lunet/t cells were grown to subconfluency on coverslips in -well plates. where indicated, cells were transfected with ng of plasmids using lipofectamine according to the manufacturer's protocol and/or infected with emcv at the specified multiplicity of infection (moi), followed by compound treatment where specified. at the indicated time points, cells were fixed with % paraformaldehyde (pfa) for min at room temperature (rt). permeabilization was done with pbs- . % triton x- for min or pbs/ . % saponin/ % bsa for min, in the case of filipin staining. cells were incubated sequentially with primary and secondary antibodies diluted in pbs containing % normal goat serum (ngs). the following primary antibodies were used for detection: mouse monoclonal anti-gm (bd biosciences), rabbit polyclonal anti-tgn (novus biologicals), mouse monoclonal anti-ergic (enzo life sciences), rabbit polyclonal anti-sec (kindly provided by b.l tang, department of biochemistry, the national university of singapore, singapore), rabbit polyclonal anti-calreticulin (sigma), rabbit polyclonal anti-ha (santa cruz), mouse monoclonal anti-ha (abcam), mouse monoclonal anti-c-myc (sigma), rabbit polyclonal anti-myc (thermo scientific), mouse anti-pi p igm (echelon biosciences), mouse monoclonal anti-dsrna (j , english & scientific consulting), mouse monoclonal anti-emcv ab (kind gift from a.g. aminev) [ ] and rabbit polyclonal anti-osbp (kindly provided by m.a. de matteis, telethon institute of genetics and medicine, naples, italy) [ ] . alexa fluor -, -conjugated igg and alexa fluor -or -conjugated igm (invitrogen, molecular probes) were used as secondary antibodies. cholesterol was stained with μg/ml filipin iii (sigma) for h at room temperature, included during the incubation with the secondary antibody. nuclei were counterstained with dapi. staining of plasma membrane or intracellular pi p was performed as described elsewhere [ ] . briefly, for pm staining, cells were fixed at rt in % pfa and . % glutaraldehyde. all subsequent steps were performed on ice. cells were blocked and permeabilized for min in buffer a ( mm pipes, ph . , mm nacl, . mm kcl) containing % ngs, mm nh cl and . % saponin. slides were incubated with primary and secondary antibodies in buffer a containing % ngs and . % saponin for h. finally, slides were post-fixed in % pfa in pbs for min. the intracellular pi p staining was performed at rt as follows: cells were fixed with % pfa, then permeabilized for min in μm digitonin in buffer a, blocked for min in buffer a with % ngs and mm nh cl and then incubated sequentially with primary and secondary antibodies in buffer a with % ngs, before post fixation in % pfa. all coverslips were mounted with fluorsave (calbiochem). images were acquired with a leica spe-ii dmi- confocal laser scanning microscope or a nikon ti eclipse microscope equipped with an andor du- emccd-camera. pi p quantification was performed for at least cells for each condition, using the imagej software as described elsewhere [ ] . to determine colocalization of sec or calreticulin with ab, images were first deconvoluted using nis advanced research . software (nikon) ( iterations) and further processed using image j as follows. individual infected cells were outlined and a mask was created, and all signal outside the mask was cropped to exclude it from the calculations. manders' colocalization coefficient was calculated for at least cells for each condition using the jacop plugin [ ] with a manually set threshold. colocalization of osbp with a in infected cells was analyzed using imagej by determining pearson's coefficient for at least cells per condition using the coloc plugin with default settings. to quantify colocalization of filipin with ab, images were first deconvoluted using nis software ( iterations), then imagej was used to select infected cells and the pearson's coefficient of colocalization for at least cells per condition was calculated using the coloc plugin with default settings. hela r cells were reverse-transfected with pmoles of sirna per well of a -well plate ( cells/well) using lipofectamine (invitrogen) according to the manufacturer's indications. scrambled sirna (allstars neg. control, qiagen) was used as a control. sirna against hpi ka (cat. no. s ) and hpi kb (target sequence: '-uguuggggc uucccugccctt- ') were from qiagen. sirna against hosbp (two sirnas mixed at : ratio, cell viability was determined in parallel with virus infection as follows. one day after seeding cells in a -well plate, the compounds were added to the cells and incubated for h. alternatively, cells were transfected with sirnas and incubated for h. subsequently, the medium was replaced with celltiter aqueous one solution reagent (promega) and optical densities were measured at nm. the obtained raw values were converted to percentage of untreated samples or samples transfected with scrambled sirnas, following correction for background absorbance. metabolic labeling of myc-tagged emcv proteins and ha-pi ka was performed as described elsewhere [ ] . briefly, huh -lunet/t cells seeded in -well plates were co-transfected with μg of plasmid encoding emcv nonstructural proteins and μg of either ptm ha-pi kiiia or an empty ptm vector (mock) using lipofectamine (invitrogen) according to the manufacturer's instructions. h later, cells were starved in methionine/cysteine-free medium for h. radiolabeling of cells was done by overnight incubation in methionine/cysteine-free medium, supplemented with mm glutamine, mm hepes, and μci/ml of express protein labeling mix (perkin elmer, boston). cells were then harvested and lysed in lysis buffer ( mm tris-cl [ph . ], mm nacl, % nonidet p- and protease inhibitors) for h on ice, followed by centrifugation at , g for min at °c. supernatants were further subjected to immunoprecipitation by a h incubation at °c with anti-c-myc rabbit polyclonal antibody (santa cruz). immunocomplexes were then captured with protein g-sepharose beads (sigma) by an additional h incubation at °c. beads were washed three times in lysis buffer, followed by elution of immunocomplexes by boiling in sample buffer, separation by polyacrylamide-sds gel electrophoresis and detection by autoradiography. for co-ip followed by western blot, cells were seeded in cm dishes and transfected with . μg of each plasmid using polyethylenimine (pei) (polysciences). immunoprecipitation was carried out as described above, but using protein a-sepharose beads (ge healthcare) and mouse monoclonal anti-c-myc (sigma) or rabbit polyclonal anti-myc (thermo scientific) antibodies. samples separated by sds-page were transferred to nitrocellulose membranes (bio-rad). membranes were incubated with the following primary antibodies: rabbit polyclonal anti-pi ka (cell signaling), rabbit polyclonal anti-pi kb (upstate), rabbit polyclonal anti-osbp (proteintech), rabbit polyclonal anti-emcv capsid (kind gift from ann palmenberg) and mouse monoclonal anti-β-actin (sigma). secondary antibodies included irdye -conjugated goat anti-mouse or irdye -conjugated goat anti-rabbit (li-cor). images of blots were acquired with an odyssey fc imaging system (li-cor). where indicated, unpaired one-tailed student's t-test or two-tailed mann-whitney test were applied as statistical analyses using the graphpad prism software. supporting information s fig. al- inhibits emcv at the step of genome replication. (a) al- is effective against emcv also at high moi. hela r cells were infected with rluc-emcv at an moi of followed by al- treatment for h, after which cells were lysed and virus replication was measured by determining the renilla luciferase activity. (b) al- blocks emcv genome replication. hela r cells were infected with rluc-emcv at an moi of . at the indicated time points, dmso, μm al- or μm dipyridamole was added to the cells and virus replication was measured by determining the renilla luciferase activity at h p.i. bars represent mean values of triplicates ± standard error of the means (sem). means were statistically compared using unpaired t tests. hela r cells were mockinfected or infected with gfp-emcv at moi . at h p.i., cells were fixed and stained with antibodies against pi p using specific protocols for detection of plasma membrane and intracellular pi p pools [ ] . nuclei were stained with dapi (blue). scale bars represent μm. the genetics of the persistent infection and demyelinating disease caused by theiler's virus saffold virus, a human theiler's-like cardiovirus, is ubiquitous and causes infection early in life molecular analysis of encephalomyocarditis viruses isolated from pigs and rodents in italy persistence of encephalomyocarditis virus (emcv) infection in piglets reproductive failure in pigs caused by encephalomyocarditis virus reproductive failure in sows following experimental infection with a belgian emcv isolate +)rna viruses rewire cellular pathways to build replication organelles rewiring of cellular membrane homeostasis by picornaviruses requirements for assembly of poliovirus replication complexes and negative-strand rna synthesis induction of membrane proliferation by poliovirus proteins c and bc membrane rearrangement and vesicle induction by recombinant poliovirus c and bc in human cells a viral protein that blocks arf -mediated cop-i assembly by inhibiting the guanine nucleotide exchange factor gbf molecular determinants of the interaction between coxsackievirus protein a and guanine nucleotide exchange factor gbf hijacking components of the cellular secretory pathway for replication of poliovirus rna viral reorganization of the secretory pathway generates distinct organelles for rna replication recruitment of pi kiiiβ to coxsackievirus b replication organelles is independent of acbd , gbf , and arf rhinovirus uses a phosphatidylinositol -phosphate/cholesterol counter-current for the formation of replication compartments at the er-golgi interface phosphatidylinositol- kinase iii beta and oxysterol-binding protein accumulate unesterified cholesterol on poliovirus-induced membrane structure a four-step cycle driven by pi( )p hydrolysis directs sterol/pi( )p exchange by the er-golgi tether osbp complex dynamic development of poliovirus membranous replication complexes the transformation of enterovirus replication structures: a three-dimensional study of single-and doublemembrane compartments oxysterol-binding protein is a phosphatidylinositol -kinase effector required for hcv replication membrane integrity and cholesterol trafficking recruitment and activation of a lipid kinase by hepatitis c virus ns a is essential for integrity of the membranous replication compartment morphological and biochemical characterization of the membranous hepatitis c virus replication compartment hepatitis c virus stimulates the phosphatidylinositol -kinase iii alpha-dependent phosphatidylinositol -phosphate production that is essential for its replication autophagy promotes the replication of encephalomyocarditis virus in host cells inhibition of cellular autophagy deranges dengue virion maturation coxsackievirus b exits the host cell in shed microvesicles displaying autophagosomal markers nonlytic viral spread enhanced by autophagy components phosphatidylserine vesicles enable efficient en bloc transmission of enteroviruses gbf , a guanine nucleotide exchange factor for arf, is crucial for coxsackievirus b rna replication differential effects of the putative gbf inhibitors golgicide a and ag on enterovirus replication a novel, broad-spectrum inhibitor of enterovirus replication that targets host cell factor phosphatidylinositol -kinase iiiβ coxsackievirus mutants that can bypass host factor pi kiiiβ and the need for high levels of pi p lipids for replication metabolism of phosphatidylinositol -kinase iiiα-dependent pi p is subverted by hcv and is targeted by a -anilino quinazoline with antiviral activity cytopathology of mengovirus infection ii. proliferation of membranous cisternae effect of mengovirus replication on choline metabolism and membrane formation in novikoff hepatoma cells differential requirements for copi coats in formation of replication complexes among three genera of picornaviridae mapping of functional domains of the lipid kinase phosphatidylinositol -kinase type iii alpha involved in enzymatic activity and hepatitis c virus replication ptdins p synthesis by pi kiiiα at the plasma membrane and its impact on plasma membrane identity determinants of membrane association for poliovirus protein ab properties of purified recombinant poliovirus protein ab as substrate for viral proteinases and as co-factor for rna polymerase dpol interaction between the '-terminal cloverleaf and ab/ cdpro of poliovirus is essential for rna replication the a protein from multiple picornaviruses utilizes the golgi adaptor protein acbd to recruit pi kiiiβ the lipid kinase phosphatidylinositol- kinase iii alpha regulates the phosphorylation status of hepatitis c virus ns a a plasma membrane pool of phosphatidylinositol -phosphate is generated by phosphatidylinositol -kinase type-iii alpha: studies with the ph domains of the oxysterol binding protein and fapp maintenance of hormone-sensitive phosphoinositide pools in the plasma membrane requires phosphatidylinositol -kinase iiialpha pharmacological and genetic targeting of the pi ka enzyme reveals its important role in maintaining plasma membrane phosphatidylinositol -phosphate and phosphatidylinositol , -bisphosphate levels immunocytochemical techniques reveal multiple, distinct cellular pools of ptdins p and ptdins( , )p( ) phosphatidylinositol -kinase iiibeta regulates the transport of ceramide between the endoplasmic reticulum and golgi targeting of golgi-specific pleckstrin homology domains involves both ptdins -kinase-dependent and-independent components the multiple roles of ptdins( )p-not just the precursor of ptdins( , )p cholesterol shuttling is important for rna replication of coxsackievirus b and encephalomyocarditis virus natural products reveal cancer cell dependence on oxysterol-binding proteins translocation of oxysterol protein to golgi apparatus triggered by ligand binding acbd -mediated recruitment of pi kb to picornavirus rna replication sites three-dimensional architecture and biogenesis of membrane structures associated with hepatitis c virus replication modulation of lipid synthesis and trafficking pathways by picornaviruses membranous replication factories induced by plus-strand rna viruses ultrastructure of the replication sites of positive-strand rna viruses architecture and biogenesis of plus-strand rna virus replication factories replication of theiler's murine encephalomyelitis viruses in bhk cells: an electron microscopic study increased long chain acyl-coa synthetase activity and fatty acid import is linked to membrane synthesis for development of picornavirus replication organelles involvement of membrane traffic in the replication of poliovirus genomes: effects of brefeldin a visualizing coronavirus rna synthesis in time by using click chemistry sars-coronavirus replication is supported by a reticulovesicular network of modified endoplasmic reticulum the lipid kinase pi kiiiβ preserves lysosomal identity gabaraps regulate pi p-dependent autophagosome:lysosome fusion cinderella story: pi p goes from precursor to key signaling molecule phosphatidylinositol , -bisphosphate is an hcv ns a ligand and mediates replication of the viral genome enteroviruses harness the cellular endocytic machinery to remodel the host cell cholesterol landscape for effective viral replication composition and three-dimensional architecture of the dengue virus replication and assembly sites phosphatidylinositol -kinases: old enzymes with emerging functions effects of picornavirus a proteins on protein transport and gbf -dependent cop-i recruitment novel perspectives for hepatitis a virus therapy revealed by comparative analysis of hepatitis c virus and hepatitis a virus rna replication west nile virus replication requires fatty acid synthesis but is independent on phosphatidylinositol- -phosphate lipids the ns a protein of hepatitis c virus is a zinc metalloprotein analyses of the radiation of birnaviruses from diverse host phyla and of their evolutionary affinities with other double-stranded rna and positive strand rna viruses using robust structure-based multiple sequence alignments and advanced phylogenetic metho role of annexin a in the production of infectious hepatitis c virus particles poliovirus proteins induce membrane association of gtpase adpribosylation factor identification of a series of compounds with potent antiviral activity for the treatment of enterovirus infections a novel probe for phosphatidylinositol -phosphate reveals multiple pools beyond the golgi a proline-rich region in the coxsackievirus a protein is required for the protein to inhibit endoplasmic reticulum-to-golgi transport cloning and synthesis of infectious cardiovirus rnas containing short, discrete poly(c) tracts encephalomyocarditis viral protein a localizes to nucleoli and inhibits cap-dependent mrna translation a guided tour into subcellular colocalization analysis in light microscopy we are grateful to raffaele defrancesco, francesco peri, petra neddermann and johan neyts for providing al- , matthew shair for osw- , gerrald hammond for the plasmid pegfp-pi ka, nihal altan-bonnet for the plasmid pgfp-pi kb, maria antonietta de matteis for the rabbit polyclonal antibody against osbp, aleksey aminev for the mouse monoclonal antibody against ab (emcv), ann palmenberg for the rabbit polyclonal antibody against emcv capsid, bor luen tang for the rabbit polyclonal antibody against sec and the center for cell imaging (faculty of veterinary medicine, utrecht university) for support with microscopy experiments. key: cord- -c wp m authors: morens, david m.; fauci, anthony s. title: emerging infectious diseases: threats to human health and global stability date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: c wp m nan historical information as well as microbial sequencing and phylogenetic constructions make it clear that infectious diseases have been emerging and reemerging over millennia, and that such emergences are driven by numerous factors (table ) . notably, to percent of new human infections likely originated in animals, disproportionately rodents and bats, as shown by the examples of hantavirus pulmonary syndrome, lassa fever, and nipah virus encephalitis [ ] [ ] [ ] . most other emerging/reemerging diseases result from human-adapted infectious agents that genetically acquire heightened transmission and/or pathogenic characteristics. examples of such diseases include multidrug-resistant and extensively drugresistant (mdr and xdr) tuberculosis, toxin-producing staphylococcus aureus causing toxic shock syndrome, and pandemic influenza [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . although precise figures are lacking, emerging infectious diseases comprise a substantial fraction of all consequential human infections. they have caused the deadliest pandemics in recorded human history, including the black death pandemic (bubonic/ pneumonic plague; - million deaths) in the fourteenth century, the influenza pandemic ( million deaths), and the hiv/aids pandemic ( million deaths so far) [ , ] . two major categories of emerging infections-newly emerging and reemerging infectious diseases-can be defined, respectively, as diseases that are recognized in the human host for the first time; and diseases that historically have infected humans, but continue to appear in new locations or in drug-resistant forms, or that reappear after apparent control or elimination [ ] . emerging/ reemerging infections may exhibit successive stages of emergence. these stages include adaptation to a new host [ ] , an epidemic/ pathogenic stage, an endemic stage, and a fully adapted stage in which the organism may become nonpathogenic and potentially even beneficial to the new host (e.g., the human gut microbiome) or stably integrated into the host genome (e.g., as endogenous retroviruses). although these successive stages characterize the evolution of certain microbial agents more than others, they nevertheless can provide a useful framework for understanding many of the dynamic relationships between microorganisms, human hosts, and the environment. it is also worth noting that the dynamic and complicated nature of many emerging infections often leaves distinctions between emerging and reemerging infections open to question, leading various experts to classify them differently. for example, we describe as ''reemerging'' new or more severe diseases associated with acquisition of new genes by an existing microbe, e.g., antibiotic resistance genes, even when mutations cause entirely new diseases with unique clinical epidemiologic features, e.g., brazilian purpuric fever [ ] . similarly, we refer to sars as an emerging disease a decade after it disappeared, and apply the same term to the related mers (middle east respiratory syndrome) b coronavirus which appeared in saudi arabia in late [ ] . the most salient modern example of an emerging infectious disease is hiv/aids, which likely emerged a century ago after multiple independent events in which the virus jumped from one primate host to another (chimpanzees to humans) and subsequently, as a result of a complex array of social and demographic factors, spread readily within the human population. aids was not recognized as a distinct entity until [ , ] , after its initial detection among certain risk groups, such as men who have sex with men, recipients of blood products, and injection drug users. it was soon apparent, however, that the disease was not restricted to these groups, and indeed, the bulk of hiv infections globally has resulted from heterosexual transmission that has been heavily weighted within the developing world, particularly sub-saharan africa where a number of factors were responsible for this rapid spread; chief among these were human movement along truck routes accompanied by a high level of commercial sex work, inadequate public health infrastructures, poverty, and social inequality. this is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. the work is made available under the creative commons cc public domain dedication. funding: both authors are employees of nih and as such our work is funded by nih from operating funds rather than grants or other awards. competing interests: the authors have declared that no competing interests exist. other examples of disease emergences [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] include sars, which emerged from bats and spread into humans first by personto-person transmission in confined spaces, then within hospitals, and finally by human movement between international air hubs. nipah virus also emerged from bats and caused an epizootic in herds of intensively bred pigs, which in turn served as the animal reservoir from which the virus was passed on to humans. the h n pandemic influenza virus emerged from pigs as well, but only after complex exchanges of human, swine, and avian influenza genes [ ] . h n influenza emerged from wild birds to cause epizootics that amplified virus transmission in domestic poultry, precipitating dead-end viral transmission to poultryexposed humans. additional examples are many [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] ; however, the variables associated with emergences are unique for each and typically complex. most of the important reemerging infectious disease agents first appeared long ago, but have survived and persisted by adapting to changing human populations and to environments that have been altered by humans. dengue virus and west nile virus (wnv), distantly related flaviviruses, serve as good examples. they have been spread by geographic movement of humans in association with the mosquito vectors for the diseases. for example, dengue came to the americas in association with the slave trade of earlier centuries. in this regard, slaves infected by mosquitoes in africa presumably brought the infection to the americas by seeding the mosquito population upon arrival [ ] . similarly, wnv came to the united states in when an infected human, bird, or mosquito came by air travel from the middle east to the western hemisphere, providing a source for introduction of infection to new world mosquitoes and birds. pathogenic strains of dengue have also spread back from southeast asia to the western hemisphere, as has a major mosquito vector, aedes albopictus. unlike most arboviruses, which are partly or completely host-restricted, wnv has become adapted to multiple mosquito and avian species, a major factor in increasing its opportunity to infect humans. the lack of additional hosts undoubtedly drove the mosquitoes that are the vectors of dengue and the dengue virus itself to favor adapting to humans and to their behaviors and environments. the association of dengue with aedes mosquitoes that live in and around human habitations mean that crowding, poor sanitation, and poverty provide ideal environments for transmission to humans [ ] . host immunity factors are also thought to be involved in the severe/fatal form of dengue known as dengue shock syndrome [ ] . other non-arboviral examples of emerging infections abound. for example, cholera has repeatedly reemerged over more than two centuries in association with global travel, changing seasons, war, natural disasters, and conditions that lead to inadequate sanitation, poverty, and social disruption. emergences of disease caused by community-and hospital-acquired clostridium difficile and methicillin-resistant staphylococcus aureus (mrsa) have been driven by increased and/or inappropriate use of antibiotics, and some hospital-acquired organisms such as mrsa have now moved into community transmission. the global emergence of plasmid-spread ndm- (new delhi b-lactamase) gram-negative pan-resistant organisms, linked to global antibiotic use and inadequate antibiotic stewardship, medical tourism, economic globalization, and other aspects of modern life, has prompted calls for development of international control mechanisms [ ] that are applicable to a number of emerging bacterial diseases in the developing and developed world. drug resistance mutations have also caused the reemergences of certain pathogens such as multidrug-resistant and extensively drug-resistant tuberculosis, drug-resistant malaria, and numerous bacterial diseases such as vancomycin-resistant enterococci. fungi have made significant contributions to disease emergence as well. in africa, cryptococcal disease has already surpassed tuberculosis as a leading cause of death [ ] . other examples of fungal emergence include comorbidities in hiv-infected individuals ( ) , cryptococcus gattii epidemics in predominantly healthy persons in the u.s. [ , ] , and a u.s. nationwide epidemic of exserohilum rostratum infections associated with contaminated pharmaceutical products [ ] . while it has become possible to eradicate certain infectious diseases (smallpox and the veterinary disease rinderpest), and to significantly control many others (dracunculiasis, measles, and polio, among others), it seems unlikely that we will eliminate most emerging infectious diseases in the foreseeable future. pathogenic microorganisms can undergo rapid genetic changes, leading to new phenotypic properties that take advantage of changing host and environmental opportunities. influenza viruses serve as a good example of emerging and reemerging infectious agents in their ability to rapidly evolve in response to changing host and environmental circumstances via multiple genetic mechanisms. new ''founder'' influenza viruses [ ] appear periodically, cause a pandemic, raise widespread population immunity, and then, in table . some major factors that underlie disease emergence and reemergence [ , ] . response to human immune pressures, evolve and persist for decades using multiple genetic evolutionary mechanisms to sustain continual immune escape. the influenza pandemic virus is one example: over the past years, its descendants have evolved continually by antigenic drift, intra-subtypic reassortment, and antigenic shift, the latter producing new pandemics in and [ ] . even the genetically complex pandemic h n influenza virus is a descendant of the virus [ ] . such continuous genetic hyper-evolution forces us to develop new influenza vaccines containing new antigens on an annual basis. in the meantime, new human diseases keep emerging. as noted, in late the novel mers coronavirus emerged in saudi arabia [ ] , and in early a new h n avian influenza virus became epizootic in eastern china, causing spillover infections of humans (as of june , ), with percent case fatality [ , ] . its pandemic potential, if any, remains to be determined. whether or not such outbreaks become more widespread, they nonetheless attract global attention and require significant international effort to monitor and contain. microbial advantages can be met and overcome only by aggressive vigilance, ongoing dedicated research, and rapid development and deployment of such countermeasures as surveillance tools, diagnostics, drugs, and vaccines. we appear to be entering a new era in which several important emerging, reemerging, and stable infectious diseases are becoming better controlled (e.g., hepatitis b, rabies, haemophilus influenzae type b, and even to some extent hiv/aids). however, our success in stopping the many new emerging diseases that will inevitably appear is not assured. we have many tools in our armamentarium, including preparedness plans and stockpiles of drugs and vaccines. but each new disease brings unique challenges, forcing us to continually adapt to ever-shifting threats [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] ] . the battle against emerging infectious diseases is a continual process; winning does not mean stamping out every last disease, but rather getting out ahead of the next one. the perpetual challenge of infectious diseases emerging infections: microbial threats to health in the united states factors and determinants of disease emergence ecology of zoonoses: natural and unnatural histories the challenge of emerging and reemerging infectious diseases emerging infections: a perpetual challenge prediction and prevention of the next pandemic zoonosis emerging infectious diseases in : years after the institute of medicine report the world must build on three decades of scientific advances to enable a new generation to live free of hiv/aids world health organization. global alert and response (gar) crossspecies virus transmission and the emergence of new epidemic diseases tracing phylogenomic events leading to diversity of haemophilus influenzae and the emergence of brazilian purpuric fever (bpf)-associated clones genome characterization of a newly discovered coronavirus associated with acute respiratory distress syndrome in humans the persistent legacy of the influenza virus dengue research opportunities in the americas the emergence of pan-resistant gram-negative pathogens merits a rapid global political response estimation of the current global burden of cryptococcal meningitis among persons living with hiv/aids genome variation of cryptococcus gattii, an emerging pathogen of immunocompetent hosts the triple threat of cryptococcosis: it's the body site, the strain, and/or the host multistate outbreak of fungal infection associated with injection of methylprednisolone acetate solution from a single compounding pharmacy -united states reconstruction of the influenza virus: unexpected rewards from the past preliminary report: epidemiology of the avian influenza a (h n ) outbreak in china drivers, dynamics, and control of emerging vector-borne zoonotic diseases key: cord- -ojyxs cp authors: gaynor, anne m; nissen, michael d; whiley, david m; mackay, ian m; lambert, stephen b; wu, guang; brennan, daniel c; storch, gregory a; sloots, theo p; wang, david title: identification of a novel polyomavirus from patients with acute respiratory tract infections date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: ojyxs cp we report the identification of a novel polyomavirus present in respiratory secretions from human patients with symptoms of acute respiratory tract infection. the virus was initially detected in a nasopharyngeal aspirate from a -year-old child from australia diagnosed with pneumonia. a random library was generated from nucleic acids extracted from the nasopharyngeal aspirate and analyzed by high throughput dna sequencing. multiple dna fragments were cloned that possessed limited homology to known polyomaviruses. we subsequently sequenced the entire virus genome of , bp, henceforth referred to as wu virus, and found it to have genomic features characteristic of the family polyomaviridae. the genome was predicted to encode small t antigen, large t antigen, and three capsid proteins: vp , vp , and vp . phylogenetic analysis clearly revealed that the wu virus was divergent from all known polyomaviruses. screening of , patients with acute respiratory tract infections in brisbane, queensland, australia, and st. louis, missouri, united states, using wu virus–specific pcr primers resulted in the detection of additional specimens that contained wu virus. the presence of multiple instances of the virus in two continents suggests that this virus is geographically widespread in the human population and raises the possibility that the wu virus may be a human pathogen. viral infections of the respiratory tract are responsible for significant mortality and morbidity worldwide [ ] . despite extensive studies in the past decades that have identified a number of etiologic agents, including rhinoviruses, coronaviruses, influenzaviruses, parainfluenzaviruses, respiratory syncytial virus, and adenoviruses, approximately % of all cases cannot be attributed to these agents, suggesting that additional respiratory pathogens are likely to exist [ ] . in fact, since , six previously undescribed viruses have been identified by analysis of clinical specimens from the human respiratory tract: human metapneumovirus [ ] , sars coronavirus [ ] , coronavirus nl [ ] , coronavirus hku [ ] , human bocavirus [ ] , and the recently described ki virus [ ] . in some instances, new molecular methods such as vidisca [ ] , pan-viral dna microarrays [ ] , and high throughput sequencing [ , ] have played key roles in the identification of these agents. the advent of these new technologies has greatly stimulated efforts to identify novel viruses in the respiratory tract and in other human disease states. viruses in the family polyomaviridae possess doublestranded dna genomes and infect a variety of avian, rodent, and primate species. to date, two polyomaviruses, bk virus and jc virus, have been unambiguously described as human pathogens. bk and jc viruses are ubiquitous worldwide, and in adult populations, seroprevalence rates approaching % and %, respectively, have been reported [ ] . although human polyomaviruses have been suggested to utilize a respiratory route of transmission, detection of bk and jc polyomavirus nucleic acids in the respiratory tract has rarely been reported [ , ] . infection with these two viruses is predominantly asymptomatic, although in the context of immunosuppression a number of syndromes have been clearly linked to these viruses. jc virus causes primary multifocal leukoencephalopathy, while bk virus has been associated with a variety of renal and urinary tract disorders, most importantly tubular nephritis, which can lead to allograft failure in renal transplant recipients and hemorrhagic cystitis in hematopoietic stem cell transplant recipients [ ] . these viruses are believed to persist in a latent phase primarily in the kidney and can periodically undergo reactivation. excretion of bk and jc viruses in urine has been reported in up to % of the general population [ , ] . besides jc and bk virus, a very recent report has described a novel polyomavirus, ki, detected in human respiratory secretions and stool [ ] . however, the pathogenicity and prevalence of this virus has not yet been established. in addition, in the late s, ; million people in the united states, and many more worldwide, may have been exposed to sv , a polyomavirus that naturally infects rhesus monkeys via contaminated polio vaccines, leading to widespread debate about whether or not sv is capable of sustained infection and replication cycles in humans [ ] . much of the interest in polyomaviruses and sv in particular derives from the transforming properties carried by the early transcriptional region of the viral genome that encodes for the small t antigen (stag) and and large t antigen (ltag). t antigen is capable of binding both p and rb proteins and interfering with their tumor suppressor functions. the early region alone is sufficient to transform established primary rodent cell lines [ ] and in concert with telomerase and ras transforms primary human cells [ ] . this has lead to controversy over whether any human tumors are associated with sv infection [ ] . we describe the identification and characterization of a novel polyomavirus initially detected by high throughput sequencing of respiratory secretions from a patient suffering acute respiratory disease of unknown etiology. the virus was detected in the respiratory secretions from an additional patients in two continents, and the complete genomes of multiple isolates were sequenced. a nasopharyngeal aspirate (npa) from a -year-old patient admitted to the pediatric ward of the royal children's hospital in brisbane with pneumonia was collected in october . the patient had no other remarkable clinical traits other than the respiratory features of pneumonia. testing of nucleic acid extracted from the npa using a panel of pcr assays for known respiratory viruses as described [ ] yielded negative results. total nucleic acid from the npa was randomly amplified and cloned as described previously [ ] . one -well plate of clones was sequenced using a universal m primer, and the resulting sequence reads were analyzed as described in materials and methods. of the reads, there were poor quality sequences that were rejected from further analysis, human sequences, six bacterial sequences, six viral sequences, and eight sequences of unknown origin that could not be classified. the bacterial sequences had greater than % nucleotide identity to known bacterial species, including haemophilus influenzae (three reads), streptococcus pneumoniae, corynebacterium pseudodiphthericum, and leifsonia xyli (unpublished data). upon further examination, the six viral reads were collapsed into three unique regions, each of which possessed only limited homology to known polyomavirus proteins (sequences available in figure s ). the highest scoring blastx hits for each of these three contigs possessed %, %, and % amino acid identity to jc virus stag, bk virus ltag, and sv vp , respectively. at the time these experiments were performed, the ki virus genome had not yet been published. subsequent analysis revealed amino acid identities of %, %, and % to ki virus for the three contigs. furthermore, three of the eight previously unclassified sequence reads were determined to have between %- % amino acid identity to ki virus vp and vp proteins by blastx analysis. based on the limited sequence homology to known viruses, we tentatively assigned the name wu to the unknown polyomavirus. the complete genome of wu was sequenced to coverage using cloned fragments of the viral genome generated by a series of pcr primers. analysis of the dna sequence revealed genomic features characteristic of polyomaviruses. first, the wu genome size of , base pairs (bp) was quite comparable to those of the primate polyomaviruses bk ( , bp), jc ( , bp), and sv ( , bp). in addition, the overall gc content of the wu genome was %, which is quite similar to the gc content of bk ( %), jc ( %), and sv ( %). the genome organization included an early region coding on one strand for stag and ltag, and a late region coding on the opposite strand for the capsid proteins vp , vp , and vp ( figure ). these two regions were separated by a regulatory region that contained typical polyomavirus features. the regulatory region contained an at-rich region on the late side of the putative replication origin. three repeats of the consensus pentanucleotide ltag binding site gaggc were present, as was one copy of the non-consensus ltag binding site taggc. while most polyomaviruses contain four copies of the consensus, baboon polyomavirus (simian agent ) is a primate polyomavirus that contains only three copies of the canonical binding sequence and one non-consensus binding site [ ] . unusual features in the wu regulatory region included the presence of two partially overlapping ltag binding sites and slightly variant spacing between the ltag binding sites as compared to sv , bk, and jc ( figure s ). in the early region, an unspliced open reading frame of amino acids was detected that possibly encodes for the stag. as the paradigm in other polyomaviruses is that stag is expressed from a spliced message, analysis of potential splice sites revealed the presence of a putative splice donor sequence just one nucleotide of the initially predicted we have identified a novel virus, referred to as wu virus, in the family polyomaviridae by screening of human respiratory secretions. two human polyomaviruses, bk and jc, were identified in and infect the majority of humans around the world. these two viruses are closely related to each other and are both are pathogenic in immunocompromised individuals. earlier this year, a third polyomavirus, ki, was described in human clinical specimens, although its pathogenicity and prevalence in humans has not yet been established. the discovery of wu virus brings the number of polyomaviruses detected in humans to four. wu differs from bk and jc significantly in its genome sequence and in its relative tissue tropism, suggesting that it is likely to have unique biological properties. this discovery raises many questions for further investigation, such as, is wu virus a human pathogen? if so, what kind of disease does it cause? where in the body does wu virus reside? at what age does infection typically occur? perhaps most importantly, there are likely to be many more as of yet unidentified viruses infecting the human body. stop codon. splicing to a downstream putative splice acceptor site would excise an intron of nucleotides and generate a slightly larger stag of amino acids ( figure s ) . while the precise carboxyl terminus of the wu stag has not yet been experimentally verified, sequence analysis revealed the presence of a highly conserved cysteine-rich motif, cx cx - cxcx cx - cscx cx wf, that was present in both of the predicted isoforms of wu stag. this motif, which is present in all stags, was perfectly conserved in wu virus with the exception of the initial cysteine residue. in all polyomaviruses, the initial ; amino acids of the nterminus of the stag and ltag are identical; the ltag is generated by alternative splicing of the early mrna transcript. in wu virus, a conserved splice donor site was identified immediately after amino acid of the early open reading frame. the position of the splice site is similar to that found in sv , bk, and jc virus, which occur after amino acids , , and , respectively. splicing to a conserved splice acceptor site would generate a predicted protein of amino acids ( table ). the predicted wu virus ltag contained conserved features common to t antigens, including a dnaj domain in the n terminus with the highly conserved hexapeptide motif hpdkgg; the lxcxe motif necessary for binding rb; a canonical dna binding domain; a zinc finger region; and conserved motifs gpxxxgkt and gxxxvnle in the atpase-p binding domain [ ] . based on comparative sequence analysis of ltags, the polyomaviruses are classified into two subclasses: a primatelike group exemplified by sv , and a mouse polyoma-like group exemplified by murine polyoma virus [ ] . using these criteria, the t antigen of wu appeared to more closely resemble the mouse polyoma-like class of virus than the primate class. first, the mouse polyoma-like viruses have insertions of varying length after amino acids and of sv as compared to the primate class. in the amino terminal domain of the wu virus ltag, multiple sequence alignment revealed the presence of a two-amino acid and a ten-amino acid insertion at these two loci, respectively. furthermore, the primate-like class typically contains an extension of the carboxyl terminus termed the host range domain that is absent in the mouse polyoma-like class. in contrast to sv , bk, jc, and baboon polyomavirus, wu virus did not appear to encode a carboxyl terminal extension ( figure s ). in addition to encoding ltag and stag, murine and hamster polyomaviruses utilize alternative splicing to generate an intermediate-sized protein referred to as middle t antigen. the wu virus early region was scanned for splicing motifs similar to known murine and hamster polyomavirus splice donor and acceptor sequences, but no obvious combination of splice sites was detected that would yield a middle t antigen sequence in the size range of known middle t antigens. in addition, sv , jc, bk, and baboon polyomavirus all encode a fourth late protein termed the agnoprotein. there was no open reading frame present in wu with any detectable homology to the known agnoproteins. thus, our sequence analysis suggests that neither middle t antigen nor agnoprotein are encoded by wu virus, although it is possible that the sequences have diverged beyond our ability to recognize the appropriate splice sites or protein products. multiple sequence alignments of the predicted stag, ltag, vp , and vp open reading frames revealed that wu virus was clearly a novel virus that is most closely related to ki virus ( figure ). neighbor-joining analysis suggested that these two viruses appear to form a new subclass of polyomaviruses. in the early region and vp protein, the wu/ki branch was most closely related to the known primate polyomaviruses bk, sv , jc, and baboon polyomavirus (figure a- c) . finally, the vp open reading frame was so divergent that its evolutionary relationship to other polyomaviruses aside from ki could not be reliably established ( figure d ). analysis of the vp amino acid sequence, which is completely contained within vp , gave similar results as vp (unpublished data). pcr primers were designed to specifically amplify wu. the initial screen used primers targeting the vp region, which possessed less than % amino acid homology to jc and bk virus to minimize the possibility of cross reactivity with the known human polyomaviruses. empirical testing of the primers on samples known to contain bk and jc confirmed that the primers did not cross react with either of these genomes (unpublished data). positives in the initial screen for wu virus were sequenced and then further confirmed by a second pcr reaction using primers targeting the end of the wu virus ltag coding sequence. all positive samples in the initial screen were confirmed using the second pair of pcr primers. a subset of samples that tested negative in the initial screen was also tested with the second pcr primer pair, and none of those samples were positive. in order to assess the prevalence of wu polyomavirus, a cohort of , respiratory specimens collected in in brisbane was examined. thirty-seven out of the , ( . %) samples tested were positive for the virus (table ). in this cohort, patients that tested positive ranged in age from months to years. the vast majority of the patients ( / ) were age and under. in patients with clear clinical evidence of respiratory tract infection, wu was the sole virus detected. strikingly, in of the positive samples, one or more additional respiratory viruses were also detected. the most common co-infections were with rhinovirus ( cases) and human bocavirus (ten cases). furthermore, in one sample, a total of four viruses (wu, bocavirus, rhinovirus, and adenovirus) were detected, and in six other samples, a total of three viruses were detected (table ) . in addition, we examined two cohorts of patients from st. louis, missouri, united states. in one set of upper respiratory specimens collected in , five out of were positive for wu virus in the pcr assay. in addition, bronchoalveolar lavage samples from patients (mostly adults) with severe acute respiratory illness were tested, yielding one positive. of the positive samples, all six were co-infected with other viruses ( table ). the age range of the positive cases varied from months to years. to assess the sequence variation within different isolates, we analyzed the -bp region encompassed by the initial screening primers for all cases ( figure ). several divergent strains were detected, including one sample that had five mutations ( %) within this region. in another case, a -bp deletion was observed. the fact that many isolates were identical in sequence was not surprising, given the relatively short length of the amplicon and the double-stranded dna nature of the genome. in addition, we sequenced the complete genome of five additional isolates from five independent patients. unfortunately, efforts to completely sequence the two most divergent isolates (based on the bp sequence, b and b ) have been unsuccessful, presumably due to low viral titers in these samples. all six complete genomes were , bp in size, and overall, there was between . % and . % sequence variation from sample to sample, well above that expected from taq pcr, ruling out the possibility that the additional positives were artifacts of pcr contamination. moreover, the majority of the observed mutations were synonymous substitutions or in non-coding regions, lending further support to the argument that these were authentic strain variants. for jc virus, the reported intratype sequence variation is of a similar magnitude, ranging between . % and . % [ ] . because bk and jc virus are frequently excreted in urine, we examined urine samples from patient cohorts in both st. louis and brisbane for the presence of wu virus by pcr. in the st. louis cohort, urine samples from adult patients participating in a study of polyomavirus infections in kidney transplant recipients were tested [ ] . for most patients, samples were tested at three time points: prior to transplant, mo post transplant, and mo post transplant, although for some patients the pre-transplant specimen was not available. zero out of samples tested were positive for the wu polyomavirus. as a control, using previously validated bk primers, we were able to amplify bk virus in a subset of these urine samples, confirming the integrity of the specimens themselves (unpublished data). similarly, from the brisbane cohort, none of the urine samples tested were positive for wu virus. we used a high throughput sequencing strategy to search for novel agents that were present in respiratory tract infections of unknown etiology. the focus of this study was on individual clinical specimens that still lacked a diagnosis after analysis with an extensive panel of diagnostic assays for known respiratory viruses. in one such patient sample, novel sequences with limited homology to known polyomaviruses were detected. complete genome sequencing and phylogenetic analysis revealed that the new virus clearly had the genomic organization typical of polyomaviruses but was divergent from all previously described polyomaviruses. in keeping with the two-letter virus names for human polyomaviruses, we have named this novel polyomavirus wu virus [ , ] . overall, the predicted amino acid sequences of wu virus proteins were most similar to the newly described ki virus ( table ) . outside of ki, wu shared only ; %- % identity to its closest relatives (table ) . detailed analysis of the viral dna sequence and genomic organization confirmed the novelty of wu virus. at all loci, wu virus was most similar to ki virus, but the degree of divergence between wu and ki was greater than the divergence between sv and bk, indicating that wu and ki are clearly distinct viruses (figure ). based on the phylogenetic analysis, it appears that wu and ki define a novel branch within the polyomaviridae family ( figure ). relative to the established polyomaviruses, some analyses suggested that the wu/ki branch might be more closely related to the primate polyomaviruses, while other features of the wu genome suggested that it might be more similar to murine polyomavirus. for example, neighbor-joining phylogenetic analysis suggested that the predicted stag, ltag, and vp open reading frames of both ki and wu were most closely related to sv , jc, bk, and baboon polyomaviruses. analysis of the vp /vp region was more equivocal, as the proteins were too divergent to reliably assess. the apparent absence of the c-terminal ''host range'' domain in the ltag and the agnoprotein open reading frame, both of which are present in the known primate polyomaviruses, suggested that wu virus was more similar to murine polyomavirus than the primate polyomaviruses by these criteria. while the evolutionary history of this virus is not clear at the moment, the totality of the analysis indicates that wu is clearly a unique virus. we detected wu in out of , ( . %) patient specimens in brisbane (excluding the original case) and in six out of ( . %) patient specimens tested in st. louis. as the positive specimens were all collected from through , it appears that wu is currently circulating, and its presence in both north america and australia suggests that the virus is geographically widespread in the human population. the age range of patients that tested positive for wu virus spanned from months to years. the majority ( %) of the cases were found in children years of age and under. of the four positive specimens from adult patients (s , s , b , and b in table ), three clearly had altered immune status. one patient was hiv-positive, one was immunosuppressed due to treatment for wegener granulo- table . doi: . /journal.ppat. .g matosis, and one was pregnant. the fourth adult patient (s ), while not obviously immunosuppressed, also suffered from liver cirrhosis, hypertension, type diabetes, and co-infection with herpes simplex virus, and required mechanical ventilation. in addition, there were two other positive patients older than years of age: a -year-old child who had previously been a bone marrow transplant recipient ( table , b ) and a -year-old child diagnosed with acute lymphoblastic leukemia ( table , the patients who yielded positive specimens suffered from a wide range of respiratory syndromes, including bronchiolitis, croup, and pneumonia as well as other clinical maladies ( table ). detection of wu virus sequences in these patients is merely the first step in assessing the potential etiologic role of wu virus in acute respiratory tract disease. it is not yet known whether wu is infectious or whether it is capable of replication in the respiratory tract. one possibility is that wu is not involved at all in respiratory disease, but rather is simply transmitted by the respiratory route. the human polyomaviruses bk and jc are hypothesized to be transmitted by the respiratory route before taking up residency primarily in the kidneys. latency in the kidneys of bk and jc is believed to be the reason that both viruses are excreted in the urine of up to % of asymptomatic individuals [ , ] . in this study, using the same pcr assays that were effective in respiratory secretions, we did not detect wu in any of the urine samples we tested. the lack of detection of wu virus in the urine may reflect sensitivity issues, a bias in the cohorts tested, or simply that wu is unlike bk and jc viruses and is not secreted in the urine. a similar tissue profile to that of wu virus has been reported in initial studies of ki virus [ ] . future experiments will aim to determine the tissue tropism of wu and whether any tissue reservoirs for wu virus exist. in the literature, there is one animal polyomavirus that has been found extensively in lung tissue. infection of suckling mice with the mouse pneumotropic polyomavirus (mppv) causes interstitial pneumonia and significant mortality. mppv also differs from other polyomaviruses in that besides the kidneys, it can also be detected in the lungs, liver, spleen, and blood of suckling mice [ ] . thus, there is precedence for an animal polyomavirus causing respiratory disease, suggesting at least the possibility that wu virus could be similarly pathogenic in humans. one striking observation from these studies is the relatively high frequency of co-infection detected in the respiratory secretions: % overall ( % in the st. louis cohort and % in the brisbane cohort). although more extensive studies are necessary to confirm the generality of this observation, this raises several intriguing non-mutually exclusive possibilities to consider: ) wu may be an opportunistic pathogen; ) wu infection may predispose or facilitate secondary infection by other respiratory viruses; and ) wu may be a part of the endogenous viral flora that is reactivated by inflammation or some other aspect of viral infection. recent studies of the prevalence of the newly identified human bocavirus have also reported higher levels of co-infection than previously described for other viruses found in the respiratory tract, with co-infection rates as high as % reported [ , ] . in addition, five of six samples positive for ki virus were reported to be co-infected with other known respiratory viruses [ ] . as detection methods improve in sensitivity and more comprehensive efforts are made to examine the diversity of viruses found in the respiratory tract, a greater appreciation for the rates of dual or multi-infection is gradually emerging. for example, the use of extensive panels of pcr assays in this study revealed that one of the positive specimens was quadruply infected; adenovirus, rhinovirus, and bocavirus and wu virus were all present. further investigations that aim to systematically define the spectrum of viruses present in the respiratory tract are clearly warranted so that the possible roles that coinfections may play in disease pathogenesis can be explored. extremely high sequence divergence was observed in the capsid proteins vp and vp of wu virus and ki virus as compared to the other known polyomaviruses. this divergence may reflect a different ''lifestyle'' for the wu/ki branch as compared to known polyomaviruses. our data demonstrating the presence of wu in respiratory secretions and its absence in urine samples suggest that the mode of transmission or the sites of persistence of wu may be distinct from the other human polyomaviruses. as such, the structure of the virion must be optimized to enable the virus to survive dramatically distinct physiological and environmental conditions. this may partially explain the observed sequence divergence in the capsid proteins. another question raised by this study relates to the potential antigenic cross reactivity of the wu capsid proteins. in terms of establishing the seroprevalence of wu itself and determining whether seroconversion accompanies acute infection with wu, it will be essential to conduct these studies with consideration for potential cross reactivity to ki, bk, jc, and sv antibodies. in addition, it is tantalizing to speculate whether serum antibodies to wu have the potential to cross react to sv -derived antigens, and if so, whether they may at least partially account for some of the studies that report the presence of sv antibodies in the human population that is too young to have suffered exposure from contaminated polio vaccination [ ] [ ] [ ] . in conclusion, we have identified and completely sequenced the genome of a novel polyomavirus. this virus appears to be geographically widespread in the human population as evidenced by the detection of distinct cases in two continents. based on preliminary analysis, wu and ki virus share some strikingly similar properties, including their complement of genes, phylogenetic relationship, and physical sites of detection in the human body. these data suggest that wu virus and ki virus define a novel branch within the polyomaviridae family with unexplored biology and pathogenicity. another implication of these results is that the diversity of viruses in this family may be far greater than currently realized. further experimentation is now underway to determine the relative pathogenicity of wu virus in humans and to understand the molecular properties of the virus. since the t antigen of wu is predicted to have transforming properties by analogy to other polyomavirus t antigens, one question currently under investigation is whether a subset of human tumors may be associated with wu. clinical specimens-respiratory secretions. brisbane cohort. a total of , specimens (predominantly npas) were collected between january , , and december , , from patients presenting to the royal children's hospital in brisbane, queensland, australia, with symptoms consistent with acute lower respiratory tract infection. st. louis cohort # . a total of bal specimens were tested. these included samples from a retrospective and a prospective collection. the retrospective specimens were from a sequential collection of bal specimens submitted routinely to the virology laboratory at st. louis children's hospital between december and august [ ] . for the present study, an effort was made to select specimens from this collection from patients with acute respiratory illness, and to exclude specimens collected as routine post-lung transplant surveillance. the prospective specimens were from an ongoing study of the etiology of severe acute respiratory illness and were collected between october and october . both collections included specimens from patients of all ages, although the large majority were from adults. st. louis cohort # . this collection was made up of respiratory specimens, mostly nasopharyngeal swabs, submitted for routine virologic testing to the virology laboratory at st. louis children's hospital between september and june . the majority of these specimens were from children. of the specimens in this collection, were selected because they had been found to be positive by fluorescent antibody staining or culture for influenzavirus a or b, respiratory syncytial virus, parainfluenza virus, rhinovirus, or adenovirus. clinical specimens-urine. brisbane cohort. urine specimens ( ) that were submitted during to the diagnostic laboratory for routine investigation were collected. these represented a diverse mixture of donors, including those from (i) sexual health clinic (n ¼ ), (ii) pediatric clinic (n ¼ ), (iii) antenatal clinic (n ¼ ), (iv) indigenous health clinic (n ¼ ), and (v) bone marrow transplant patients (n ¼ ). the st. louis urine specimens were from a study of polyomaviruses in adult renal transplant recipients [ ] . a total of individuals were enrolled in the study between december and october . from each patient, up to three specimens were tested, including a specimen obtained before the transplant and specimens obtained at and mo after transplantation. diagnostic testing of clinical specimens for known respiratory viruses. brisbane cohort. nucleic acids were extracted from . ml of each specimen using the high pure viral nucleic acid kit (roche diagnostics australia, http://www.rochediagnostics.com.au) according to the manufacturer's instructions. pcr assays for known respiratory viruses were performed as described [ ] . st. louis cohort. all respiratory specimens were tested originally by fluorescent antibody staining using a panel of monclonal antibodies directed against influenza a and b, respiratory syncytial, parainfluenza - , and adenoviruses (simulfluor respiratory screen; chemicon, http://www.chemicon.com). specimens that were negative were also cultured using cell culture systems that could detect the same group of viruses plus rhinoviruses, cytomegalovirus, and herpes simplex virus. total nucleic acid extracts were purified using a qiagen m instrument (http://www.qiagen.com). nucleic acid extracts were tested for a panel of respiratory viruses using the eragen multicode-plx respiratory virus panel (eragen biosciences, http:// www.eragen.com), a multiplex pcr assay that detects the following viruses: influenza a and b, respiratory syncytial virus a and b, parainfluenza - , human meatpneumovirus, adenovirus subgroups b, c, and e, rhinoviruses, and coronaviruses oc , e, and nl . library construction and shotgun sequencing. samples were prepared in the following manner for high throughput sequencing analysis. a total of ul of neat npa sample was thawed and directly treated with dnase i (fermentas, http://www.fermentas.com) for min at c. total nucleic acid was extracted using the masterpure complete dna and rna purification kit (epicentre biotechnologies, http://www.epibio.com). then, ng of total nucleic acid was randomly amplified using the rdab protocol exactly as described [ ] . rna in the total nucleic acid preparation was converted to cdna by reverse transcription with primer-a ( gtttcccagtcacga-tannnnnnnnn). two rounds of random priming with primer-a and extension with sequenase (united states biochemical, http:// www.usbweb.com) enabled second strand cdna synthesis as well as random priming of dna originally present in the total nucleic acid sample. amplicons were then generated via cycles of pcr using primer-b ( gtttcccagtcacgata) with a cycling profile of: c s; c s; c s; c s. the primer-b-amplified material was topo cloned into pcr . (invitrogen, http://www. invitrogen.com) and transformed into bacteria, and white colonies were picked into -well plates. dna was purified by magnetic bead isolation and sequenced using standard big dye terminator (v . ) sequencing chemistry. reaction products were ethanol precipitated, resuspended in ul of water, and loaded onto the abi xl sequencer. analysis of shotgun sequences. sequences were assessed for quality using phred [ ] , and reads that contained less than contiguous bases with a score of phred or greater were rejected. the remaining reads were analyzed in the following steps: ) reads were aligned to the human genome using blastn with an e À cutoff; ) remaining reads were aligned to a bacterial database using blastn with an e À cutoff; and ) remaining reads were aligned to the viral refseq protein database using blastx with an e À cutoff [ ] . complete genome amplification and sequencing. the wu genome derived from the index case was sequenced to coverage using six unique pairs of pcr primers for the amplification. amplicons were cloned into pcr . and sequenced using standard sequencing technology. all primers used for amplification and sequencing are listed in table s and their positions depicted in figure s . additional complete genomes were sequenced to at least coverage using the same primers listed in table s . completed nucleic acid prevalence studies. for all pcr assays, standard precautions to avoid end product contamination were taken, including the use of pcr hoods and maintaining separate areas for pcr set up and analysis. for initial screening of wu virus, pcr primers ag tgttacaaatagctgcaggtcaa and ag gctgcataatggggagtacc were used with accuprime hot start taq (invitrogen) to amplify ul of template using the following program: cycles of c s; c s; c s. for every samples tested, seven notemplate negative controls were interspersed between the actual samples. products were visualized following electrophoresis on % agarose gels. the resulting -bp amplicon was sequenced directly in both directions using primer ag and ag . these sequences have been deposited in genbank (see supporting information for accession numbers). secondary confirmation was performed using primers ag tgtttttcaagtatgttgcatcc and ag cacccaaaagacacttaaaagaaa that generate a -bp amplicon in the end of the ltag coding region. the same cycling profile of cycles of c s; c s; c s was used. for detection of both bk and jc viruses, primers ag agtctt-tagggtcttctacc and ag ggtgccaacctatggaa-cag were used with a profile of cycles of c s; c s; c s. figure s . raw sequence data from high throughput screening a) the initial six shotgun reads with homology to polyomaviruses. b) the three contigs derived from the six reads. the consensus tag binding motif is gaggc. the known primate polyomaviruses sv , jc, bk, and baboon polyomavirus all have four copies of the copies of the binding site oriented as shown above for sv (nc_ ). the first nucleotide of the third copy of the consensus tag binding site is defined as nucleotide for wu and sv . differences between sv and wu virus are ) one of the tag binding sites in wu virus appears to be a non-canonical taggc; ) the second and third consensus tag binding sites in wu virus overlap; and ) the nucletoide spacing between the tag binding sites in wu virus varies from the prototype sv as shown. shown in blue is the polya/t tract that is commonly found to the late side of the origin in polyomaviruses found at doi: . /journal.ppat. .sg ( kb pdf). genbank) protein sequences used in this paper are as follows: ltag: african green monkey (np_ ) bk (yp_ ); bovine (np_ ); budgerigar (np_ ); crow (yp_ ) murine (np_ ); murine pneumotropic (np_ ) sv (np_ ) stag: african green monkey (np_ ) bk (yp_ ); bovine (np_ ); budgerigar (np_ ); crow (yp_ ) murine (np_ ); murine pneumotropic (np_ ) vp : african green monkey (np_ ) bk (yp_ ); bovine (np_ ); budgerigar (np_ ); crow (yp_ ) murine (np_ ); murine pneumotropic (np_ ) vp : african green monkey (np_ ) bk (yp_ ); bovine (np_ ); budgerigar (np_ ); crow (yp_ ) murine (np_ ); murine pneumotropic (np_ ) wu complete genome sequences have been deposited under accession numbers ef -ef . vp partial sequences have been deposited under accession numbers global burden of acute respiratory infections in children: implications for interventions the common cold a newly discovered human pneumovirus isolated from young children with respiratory tract disease a novel coronavirus associated with severe acute respiratory syndrome identification of a new human coronavirus characterization and complete genome sequence of a novel coronavirus, coronavirus hku , from patients with pneumonia cloning of a human parvovirus by molecular screening of respiratory tract samples identification of a third human polyomavirus viral discovery and sequence recovery using dna microarrays seroepidemiology of the human polyomaviruses the role of bk virus in acute respiratory tract disease and the presence of bkv dna in tonsils detection of bk virus dna in nasopharyngeal aspirates from children with respiratory infections but not in saliva from immunodeficient and immunocompetent adult patients association of bk viruria with hemorrhagic cystitis in recipients of bone marrow transplants incidence of bk virus and jc virus viruria in human immunodeficiency virus-infected and -uninfected subjects detection of bk virus and jc virus dna in urine samples from immunocompromised (hiv-infected) and immunocompetent (hiv-noninfected) patients using polymerase chain reaction and microplate hybridisation simian virus and human disease sv small t antigen enhances the transformation activity of limiting concentrations of sv large t antigen creation of human tumour cells with defined genetic elements is there a role for sv in human cancer? frequent detection of human rhinoviruses, paramyxoviruses, coronaviruses, and bocavirus during acute respiratory tract infections complete nucleotide sequence of polyomavirus sa common and unique features of t antigens encoded by the polyomavirus group five complete genomes of jc virus type from africans and african americans incidence of bk with tacrolimus versus cyclosporine and impact of preemptive immunosuppression reduction new human papovavirus (b.k.) isolated from urine after renal transplantation cultivation of papova-like virus from human brain with progressive multifocal leucoencephalopathy effect of host age on experimental k virus infection in mice evidence of human coronavirus hku and human bocavirus in australian children the association of newly identified respiratory viruses with lower respiratory tract infections in korean children serological evidence of sv infections in hiv-infected and hiv-negative adults studies of neutralising antibodies to sv in human sera prevalence and stability of human serum antibodies to simian virus vp virus-like particles detection of severe human metapneumovirus infection by real-time polymerase chain reaction and histopathological assessment base-calling of automated sequencer traces using phred. i. accuracy assessment gapped blast and psi-blast: a new generation of protein database search programs we would like to thank monique gaudreault-keener for technical support and rakesh nagarajan and sunita koul for assistance with sequence analysis.author contributions. mdn, tps, and dw conceived and designed the experiments. amg, dmw, imm, and gw performed the experiments. amg, mdn, sbl, gas, tps, and dw analyzed the data. mdn, dcb, gas, and tps contributed reagents/materials/analysis tools. dw wrote the paper.funding. this work was supported in part by national institutes of health (nih) grant u ai to the midwest regional center of key: cord- - th jiq authors: qing, jie; wang, yaxin; sun, yuna; huang, jiaoyan; yan, wenzhong; wang, jinglan; su, dan; ni, cheng; li, jian; rao, zihe; liu, lei; lou, zhiyong title: cyclophilin a associates with enterovirus- virus capsid and plays an essential role in viral infection as an uncoating regulator date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: th jiq viruses utilize host factors for their efficient proliferation. by evaluating the inhibitory effects of compounds in our library, we identified inhibitors of cyclophilin a (cypa), a known immunosuppressor with peptidyl-prolyl cis-trans isomerase activity, can significantly attenuate ev proliferation. we demonstrated that cypa played an essential role in ev entry and that the rna interference-mediated reduction of endogenous cypa expression led to decreased ev multiplication. we further revealed that cypa directly interacted with and modified the conformation of h-i loop of the vp protein in ev capsid, and thus regulated the uncoating process of ev entry step in a ph-dependent manner. our results aid in the understanding of how host factors influence ev life cycle and provide new potential targets for developing antiviral agents against ev infection. cyclophilins (cyps) are key cellular factors that function in numerous cellular processes, including transcriptional regulation, immune response, protein secretion, and mitochondrial function [ ] . cyps possess peptidyl-prolyl cis-trans isomerase activity and have high affinity for the immunosuppressant cyclosporine a (csa). cyclophilin a (cypa) is a key member of the cyp family and was first shown to mediate the immunosuppressive function of csa through the formation of a csa-cypa complex. this complex binds to and inhibits the function of the phosphatase calcineurin, which normally functions to dephosphorylate nf-at, a transcription factor important for t cell activation [ ] . cypa is also known to play critical roles in the proliferation of a number of viruses, including human immunodeficiency virus type (hiv- ), influenza virus, hepatitis c virus (hcv), vesicular stomatitis virus (vsv), vaccinia virus, severe acute respiratory syndrome coronavirus (sars-cov), rotavirus (rv) and human papillomavirus (hpv), by interacting with viral proteins or facilitating ifn-b production [ , ] . cypa was first shown to be incorporated into hiv- virions through its interaction with the capsid protein (ca), and the interaction between newly synthesized hiv- ca and cypa is required for hiv- to induce dendritic cell maturation [ , ] . cypa also interacts with other hiv- proteins, such as vpr and p , to regulate hiv infection [ , ] . cypa was further revealed to interact with extracellular cd , which is the main receptor for cypa on the cell membrane of human leukocytes, and this interaction can induce the phosphorylation of hiv- matrix protein to regulate the liberation of the reverse transcriptase complex into cytoplasm during an early stage of hiv- infection or can function in hiv- attachment to host cells [ ] . but a recent research showed that cypa stabilized the hiv- capsid and antagonizes hiv- uncoating in vitro, indicating the versatile roles of cypa in hiv- infection [ ] . moreover, several lines of evidences revealed that cyps play crucial roles in hcv life cycle. cypb was first reported to be important for hcv replication [ ] , but later studies showed that cypa, but not cypb, was required for hcv infection in vitro [ ] [ ] [ ] [ ] . cypa was reported to function in the replication of hcv by increasing the affinity of the hcv polymerase ns b for viral rna to enhance hcv replication [ ] , or by binding to the hcv ns a protein to aid in viral replication [ , ] . furthermore, cyps were demonstrated to play an essential role in hpv infection by facilitating conformational changes in capsid proteins of hpv, resulting in exposure of the nterminus of l protein, and the dissociation of l pentamers from recombinant hpv l /l complexes in a ph-dependent manner [ , ] . enterovirus- (ev ), a member of the picornaviridae family, is one of the major causative agents of hand-foot-and-mouth disease (hfmd) in pan asia-pacific region and results over eight millions of infections and three thousands of dead cases since [ , ] . the genome of ev contains a single-stranded, positivesense rna (+ssrna) and encodes a polypeptide with a molecular weight of approximate kda [ ] . this polyprotein is initially processed into one structural (p ) and two non-structural (p and p ) regions and then undergoes proteolytic cleavage into various precursors, ultimately resulting in mature proteins. among them, p is further proteolyzed into vp to vp to form the viral capsid, while p and p are processed into replicase proteins. for a productive infection, virions must uncoat and release viral genome into host cytoplasm, following the successful bindings with functional receptors. enteroviral uncoating process involves sequential capsid alterations by conformational changes [ ] . during uncoating, mature particles with sediment coefficient of s are converted to the uncoating intermediate a particles with sediment coefficient of s, and subsequent empty s particles representing the final production of the entry process [ ] . the s particles are empty particles that have shed genomic rna, whereas the s particles retain the full complement of genomic rna but lack some or all of their content of vp and have externalized most of the n-terminal extension of vp that is normally inside the virions [ ] . the involvement of host cellular factors plays essential roles in virus proliferation. however, the knowledge of how ev utilizes host factors in its life cycle is limited. only two extracellular membrane proteins, human p-selectin glycoprotein ligand- (psgl- ) [ ] and scavenger receptor b (scarb ) [ , ] , as well as heparan sulfate (hs) [ ] , were recently identified as functional receptors for ev infection. another result suggests that the binding of ev to human annexin ii on the cell surface enhanced viral entry and infectivity, especially at a low infective dose [ ] . interestingly, scarb was reported to be the exclusive uncoating receptor to trigger conversion of s particles to other forms during uncoating process at acidic condition, resulting in the releasing of viral genome [ ] . here we used cypa inhibitors as bioprobes to show that cypa played an essential role in ev proliferation. we also elucidated the mechanism by which cypa interacted with and modified the conformation of ev vp h-i loop, and thus regulated the uncoating process of ev entry. this cypa-ev capsid functional association not only provides information to understand the cellular factors used in ev infection, but also presents a new promising potential for the development of antiviral therapeutics. our compound collection, which includes chemically synthesized compounds, was screened by using rhabdomyosarcoma (rd) cells infected with the ev virus strain anhui . this screen identified compound hl p (figs. a) as a potent inhibitor of viral proliferation, with an ec value of nm, by measuring ev virus rna through quantitative rt-pcr (qrt-pcr) (fig. c) . no significant cytotoxicity was observed from compound hl p at concentrations below mm, as demonstrated by the wst- -based assay (fig. d) , indicating that the inhibition of ev proliferation was specific. because compound hl p was previously reported to function as a cypa inhibitor [ ] , we next selected csa (fig. b) , a well-known cyp inhibitor and clinical immunosuppressant drug with antiviral effects, to suppress hiv- and hcv replication and to check whether other cypa inhibitor can also inhibit ev replication. the results revealed that csa clearly impaired ev proliferation with an ec value of . mm (fig. c) ; however, csa had a slightly higher cytotoxicity than hl p (fig. d) . because cyps are known to be involved in the viral life cycle, our interest in identifying host factors in the ev life cycle and antiviral agents prompted us to initiate further investigations to study the working mechanism of cypa in the ev life cycle and the inhibitory mechanism by which cyp inhibitors block ev replication. cypa plays a critical role in ev virus proliferation over ten subfamilies have been identified in the cyp family to date, among which cypa and cypb are the most abundant subtypes [ ] . to clarify which type of cyp is most essential for ev proliferation, we next used the rna interference (rnai) method to investigate the impact of cypa or cypb on ev virus proliferation. we first introduced short hairpin rnas (shrnas) that were designed to recognize the non-coding region of cypa (sh-cypa) or cypb (sh-cypb) by lentiviral vectors into rd cells to downregulate endogenous cypa and cypb expression [ ] . the expression of glyceraldehyde- -phosphate dehydrogenase (gapdh), a housekeeping gene used as an internal control, was not downregulated (fig. a) . we obtained stable knockdown cell lines through resistance gene screening and then infected these shrna-rd cells by applying an ev -gfp virus with a multiplicity of infection (moi) of . . the results of the flow cytometric studies revealed that . % of the rd cells with negative control shrna (rd-sh-control) were infected with the ev -gfp virus, and the infection ratio was decreased to . % in the rd-sh-cypa cells. however, the infection rate remained at . % in the rd-sh-cypb cells, suggesting that cypa dominantly impacted ev infection (fig. b) . moreover, when infected with ev at a multiplicity of infection (moi) of , the ev rna level in the cypa knockdown cells was diminished to approximately % of the levels observed in the rd-sh-control cells, whereas the cypb knockdown did not result in this reduction (fig. c) . we also infected the rd-sh-cypa and rd-sh-cypb cells with ev at enterovirus (ev ) is the major causative agent of handfoot-and-mouth disease (hfmd) in asia-pacific region and caused over one million infection cases and nine hundred deaths in the year of in china mainland. ev is known to infect the young children for the sake of their undeveloped immune system. unlike other enterovirus (e.g. coxsackievirus), ev could cause severe aseptic meningitis, encephalitis, myocarditis, and acute flaccid paralysis, thus leading to high fatality rates. there is no clinically applied therapeutics. in this work, we used cypa inhibitors as bioprobes to show that cypa played an essential role in ev proliferation. we also elucidated the mechanism by which cypa interacted with the ev vp h-i loop and functioned as an uncoating regulator in ev entry step. since there are several non-immunosuppressive cypa inhibitors, e.g. nim- and debio- , have been reported to show antiviral potency, our results provide a potential way to discover clinical therapeutics against ev infection. mois of . and , respectively, and the results revealed a similar finding as the one caused by ev infection at an moi of (fig. d ). furthermore, the expression level of ev vp protein, which is the major component of the ev capsid [ , ] , was significantly reduced by the knockdown of cypa, but not cypb, which is consistent with the impact of cypa reduction on the ev rna level (fig. e, left half ). an interesting observation is that the cypb knockdown resulted in a small increase in the ev rna replication and vp expression (figs. c and e). to verify the impact of cypa in the different cell lines used for ev infection, we generated an additional stable cypa knockdown huh . . cell line (huh . . -sh-cypa) and observed the ev infections (at moi of ) in huh . . -sh-cypa and huh . . cells with the negative control shrna (huh . . -shcontrol). the results demonstrated that the decreased ev rna replication (fig. f ) and vp expression (fig. g ) were similar to those in the rd cell lines. taken together, all these data suggested that the loss of cypa function is specifically associated with the inhibition of ev proliferation. previous studies suggested that cypa was upregulated during virus infection and is correlated with the final results of the infection [ , , ] . similarly, the expression of host cell cypa was upregulated in the rd cells following ev infection (fig. h ). we also found that ev infection led a very minor alteration of secreted cypa in the supernatant of rd-sh-control cells, and very little cypa could be detected in the rd-sh-cypa culture with or without ev infection (fig. i ). this finding indicated that cypa was present in host cells, but not those that were secreted in the supernatant, and cypa is upregulated following ev infection. cypa was found to interact with different viral proteins and affect different stages of the viral life cycle through distinct mechanisms [ ] . to define the working target of cypa in the ev virus, we generated an ev virus that was resistant to a cypa inhibitor through multiple cell culture passages in the presence of compound hl p . sequence analyses of the entire genome of multiple resistant viruses identified only a single t-to-c mutation at nucleotide position , of the ev genome (table s ). this mutation translated into a single amino acid substitution of a serine to a proline at residue vp - (all residue numbers correspond to the residue in the sequence of the vp protein, not in the polypeptide), which is located in the h-i loop of vp [ ] . to confirm that the vp -s p mutant was the mutation that contributed to the resistant phenotype of the selected mutant virus, we engineered a recombinant mutant ev virus (s p-ev ) from the wild type ev (wt-ev ) strain anhui through the introduction of a single serine-to-proline substitution at the vp - position and investigated the sensitivity of the s p-ev virus to compound hl p or csa in comparison with wt-ev . the ec value of compound hl p on s p-ev proliferation ( . mm) was approximately -fold higher than the ec value for wt-ev , and the ec value of csa was -fold higher for the s p mutated virus (table s ) . moreover, following an infection with the drug-resistant s p-ev recombinant virus, the ev rna level in the rd-sh-cypa cells was approximately % of the levels in the rd-shcontrol cells. this value was much higher than the values observed in rd-sh-cypa cells that were infected with wt-ev virus (approximately %), suggesting that the vp -s p mutant rescued ev replication. the expression level of ev vp protein also consistently recovered to normal levels following infection with the s p-ev virus (fig. e, right half) . these data again revealed that the vp -s p mutant is specifically associated with resistance to the cypa inhibitor. cypa has peptidyl-prolyl cis-trans isomerase activity to facilitate the conformational modification of proline residue. by examining the protein sequence of ev vp , we found that there is only one proline residue located close to the vp -s position, i.e., vp -p . we hypothesized that the resistant mutation from a serine residue to a proline residue at the vp - position may increase the binding affinity of vp to cypa and help the virus escape from drug treatment and the depletion of the endogenous cypa. to clarify the mechanisms underlying the cypa regulation of the ev life cycle, we analyzed the molecular interaction of cypa with ev virions or the vp h-i loop by using a gst pull-down assay. we first checked whether recombinant cypa protein could associate with the ev virions (fig. ). by using a gst-tagged cypa as a probe (fig. a) , we demonstrated that recombinant cypa protein was clearly bound to wt-ev , and the interaction of cypa with the ev virion was increased by substituting with a proline residue at vp -s (fig. b, upper panel) . however, when we replaced both s and p with alanine residues, the mutated virus, i.e., s a/p a-ev , cannot bind with cypa (fig. b, bottom panel) . this interaction between cypa and the ev virion was also reduced in a dose-dependent manner after treating with csa and can almost be abolished at a concentration of mm csa (fig. c, upper panel) . additionally, the s p mutant rescued the interaction between cypa and ev under csa treatment (fig. c, bottom panel) . we further demonstrated that recombinant cypa protein not only bound to wt-ev strain anhui but also to other strains of the wt-ev virus (fig. d) . we we also used nmr spectra to demonstrate that recombinant cypa binding to chemically synthesized ev vp h-i loop peptides caused chemical shift changes, suggesting that cypa catalyzed the correct cis-trans reaction of the vp h-i loop (fig. s ). moreover, a reported catalytic-defective mutant of cypa called h q [ ] eliminated the interaction between cypa and ev virions (fig. f) . a similar attenuation of the interaction between the cypa h q mutant and virions was also reported in hiv- and hcv [ , ] . taken together, all these results revealed that cypa functioned directly at the h-i loop of ev vp , and the replacement of serine with proline at the vp -s position could increase the binding affinity of cypa with ev virions. cypa functions in the entry step of ev infection s is located in the h-i loop of the vp protein of the ev virus, and several loop regions of the vp protein are known to play critical roles in the entry step during picornavirus infection [ ] . therefore, we hypothesized that cypa may also act in the entry step of the ev life cycle. to verify this hypothesis, we first infected rd cells with ev and treated them with mm compound hl p at , , , , , , and h post-infection (hpi), in which hpi indicates the supply of a virus infection inhibitor. the results showed that the inhibition of ev by hl p represented a clear dependence on the treatment time. the hl p treatments at to hpi showed the inhibition of ev replication, whereas the anti-ev effect of the treatments after hpi was significantly attenuated (fig. a) . we further transfected an ev subgenomic replicon rna lacking the p region in the rd-sh-control and rd-sh-cypa cells and found that the ev rna replication inside the host cells was not affected by the downregulation of cypa (fig. b ). these results indicated that cypa affected the early step, but not genome replication, during ev infection. by detecting viral rna at different time points in rd cells that had been infected with the ev virus, we found that the amount of ev rna in the rd-sh-cypa cells decreased to less than % of that in the rd-sh-control cells at hpi (fig. c , start point), and this reduction was reversed when the rd-sh-cypa cells were infected with s p-ev virus (fig. d ). when we detected ev vp expression, we found that vp expression can be clearly and consistently attenuated in ev -infected rd-sh-cypa cells in comparison with ev -infected rd-sh-control cells from hpi (fig. e) . moreover, the augmentation of ev virus rna in rd-sh-control cells indicated that the replication of ev rna began at hpi (fig. c , black line); by contrast, this stage was obviously delayed to hpi in the rd-sh-cypa cells (fig. c , red line). however, when we infected rd or rd-sh-cypa cells with s p-ev , the growth curves revealed a similar curve, indicating that the vp -s p mutant confers resistance to cypa depletion (fig. d ). when we checked the viral titers in the culture, we found that the viral titers in the supernatant were not clearly altered in ev -infected rd-sh-cypa and rd-sh-control cells (fig. f) . we also infected rd cells by using wt-ev with a mm hl p treatment and measured the viral titers in the supernatant at different hpis (fig. a) . the results showed that infectious viral production was affected by the inhibitor at - , - and - hpi, but not as significantly as the impact on the intracellular viral genome at the same hpi. we thus speculated that cypa depletion blocked viral entry during re-infection and left more viruses in the culture. we further examined the internalization of ev by using immunofluorescence (fig. g) . the rd-sh-control and rd-sh-cypa cells were infected with wt-and s p-ev , and endogenous cypa and ev vp proteins were analyzed by immunofluorescence. in the rd-sh-control cells infected with wt-ev , ev vp was distributed throughout the cytoplasm at hpi, which was indicative that the virus particle internalization and localization with cypa was random (fig. g, panel a-c) . the downregulation of cypa in the rd-sh-cypa cells was first confirmed ( fig. a) . in rd-sh-cypa cells infected with wt-ev , the localization of wt-ev was restricted to the cytoplasm of the perimembrane region at hpi (fig. g , panel d-f). we can also observe the colocalization of ev vp with cypa, suggesting that the knockdown of cypa inhibited the internalization of ev and cypa was accumulated around ev virions. by contrast, the internalization of ev was rescued by the s p-ev mutant (fig. g , panel g-i). a similar observation was also reported in the hpv pseudovirus in the presence of the cypa inhibitor [ ] . these data indicated that cypa depletion inhibited the internalization of ev into the host cells. treating with cypa did not enhance the binding of ev virions to functional receptors the entry of ev can be further divided into the following two processes: ) receptor binding and ) uncoating to release the viral genome [ ] . to demonstrate the exact function of cypa in ev entry, we first checked the effect of cypa downregulation in the binding of ev virions to host cells. the results showed that ev binding to host cells was attenuated by cypa knockdown (fig. a ) and could be rescued by the substitution of s with a proline residue (from % attenuation to % attenuation) (fig. b) . we then checked the binding affinity of three reported ev functional receptors, i.e., scarb , psgl- and hs, for the wt-ev virions without or with cypa treatment. the results revealed that cypa treatment did not lead to obvious upregulation in the binding with ev functional receptors (figs. c- e). together with the result showing that cypa directly interacts with the ev virion, the cypa treatment is not likely to enhance the binding to all reported receptors, and the attenuation of ev virions that are binding to rd-sh-cypa and compensation by s p mutation are likely to resulted in an interaction change between the virions and cypa located at the cell membrane. the data for the ev genome were expressed as the percentage of ev rna from the untreated control cells; the mrna level of gapdh was used as an internal control. the data for ev viral titers in the supernatant were measured as previously described. (b) the ev subgenomic replicon rna was transfected into rd-sh-control and rd-sh-cypa cells, and the luciferase levels were quantified by measuring the firefly luciferase activity in relative luminescence units at hpi; the firefly luciferase activity of pnl - was used as an internal control. the growth curves of wt-ev (c) and s p-ev (d) in rd-sh-control or rd-sh-cypa cells at an moi of . the ev infection was quantified by using qrt-pcr to detect the ev rna in cypa is an uncoating regulator of ev entry the conversion from ev s particles to s particles can be induced by uncoating scarb under an acidic condition [ ] . however, recent biochemical and structural studies have suggested that simply heating the s particles near uc induces virus expansion and rna genome release, and heating over uc leads to the subsequent protein melting of virions during uncoating [ ] . we next used a previously reported virion flotation assay [ ] , which is used to detect the conversion of s particles into other forms during viral uncoating, to check whether the uncoating process of ev entry could be affected by cypa. to be consistent, ev virions that were treated at uc exhibited a smaller shift (fig. f , blue line) from the native peak ( s) (fig. f , black line) after ultracentrifugation in a . - . g/ml discontinuous cscl gradient, whereas the virions that were treated at uc exhibited a much larger shift (fig. f, red line) . we next incubated s virions with mg of recombinant cypa followed by incubation at uc for h at ph . and the rd-sh-control or rd-sh-cypa cells at different times post infection, and the mrna level of gapdh was used as an internal control. the data represent the means of three independent experiments. error bars represent the sem. (e) the ev vp expression was also checked in the ev infected rd-sh-control and rd-sh-cypa cells by immunoblotting analysis. (f) viral titers in the supernatant were measured at different times post infection. (g) the subcellular localization of cypa and ev . immunofluorescence analysis was performed on rd-sh-control and rd-sh-cypa cells infected with wt-or s p-ev at an moi of . at hpi, the cells were fixed and stained with anti-cypa (panels a, e and i, green) or anti-ev vp (panels c, g, and k, red) antibodies, and dapi was used to visualize the nuclei (panels b, f, and j, blue). panels a-d, e-h, and i-l show the same cells. the merged images are shown in panels d, h, and l, respectively. doi: . /journal.ppat. .g ph . , respectively, before subjecting them to ultracentrifugation at , rpm for h at uc. the results revealed that cypa cannot trigger the conversion of s particles at ph . and ph . (fig. g) . on the contrary, when a mixture of s particles and cypa was incubated at uc at ph . for h, the shift from s particles was distinct (fig. h, black and red lines) . moreover, the catalytic-defective mutant of cypa, namely h q, was incubated with s particles at uc under ph . for h, the shift in virions was completely eliminated (fig. h, blue line) . all these results support the idea that cypa can regulate the uncoating process of ev entry in a phdependent manner, which plays a similar role as the only ev uncoating receptor, or scarb [ ] . the s p mutation in ev vp decreased viral fitness but conferred cypa inhibitor resistance to study the fitness of the mutation in the vp h-i loop, we transfected rd cells with rna transcripts of ev recombinants containing the coding mutations and generated recombinant viruses, which were designated as s p-ev , s a-ev , p a-ev , s p/p a-ev , and s a/p a-ev (fig. ). in comparison with the wt-ev , s a-ev and s p/p a-ev had almost equal supernatant ev infectivity titers (p = . or p = . , respectively). p a-ev and s a/p a-ev had slightly lower ev infectivity titers (p = . or p = . , respectively) (fig. a ). this finding indicated that a proline residue located in the h-i loop acts in the interaction of cypa and ev , leading to the correct conformation of viral capsid and further virus uncoating. it is notable that the supernatant infectivity titers of s p-ev were . log lower than those of wt-ev (p = . , fig. a ). the intracellular growth curve showed that s p-ev growth is also slower than that of wt-ev (fig. c) . however, when we infected rd cells with s p-ev and wt-ev viruses under a mm hl p treatment, we found that the growth of s p-ev was much better than that of wt-ev (fig. d ). this finding is consistent with the results of the infection by wt-ev or s p-ev in rd-sh-cypa cells (figs. c and d) . together, these results suggested that replacing s -vp with a proline residue decreased viral fitness but conferred resistance to cypa inhibitors or caused a cypa loss of function. the results we report here demonstrate that the cypa host factor played a crucial role in the uncoating process during the entry step of ev infection, and the action site of cypa was mapped to the h-i loop of capsid protein vp . an analysis of all ev sequences in genbank showed that the action position of cypa in the ev vp h-i loop was strictly conserved in all ev genotypes and stains (fig. a) , either in the protein sequence or the gene codon. however, a comparison of several representative strains of coxsackie virus (cv), poliovirus (pv) and ev suggest that this position is not conserved among ev , cva , cvb and pv (fig. a ). the dependence of the proliferation of other enteroviruses or picornaviruses on the host factors must be further defined. the crystal structure of mature ev particles [ ] revealed that the vp h-i loop is a mostly solvent-exposed region at the surface of the virus particle (fig. b ) and usually functions in receptor binding or uncoating. among all reported ev functional receptors, scarb is the only one that can mediate both attachment to the host cell and uncoating [ ] . psgl- cannot induce the conversion from mature s particles to other forms during the viral uncoating process [ ] . in a recent result, nishimur et al. reported that the h-i loop of vp plays an essential role in ev recognition through one of its functional receptors, namely psgl- , and they demonstrated that the substitution of vp -k and k , which are located in the vp h-i loop, significantly attenuated virus binding to psgl- [ ] . they also indicated that the vp e residue modulates the orientation of vp k and thus regulates the exposure of the positively charged lysine side chain, which in turn regulates receptor binding [ ] . moreover, tan et al. showed that ev binds to heparan sulfate on the cell surface, and they suggested that heparan sulfate may bind to the positively charged amino acids (including vp -k , k , and r ) that form a cluster around the five-fold symmetry axis [ ] . these findings suggested that the lysine residues at the vp - and positions play essential roles in the binding of the ev virus to variable receptors. interestingly, these two lysine residues are surprisingly very close to the cypa action site in the vp h-i loop, which is vp -s (fig. b) . in a very recent result, lee et al. reported an anti-ev neutralizing antibody called ma - , which has epitopes at the fivefold vertex that cover the vp h-i loop [ ] ; this study supports the critical role of the correct h-i loop conformation in the ev entry step. by contrast, the binding site of the ev virion to scarb was mapped at a canyon of vp around residue q [ ] , which is far away from s -vp . we observed that ev virion binding to scarb was not enhanced, but slightly decreased by cypa treatment, suggesting that the cypa function in scarb -mediated ev entry could be more complicated. taken together, we propose that cypa plays a role as an uncoating regulator by altering the conformation of the h-i loop in vp during the ev entry through the psgl- or hsmediated pathway, and cypa has different impacts on the entry of ev through various functional receptors. another interesting observation is that cypa mediated ev uncoating most distinctly at ph . , but not at ph . and . . during virus internalization, endosome acidification increases during maturation, at values ranging from ph . to . in early endosomes to ph . to . in late endosomes [ , ] . because scarb was previously shown to mediate ev uncoating most efficiently at ph . [ ] , we propose that cypa acts to mediate ev uncoating before scarb during the maturation of late endosomes. with the increasing maturation of late endosomes and acidification, the ev uncoating regulator transfers from cypa to the next one, namely scarb . a similar observation was also found for the cypa study in hpv entry. cypa treatment induced the release of capsid protein l from l in a ph-dependent manner, in which l dissociation from l was most efficient at ph . , less efficient at ph . , and undetectable at ph . and . [ ] , suggesting a complicated process during virus uncoating in endosomes. furthermore, cypa showed complicated impacts on the hiv- life cycle; cypa is necessary for hiv- infection [ , , ] but also blocks hiv- uncoating [ ] , as revealed in previous reports. we cannot simply exclude the possibility that cypa may not only be associated with ev entry but might also affect other intracellular steps of ev protein translation, assembly or secretion in addition to its effects on the entry step. in our results, we actually found that the s p-ev virus proliferated more slowly than the wt-ev without cypa inhibitor treatment, although the s p mutant in vp can enhance the interaction between virions and cypa. moreover, when we infected rd-sh-cypa cells with wt-ev , although the intracellular genome rna and vp protein level were much lower than that of wt-ev -infected rd-sh-control cells, the supernatant virus titer exhibited no significant difference. interestingly, the downregulation of cypb did not attenuate but actually increased ev rna replication and vp expression (figs. c and d), and the expression of endogenous cypb was also upregulated by ev infection (fig. h) . moreover, the s p mutant recovered both the rna replication and protein expression of ev in rd-sh-cypa cells, but presented discrepancies in rd-sh-cypb cells, i.e., slightly increased rna replication but decreased protein expression (figs. c and e). interestingly, two recent works revealed that cyps inhibited the proliferation of hiv- virus, which is in opposition to the previously identified positive function of cyps in the hiv- life cycle [ , , ] . all these findings indicated that cyps may have multiple functions in the ev life cycle and may have additional (or opposing) effects on viral assembly and secretion. this finding requires further validation. the work we describe here highlights the new function of cypa as an uncoating regulator for ev proliferation by facilitating the conformational shift of the vp h-i loop. our results significantly increase our understanding of virus-host interactions and provide an additional target of action for csa-derived antivirals without immunosuppressive activity that are currently in clinical trials for treating ev -infection. rd cells (a human embryonal rhabdomyosarcoma cell line) were purchased from atcc and the huh . . cells were kindly given by jin zhong (institute pasteur of shanghai, chinese academy of science). the cells were grown in dulbecco's modified eagle's medium (dmem) (gibco) supplemented with % fetal bovine serum (fbs) (gibco) at uc in a humidified incubator with % co . the plasmids containing human ev strain anhui (gq . ) and brcr (u ) were kindly provided by prof. bo zhang from the wuhan institute of virology. plasmids containing human ev strain sk-ev (ab . ) and ev -gfp, which contains a gfp reporter gene that is inserted into the sk-ev genome, were donated by prof. satoshi koike (tokyo metropolitan institute of medical science). the plasmid with ev subgenomic replicon rna was given by prof. wenhui li (national institute of biological sciences [ ] ). the pnl - plasmid was given by prof. linqi zhang (school of medicine, tsinghua university). the ev viruses were amplified in rd cells, quantified by making a determination of the % tissue culture infective dose (tcid ) per ml in rd cells as previously described [ ] , and used for all experiments. a mouse anti-ev monoclonal antibody against vp (abcam, f , cat #ab ) was used to detect the virus in all experiments. rabbit anti-cypa monoclonal antibody (cat #ab - ) and rabbit anti-cypb monoclonal antibody (cat #ab ) were purchased from abcam. the anti-gapdh monoclonal antibody and anti-gst monoclonal antibody were purchased from jiamei, china. the secondary antibodies used for western blot analysis and immunofluorescence were purchased from southern biotech (hrp-conjugated goat anti-mouse igg(h+ l)), cowin bioscience (hrp-conjugated goat anti-rabbit igg), santa cruz (pe-conjugated goat anti-mouse igg), and life technologies (donkey anti-rat igg (h+l)). the cyclophilin a inhibitor known as csa was purchased from sigma, and hl p compound was generously provided by prof. jian li [ ] . the inhibitors were initially dissolved in dmso, and stock solutions were stored at uc. immediately before addition, these compounds were diluted to the desired concentrations in dmem with % fbs. trizol reagent and a super script iii first-strand synthesis system for rt-pcr kit were purchased from invitrogen. a mega script t high yield transcription kit was purchased from ambion. a quantitect sybr green rt-pcr kit was purchased from qiagen. a cell viability and proliferation assay (wst- ) was purchased from roche. rna transcripts and the ev subgenomic replicon were obtained by using the mega script t high yield transcription kit (ambion), and the dna that was linearized by sali or xbali (neb) digestion was used as a template according to the manufacturer's protocol. in vitro transcribed rna was transfected into rd cell monolayers in mm mm dishes with lipofectamine (invitrogen), and the cells were then incubated at uc in ml dmem containing % fbs per dish. the cytopathic effects (cpe) of rd cells were observed at h post transfection. when % of the cells exhibited cpe, the cell supernatants were then collected by centrifugation at , rpm for min, and the target viruses were stored at - uc. the virus titers were determined by using endpoint dilution assays (epda), with focus-forming units (ffu) as the read-out [ ] . in brief, the measurement was performed by seeding rd cells per well in -well microtiter plates. after overnight culture, the ev viruses were serially diluted -fold with dmem containing % fbs ( -to -fold dilutions) and added to rd cell. the plates were then incubated at uc in % co . cpe was observed under the microscope after to days post infection or the gfp expression level was monitored under a fluorescence microscope after days post infection. the virus titer, which was expressed as the tcid , was determined by epda. total cellular rna was isolated with trizol reagent according to standard protocols. the following primer sequences were used for qrt-pcr: gapdh, forward primer -cccactcctc-cacctttgacg- , reverse primer -caccaccctgttg-ctgtagcca- , ev -utr forward primer -tgaatg-cggctaatcccaact- , and reverse primer -aagaaa-cacggacacccaaa g- . qrt-pcr was performed with a quantitect sybr green rt-pcr kit (qiagen), and the ev and gapdh transcript levels were determined by ddct methods. to determine the amount of purified ev virions, viral rna was extracted from ml of pbs buffer containing ev virions by using trizol ls reagent (invitrogen). to determine the amount of ev virions in the cscl fractions, viral rna was extracted from ml of the cscl fraction containing ev virions with an additional ml of nuclease-free water by trizol ls reagent (invitrogen). a puc -ev ah plasmid was used as a standard sample to generate a standard curve ranging from - copies/ml. ev rna copies were quantified by using the quantitect sybr green rt-pcr kit (qiagen). the antiviral activities of the compounds were determined by using a qrt-pcr-based assay with the ev virus and rd cells. in brief, , rd cells were seeded in each well of the -well tissue culture plates and allowed to attach in complete culture medium overnight. the culture medium was replaced with medium containing serially diluted compounds in the presence of % fbs and . % dmso. after h, the rd cells were infected with ev at the multiplicities of infection (mois) indicated in the figure legends, and the compounds were added at the indicated concentrations. total cellular rna was isolated by using trizol reagent according to standard protocols at hpi. the qrt-pcr assay was performed as described above. the ev and gapdh transcript levels were determined by ddct method. the ic value represents the concentration of the compound at which the ev rna level in the rd cells was reduced by %. to monitor the cytotoxic effects of the compounds, the viability of the rd cells was determined after h of compound treatment; the viability was determined in -well tissue culture plates by using cell proliferation reagent wst- (roche). each data point represents the average of three replicates. the ec and cytotoxicity values were plotted by using graphpad prism software. rd cells were seeded at cells/well in -well plates. on the following day, the medium was removed and replaced with dmem containing % fbs and . mm hl p ; . % dmso was used as a control. after h, ev strain anhui was used to infect rd cells at an moi of . in complete medium containing the inhibitors. over the course of selection, the rd cells were split when they reached - % confluence. fresh complete medium containing inhibitors was added when the cell cultures were split. viral replication in the presence of compound hl p was monitored by determining the cytopathic effects (cpe) at each passage. the viruses demonstrated apparent cpes after approximately to days after ev infection in the medium containing inhibitors or after days in the medium containing . % dmso. the cell supernatants were then collected following centrifugation at , g for min and were stored at uc as ev -p (passage ) virus. the rd cells were then treated with cyclophilin a inhibitors for h and were infected with the ev -p virus under the same conditions described above. the experiment was repeated for cycles, and the cell supernatants were collected as ev -p (passage ) virus. the rd cells were lysed with trizol reagent. the cypa and cypb stable knockdown rd or huh . . cell lines were produced as previously described, with some modifications [ ] . the following shrna sequences were used in this study: nc, -ttctccgaacgtgtgtcacgtttc- ; cypa, -ctggattgcagagttaagttta- ; and cypb, -gccgggtgatctttggtctctt - . shrna recombinant lentiviruses (lv -nc, lv-cypa, lv-cypb) were produced by shanghai genepharma, and the virus titers were determined to be tu/ml. rd or huh . . cells were infected at an moi of . the shrna recombinant lentivirus was incubated with mg/ml polybrene to enhance the lentivirus infection. all knockdown cell lines were confirmed at h post infection by western blot analysis. for the stable knockdown cell lines, the rd or huh . . cells were incubated in selection medium containing mg/ml puromycin (invitrogen) beginning h after transduction, and the cypa and cypb knockdowns were stable after approximately two weeks of cell culture. infection and immunofluorescence assays after an sirna knockdown of cyps rd cells or cypa knockdown cells were grown on cover slips until the cells reached % confluence; the cells were then infected with the ev virus. the cells were washed with pbs at the indicated times post infection and fixed with % paraformaldehyde for min at room temperature, washed, and permeabilized with . % triton x- in pbs for min. the cells were then washed and blocked with % normal goat serum in pbs for min, followed by a h incubation with primary antibodies ( : dilution) at room temperature. after three washes with pbs, the cells were incubated with fitc-or pe-conjugated secondary antibodies (a : dilution) for h. after extensive washing with pbs, the cell nuclei were stained with dapi. images were captured by using a confocal microscope (olympus fluoview fv confocal microscope operated by fluoview software). the same microscope settings and exposure times were used within the individual experiments. the genes encoding the h-i loop of ev vp (residues -gsskskypl- ), the h-i loop with s p substitution (residues -gsskpkypl- ), human cypa and the catalytic-defective mutant cypa h q were cloned into the pgex- p- expression vector with a gst tag fused at the n-terminus according to a general protocol. the accuracy of the insert was verified by sequencing. the plasmids were transformed into e. coli bl (de ) cells, and the transformed cells were cultured at uc in lb media containing mg/l ampicillin. after the od reached . , the culture was cooled to uc, and recombinant protein expression was induced. after overnight induction, the cells were harvested by centrifugation. the pellets were then resuspended in lysis buffer containing mm tris-hcl (ph . ) and mm nacl, followed by homogenization using an ultra-high-pressure cell disrupter (jnbio, guangzhou, china) at uc. the insoluble material was removed by centrifugation at , g. the supernatant was then loaded twice onto a gst column preequilibrated with lysis buffer. after loading, the gst column was washed with at least column volumes of lysis buffer to remove the unbound protein. the beads containing recombinant gst- s, gst- p, gst-cypa or gst protein were added to eppendorf tubes and stored at uc until use. to obtain recombinant human cypa without a gst tag, the gst tag on cypa was removed by overnight incubation with prescission protease, and the target proteins were eluted with lysis buffer. the eluted target proteins were further purified by superdex- gel filtration chromatography (ge healthcare) to remove any contamination. the fractions were analyzed with sds-page, and the final purity was over %. for the immunoblot analysis, the cells were lysed in a lysis buffer containing mm tris-hcl (ph . ), mm nacl, % nonidet p- , . % sds, mm edta, and protease inhibitors; the protein concentrations of the lysates were determined with a spectrophotometer. the proteins were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (sds-page) and transferred to nitrocellulose membranes (millipore). the membranes were blocked for h with % nonfat dry milk solution in tris-buffered saline. the membranes were then blotted with specific primary antibodies, followed by incubation with secondary antibodies conjugated to horseradish peroxidase. the proteins were visualized by chemiluminescence by using a clarity western ecl substrate (bio-rad). to allow the pull-down assays to detect the interaction between cypa and the ev virion, we incubated ml of the ev strain (anhui , sk-ev , s a/p a-ev and brcr) ( ticd ) with ml of glutathione-sepharose beads containing gst ( mg) or gst-cypa ( mg) in ml of immunoprecipitation buffer ( mm tris-hcl, ph . , mm nacl, % nonidet p- , mm edta, and protease inhibitors) overnight at uc. the beads were then washed three times with pbs, and the complexes were eluted from the glutathione-sepharose beads with reduced glutathione (gsh) solution. we then moved the supernatant to new eppendorf tubes and added sds loading buffer. we then incubated the supernatants in boiling water for min and subjected the samples to % sds-page followed by western blot analysis with mouse antibodies against ev -vp or gst. to detect the interaction between cypa and gst- s or gst- p, recombinant human cypa was expressed, purified, and concentrated to mg/ml. recombinant human cypa ( mg) was incubated with ml of glutathione-sepharose beads containing gst ( mg), gst- s ( mg), or gst- p ( mg) in ml of immunoprecipitation buffer as described above, and the reactions were incubated overnight at uc. we then washed the beads three times with pbs and eluted the complexes from the glutathionesepharose beads with reduced gsh solution. the samples were then subjected to western blot analysis, as described above, by using mouse antibodies against gst or cypa. the bands were quantified by imagej software. the time of addition effect was examined for hl p . rd cells ( . per well in ml of % fbs-dmem medium) were cultured at uc under % co in -well plates overnight. the cells were subsequently treated with mm hl p either concurrent with the wt-ev ( h) at an moi of or at intervals of , , , , , , and hpi. after incubating at uc for h, the antiviral activity was determined by measuring the percentage of ev rna from the untreated control cells, and the mrna level of gapdh was used as an internal control. the supernatant virus titer, which was expressed as the tcid , was determined by epda. the virus binding assay was performed by using a previously reported protocol with some modifications [ ] . in brief, rd cells were seeded at rd cells/well in -well plates. the following day, the culture medium was removed and the cells were washed once with cold phosphate-buffered saline (pbs). after that, ml of binding buffer (pbs containing % bsa and . % sodium azide) was added to the cells on ice and incubated for min; the supernatant was subsequently removed from the cells. the ev stocks (strain anhui ) ( tcid /ml) were prepared as previously mentioned. the ev virus was diluted in ml of dmem complete medium (dilution fold = : [ tcid ], or : [ . tcid ]) per well and added to the cells. after h of incubation on ice, the unbound virus was removed by three wash steps with ml of pbs, and the cells were lysed in the wells with ml of trizol. viral rna was extracted and detected by qrt-pcr. the virus binding assays were systematically performed in duplicate, and two individual experiments were performed for each condition. binding assays of viruses with hs, psgl- or scarb the virus binding assay was performed according to a previously reported protocol [ , ] . to detect the influence of cypa on the hs and ev virus interaction, ml of ev strain anhui ( ticd ) was incubated with or without cypa at uc for h. the supernatant was added to a ml hitrap heparin hp column (ge healthcare, sweden) that was previously equilibrated with binding buffer ( . m tris-hcl and . m nacl [ph . ]) at a flow rate of approximately . ml/min. after loading, the two columns were washed with at least column volumes of binding buffer to remove the unbound virus. the bound viral particles were eluted by using elution buffer ( . m tris-hcl and m nacl [ph . ]). fractions of ml were collected, and the ev rna was isolated and quantified by using the qrt-pcr method described above. purified cypa was also uploaded to the same hitrap heparin hp and did not show any detectable interaction between cypa and the column (data not shown). to detect the cypa influence on the psgl- and ev virus interaction, ev strain anhui ( ticd ) was incubated with or without recombinant human cypa ( mg) in ml of dmem for h at uc. human psgl- -fc ( mg, r&d systems) or human igg vrc fc ( mg) (a control), were then added to the assays, and the reactions were incubated for h at uc. protein g-agarose ( ml, roche) was then added to the mixture, and the tubes were shaken overnight at uc. to detect the cypa treatment effect of the scarb and ev virus interaction, ev strain anhui ( ticd ) was incubated with or without recombinant human cypa ( mg) in ml of dmem for h at uc. scarb at a mg quantity was added to the assays, and the reactions were incubated for h at uc. ni-nta beads ( ml) were then added to the mixture, and the tubes were shaken overnight at uc. all the beads were washed three times with pbs and then eluted with ml of elution buffer containing mm tris-hcl, ph . , mm nacl and m imidazole. the ev rna in the supernatant was isolated by trizol ls reagent and quantified by using the qrt-pcr method described above. the purification of wt-ev virions was performed by using a previously reported protocol with modifications [ ] . in brief, rd cells in five t cell culture flasks were infected with ev (anhui strain) at an moi of . and cultured in % fbs. when % of the cells exhibited cpe, the supernatant was collected and concentrated by filtration through a kda-cutoff centrifugal filter (millipore). the concentrated virus was mixed with . g/ml cscl at a volume ration of : and loaded on the middle of a cscl gradient ( . g/ml, . g/ml, . g/ml, . g/ml, and . g/ml, discontinuously) followed by ultracentrifugation at , rpm for h at uc in a beckman sw ti rotor. after being dialyzed with pbs, the purified ev virus was quantified and stored at - uc. the rna copies of purified ev virus was quantified as previously described. a virion flotation assay of the cscl density gradient centrifugation fifty ml of purified ev virus ( genome copies) was incubated with mg of purified wt cypa or catalytic-defective mutant cypa h q in pbs containing . % bsa in a total volume of ml. for the low ph treatments, hcl was added to the mixture to bring the ph values to . , . and . . the mixture was then incubated at uc for h and subsequently applied to a . - . g/ml discontinuous cscl gradient, which was then ultracentrifuged at , rpm for h at uc in a beckman sw ti rotor. the samples were then analyzed by qrt-pcr. the transition states of viral particles during uncoating was performed in vitro with native s virions by heating for min in a low salt buffer containing mm cacl , mm hepes, ph . at uc and uc. figure s recombinant cypa catalyzed the cis-trans reaction of chemically synthesized peptides. the shifts in nmr spectra indicated the conformational peptidyl-proline change [ ] [ ] [ ] . at mhz h nmr spectra of peptides at uc and ph . , the concentration of each peptide is mm. s without cypa treatment (a), p without cypa treatment (c), or s (b) and p (d) with cypa treatment (the concentration was mm) for h at uc. the signal marked here is from the orthoproton signals from the p-nitroanilide of peptides, which represents the signals from the cis isomer (the chemical shift is . ppm) or the trans isomer (the chemical shift is approximately . ppm). the integral area of the respective peaks is used as the upper numerical value. the s peptide sequence is gsskskypl, and p peptide sequence is gsskpkypl. (tif) table s vp amino acid changes that emerged during selection with compound hl p . r- , r- , and r- represent three individual selection experiments for cypa inhibitor-resistant virus. (doc) table s hl p or csa resistance levels for sitedirected changes that were engineered into the ev virus. the s p substitution in ev vp confers resistance to hl p . the rd cells were incubated for h with various concentrations of compound hl p ( . to mm) and csa ( . to mm) and then infected with wt-ev virus anhui or s p ev at an moi of . the ev rna levels were quantified by rt-qpcr. each data point represents the average for three replicates. text s supporting information for the nmr measurement of cypa activity. the pplase activity of recombinant activity was measured as in a previously described nmr method [ ] [ ] [ ] . in brief, the recombinant cypa protein used in the nmr experiments was diafiltered into mm phosphate buffer, ph . , and concentrated at a stock concentration of mm. the substrate peptides were chemically synthesized and dissolved to a concentration of mm in buffer containing mm sodium phosphate, ph . . the recombinant cypa protein (at a final concentration of mm) was added to the substrate peptides and the mixture was incubated for h at uc. a % d o solution was used as a lock sample in the nmr spectrometer. aliquots were added to the mm nmr tube. h nmr measurements were performed at . mhz, uc, on a bruker- mhz nmr spectrometer, and the acquired data were processed with mestrenova software. (doc) calcineurin is a common target of cyclophilin-cyclosporin a and fkbp-fk complexes cyclophilin a and viral infections target cell cyclophilins facilitate human papillomavirus type infection functional association of cyclophilin a with hiv- virions specific incorporation of cyclophilin a into hiv- virions structural characterization of the hiv- vpr n terminus: evidence of cis/trans-proline isomerism binding of the human immunodeficiency virus type gag polyprotein to cyclophilin a is mediated by the central region of capsid and requires gag dimerization cd facilitates hiv- infection by interacting with virus-associated cyclophilin a the host proteins transportin sr /tnpo and cyclophilin a exert opposing effects on hiv- uncoating cyclophilin b is a functional regulator of hepatitis c virus rna polymerase cyclophilin a is an essential cofactor for hepatitis c virus infection and the principal mediator of cyclosporine resistance in vitro essential role of cyclophilin a for hepatitis c virus replication and virus production and possible link to polyprotein cleavage kinetics critical role of cyclophilin a and its prolyl-peptidyl isomerase activity in the structure and function of the hepatitis c virus replication complex cyclosporine a inhibits hepatitis c virus nonstructural protein through cyclophilin a hepatitis c virus ns a protein is a substrate for the peptidyl-prolyl cis/trans isomerase activity of cyclophilins a and b identification of residues required for rna replication in domains ii and iii of the hepatitis c virus ns a protein cyclophilins facilitate dissociation of the human papillomavirus type capsid protein l from the l /dna complex following virus entry enterovirus vpg uridylation uses a two-molecular mechanism of d polymerase current progress in antiviral strategies crystal structure of enterovirus rna-dependent rna polymerase complexed with its protein primer vpg: implication for a trans mechanism of vpg uridylylation molecular determinants of enterovirus viral entry: cleft around gln- on vp protein interacts with variable region on scavenge receptor b human p-selectin glycoprotein ligand- is a functional receptor for enterovirus scavenger receptor b is a cellular receptor for enterovirus structure of limp- provides functional insights with implications for sr-bi and cd enterovirus uses cell surface heparan sulfate glycosaminoglycan as an attachment receptor annexin ii binds to capsid protein vp of enterovirus and enhances viral infectivity discovering potent small molecule inhibitors of cyclophilin a using de novo drug design approach structural perspective on the formation of ribonucleoprotein complex in negative-sense single-stranded rna viruses cyclophilin a interacts with influenza a virus m protein and impairs the early stage of the viral replication cyclophilin a restricts influenza a virus replication through degradation of the m protein crystal structure of human enterovirus active site mutants of human cyclophilin a separate peptidyl-prolyl isomerase activity from cyclosporin a binding and calcineurin inhibition the isomerase active site of cyclophilin a is critical for hepatitis c virus replication a sensor-adaptor mechanism for enterovirus uncoating from structures of ev more-powerful virus inhibitors from structure-based analysis of hev capsid-binding molecules enterovirus binding to psgl- on leukocytes: vp - acts as a molecular switch to control receptor interaction a strainspecific epitope of enterovirus identified by cryoem of the complex with fab from neutralizing antibody endosome maturation virus entry by endocytosis preferential chemotaxis of activated human cd + t cells by extracellular cyclophilin a structural and functional analysis of prehistoric lentiviruses uncovers an ancient molecular interface single amino acid changes in the virus capsid permit coxsackievirus b to bind decayaccelerating factor hiv- evades innate immune recognition through specific cofactor recruitment target cell type-dependent modulation of human immunodeficiency virus type capsid disassembly by cyclophilin a characterization of a vero celladapted virulent strain of enterovirus suitable for use as a vaccine candidate robust hepatitis c virus infection in vitro identification of site-specific adaptations conferring increased neural cell tropism during human enterovirus infection cyclophilin and peptidyl-prolyl cis-trans isomerase are probably identical proteins peptidylproline cis/trans isomerases peptidyl-prolyl cis-trans isomerase activity as studied by dynamic proton nmr spectroscopy we gratefully acknowledge prof. linqi zhang from tsinghua university for his great and generous support to this work. we also thank prof. li yu from tsinghua university for his critical suggestions. key: cord- -csro ks authors: sigalov, alexander b. title: novel mechanistic insights into viral modulation of immune receptor signaling date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: csro ks nan to successfully infect, replicate, and persist in the host, viruses have evolved numerous strategies to take control of multiple cellular processes, including those that target transmembrane (tm) signal transduction mediated by immune receptors. despite tremendous advancement in recent years, the exact molecular mechanisms underlying these critical points in viral pathogenesis remain unknown. in this opinion, based on a novel model of immune signaling, the signaling chain homooligomerization (school) model, i suggest specific mechanisms used by different viruses such as human immunodeficiency virus (hiv), cytomegalovirus (cmv), severe acute respiratory syndrome coronavirus (sars-cov), herpesvirus saimiri (hvs), human herpesvirus (hhv- ), etc., to modulate the host immune response mediated by members of the family of multichain immune recognition receptors (mirrs). i also demonstrate how the school model, together with the lessons learned from viral pathogenesis, can be used practically for rational drug design and the development of new therapies for immune disorders. in mirrs, the recognition domains and signaling sequences containing immunoreceptor tyrosine-based activation motifs (itams) are located on separate subunits bound together by noncovalent tm interactions ( figure a ) [ ] [ ] [ ] . based on a novel biophysical phenomenon, the homointeractions of intrinsically disordered proteins [ , ] , the school model of mirr signaling [ , [ ] [ ] [ ] uncovers the molecular mechanisms by which clustering of the extracellular recognition domains leads to receptor triggering. the model suggests that mirr engagement leads to receptor oligomerization coupled with a multi-step structural reorganization driven by the homooligomerization of signaling subunits ( figure b) . importantly, this model is based on specific protein-protein interactions-biochemical processes that can be influenced and controlled, providing a promising drug design approach [ ] . within the model, specific blockade or disruption of tm interactions causes a physical and functional disconnection of the mirr subunits ( figure c ) [ , , , ] . antigen stimulation of these ''predissociated'' receptors leads to reorientation and clustering of the recognition but not signaling subunits. as a result, signaling oligomers are not formed, itam tyr residues do not become phosphorylated, and the signaling cascade is not initiated ( figure c ). in contrast, this ''predissociation'' does not prevent the formation of signaling oligomers when signaling subunits are clustered by specific antibodies that trigger cell activation (not illustrated). predicted and molecularly explained by the school model [ , [ ] [ ] [ ] [ ] , manipulation of mirr signaling is performed by numerous unrelated viruses throughout their life cycle. in this context, the ability viruses have developed over centuries of evolution [ , ] to modulate t cell receptor (tcr) signaling plays a crucial role in viral pathogenesis. for t lymphotropic viruses, the virus may inhibit tcr signaling to disarm the receptor and successfully enter the cell [ , , , , ] . a similar strategy can be used by the virus to persist in the cell until it reactivates and produces infectious particles. for other viruses, modulation of tcr signaling can be used to inhibit the t cell response to infected cells [ , , , ] . structurally, tcr is a member of the mirr family and has the a and b antigen-binding subunits that are bound by electrostatic tm interactions with three signaling homo-and heterodimers: ff, cd ed, and cd ec ( figure a ) [ , ] . as suggested by the school model [ , , , ] , these interactions are not only promising thera-peutic targets, but also represent an important point of viral attack. tm peptides capable of inhibiting tcr-mediated cell activation were first reported in [ ] . the vast majority of findings were reported for the tcr core peptide (cp), a synthetic peptide corresponding to the sequence of the tcra tm domain (tmd) and known to interact with the tmds of cd de and f [ , ] . interestingly, t cell activation via anti-cd antibodies is not affected by this peptide ( table ) . as shown, tcr cp might be a proper treatment for human t cell-mediated dermatoses substituting for corticosteroids [ , ] . however, despite extensive studies [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , the mode of action of this clinically relevant peptide was not explained until when the school model was first introduced [ ] . recently, inhibition of t cell activation has been reported for the fusion peptide (fp) found in the n terminus of the hiv envelope glycoprotein (gp ) [ , ] . these data are the first to demonstrate that not only does fp function to fuse the virion with the host cell membrane [ , ] , but it also has immunomodulatory activity. the peptide inhibits antigen-but not anti-cd stimulated t cell activation in vitro and has immunosuppressive activity in mice [ ] ( table ) . similar to tcr cp [ , , , ] , hiv fp has been suggested for the treatment of t cell-mediated pathologies [ ] . however, the mode of action of this peptide remained unexplained until when the school model was first applied to this area [ ] . considering the similarity between fp and cp in patterns of immunomodulatory activity (table ) and in their having two electropositive residues in their primary sequences, the school model reasonably suggests a similar mode of action for these peptides ( figure ) [ , , , - ]. briefly, cp and fp compete with tcra for binding to cd de and ff, resulting in functional disconnection of these subunits ( figure c ). this prevents formation of cd de and f signaling oligomers and thus inhibits t cell activation upon stimulation with antigen but not anti-cd antibodies ( figure d ), thereby suggesting a molecular explanation for the use of okt antibodies in hiv therapy to augment immune activation [ ] . interestingly, the school mechanism is the only one consistent with all of the experimental data on the immunomodulatory action of hiv fp and tcr cp reported so far [ , , [ ] [ ] [ ] [ ] [ ] [ ] . charge distribution patterns for fusion protein regions are surprisingly conserved in many unrelated viruses and show similarities to those for tcr cp and hiv fp ( figure e ). thus, it is highly probable that these proteins would also target the tcr tm interactions using the school mechanism. exploratory sequence investigation of fps from sars-cov, lassa virus (lasv), lymphocytic choriomeningitis virus (lcmv), mopeia virus (mopv), and tacaribe virus (tacv) reveals a close similarity in the positioning of the electropositive residues ( figure e) . intriguingly, analysis of other unrelated viruses has yielded similar correlations in primary structure and function. earlier studies have reported an inhibitory effect on lymphocyte proliferation by cks- peptide, a synthetic mer peptide with sequence corresponding to a highly conserved region of tm proteins of human and animal retroviruses, including the tm protein gp of human t lymphotropic virus type (htlv- ) [ ] [ ] [ ] . interestingly, peptides corresponding to regions of hiv tm protein gp homologous to the highly conserved and immunosuppressive sequence contained within the tm proteins p e and gp of animal and human retroviruses, respectively, also have been reported to inhibit lymphoproliferation , shown as a simplified axial view) and prevents formation of signaling oligomers upon antigen but not antibody stimulation, thus inhibiting antigen-mediated but not anti-cd -mediated tcr triggering and cell activation (d). primary sequence analysis of proven and predicted immunomodulatory sequences of viral fusion protein regions and other domains shows a similarity in the charge distribution pattern with two essential positively charged residues (shown in blue) spaced apart by three to four or seven to eight amino acids (e), suggesting a similarity of mechanisms used by diverse viruses in their pathogenesis to modulate the host immune response. note: although the three-dimensional structures of the analyzed sequences within the cell membrane are not known, it might be assumed that these sequences may adopt a helical conformation upon membrane binding. thus, helical wheel projections are used for illustrative purposes only; the suggested mode of action does not depend on a particular secondary structure of the sequences. [ , ] . recently, filoviral -mer peptides corresponding to a -amino acid domain in filoviral glycoproteins that resembles an immunosuppressive motif in retroviral envelope proteins have been demonstrated to inhibit tcr-mediated cell activation [ ] . in all peptides, a striking similarity is observed in the charge distribution patterns with the positioning of the essential positively charged residues almost identical to that for the hiv gp fp ( figure e ), suggesting again a similarity in their mode of action. this clearly demonstrates that different viruses have adopted similar mechanisms of specifically targeting tcr, disrupting receptor architecture, and suppressing the immune system. importantly, by virtue of the acquired insight into this conserved structural motif, expanded predictions, hypotheses, and conclusions can be derived to being answering the question of whether shared tcr-targeted strategies represent a conserved function or a convergent tactic of divergent viruses. the generality of the school model suggests that tm interactions of other mirrs can also represent a point of viral attack. as reported [ ] , the recognition of the human cmv tegument protein pp by nkp , the natural killer (nk) cell-activating receptor, does not lead to nk cell activation but instead results in a general inhibition mediated by the dissociation of the nkp -f complex and a loss in the ability of cells to kill virus-infected cells. within the context of the school model, pp may target the tm interactions between nkp and f, leading to functional disconnection of f in a manner similar to the action of tcr cp and hiv fp (figure ) . tm interactions can be targeted not only from outside but also from inside the cell. recently, it has been shown that the hhv- u protein downregulates tcr surface expression and that u -expressing t cells are resistant to activation by antigenpresenting cells [ ] . by controlling lymphocyte activation that is often accompanied by herpesvirus reactivation, the virus might prevent its own reactivation and persist in a latent state, which is less prone to immune recognition [ ] . in this context, u can serve to maintain equilibrium between the virus and its host by keeping hhv- titers low enough that they do not cause massive immune activation [ ] . tcr downregulation activity also has been reported for the highly conserved membrane-proximal sequence of the tyrosine kinase-interacting protein (tip) of hvs [ , ] . notably, primary sequences of hhv- u - and hiv fp exhibit a similar pattern with two arg residues spaced apart by eight amino acids ( figure e ). the positioning of the essential electropositive residues is remarkably conserved in hvs tip - , the relevant domain of the two-in-one (tio) protein of herpesvirus ateles (hva) and htlv- gp ( figure e ). thus, the school mechanisms similar to those applied for tcr cp and hiv gp fp (figure ) can be used by hhv- and other viruses in their arsenal of immune evasion tactics. importantly, as predicted, the viral agents prevent only antigen-but not antibodyspecific, t cell activation ( figure d ). indeed, anti-cd antibodies activate hhv- -infected t cells, resulting in a large increase of viral replication [ , ] . interestingly, increase of viral replication induced by okt -mediated activation of hiv-infected t cells is currently used for purging of the latent hiv- reservoirs in vivo [ ] , thus suggesting a potential generality of the school mechanismbased antiviral approaches. there are several important lessons that we can learn from the molecular mecha-nisms of viral pathogenesis. first, using modern methodologies [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , it is possible to design and produce tm agents that are able to modulate the immune response as specifically and effectively as viruses do. second, as predicted, tcr cp and many different immunomodulatory viral sequences affect similar tcr-tm interactions, suggesting that general principles of designing tm peptides might be readily used at this stage [ , ] . third, antibodies to mirr signaling subunits can be used to modulate the affected immune cell response during viral infection. fourth, considering our selective ability to functionally disconnect any particular tcr signaling subunits [ , , , ] , we can use the relevant peptides as a powerful tool to dissect fine mechanisms of viral pathogenesis. finally, two unrelated viruses, hiv and human cmv, use a similar mode of action to modulate the host immune response mediated by two functionally different mirrs, tcr and nkp , thus suggesting that similar general mechanisms can be or are used by other viral and possibly non-viral pathogens. in conclusion, rather than targeting virus-specific proteins or processes, it would be advantageous to transfer therapeutic strategies that target redundant processes found among a number of viruses. in addition, as demonstrated by the similar function of natural hiv fp and synthetically derived clinically relevant tcr cp, viral immune evasion strategies can be transferred to therapeutic strategies that require similar functionalities. viruses represent years of evolution and the efficiency and optimization that come along with it. therefore, viral functions should not only be studied as foreign processes but as efficient strategies that we can use in our own attempts at immune evasion or immunomodulation. human natural killer cell activating receptors transmembrane helical interactions and the assembly of the t cell receptor complex multi-chain immune recognition receptors: spatial organization and signal transduction homooligomerization of the cytoplasmic domain of the t cell receptor zeta chain and of other proteins containing the immunoreceptor tyrosine-based activation motif binding of intrinsically disordered proteins is not necessarily accompanied by a structural transition to a folded form multichain immune recognition receptor signaling: different players, same game? immune cell signaling: a novel mechanistic model reveals new therapeutic targets signaling chain homooligomerization (school) model disruption of proteinprotein interactions: towards new targets for chemotherapy transmembrane interactions as immunotherapeutic targets: lessons from viral pathogenesis multichain immune recognition receptor signaling: from spatiotemporal organization to human disease interaction between hiv gp fusion peptide and t cell receptor: putting the puzzle pieces back together viral pathogenesis, modulation of immune receptor signaling and treatment viral modulation of t-cell receptor signaling the organizing principle in the formation of the t cell receptor-cd complex t-cell antigen receptor transmembrane peptides modulate t-cell function and t cell-mediated disease t cell receptor mimic peptides and their potential application in t-cellmediated disease therapeutic application of t cell receptor mimic peptides or cdna in the treatment of t cellmediated skin diseases kinetic and conformational properties of a novel t-cell antigen receptor transmembrane peptide in model membranes the mode of anti-arthritic peptide delivery impacts on the severity and outcome of adjuvant induced arthritis t-cell antigen receptor assembly and cell surface expression is not affected by treatment with t-cell antigen receptor-alpha chain transmembrane peptide therapeutic application of transmembrane t and natural killer cell receptor peptides peptides in the treatment of inflammatory skin disease t cell antigen receptor (tcr) transmembrane peptides colocalize with tcr, not lipid rafts, in surface membranes t-cell antigen receptor peptides inhibit signal transduction within the membrane bilayer lipidation and glycosylation of a t cell antigen receptor (tcr) transmembrane hydrophobic peptide dramatically enhances in vitro and in vivo function hydrophobic transmembrane-peptide lipid conjugations enhance membrane binding and functional activity in t-cells t-cell antigen receptor-alpha chain transmembrane peptides: correlation between structure and function hiv- fusion peptide targets the tcr and inhibits antigen-specific t cell activation t-cell inactivation and immunosuppressive activity induced by hiv gp via novel interacting motif identification of the fusion peptide of primate immunodeficiency viruses detection of a fusion peptide sequence in the transmembrane protein of human immunodeficiency virus okt and il- treatment for purging of the latent hiv- reservoir in vivo results in selective long-lasting cd + t cell depletion inhibition of lymphocyte proliferation by a synthetic peptide homologous to retroviral envelope proteins identification, using synthetic peptides, of the minimum amino acid sequence from the retroviral transmembrane protein p e required for inhibition of lymphoproliferation and its similarity to gp of human t-lymphotropic virus types i and ii inhibition of lymphoproliferation by a synthetic peptide with sequence identity to gp of human immunodeficiency virus type implication of a retrovirus-like glycoprotein peptide in the immunopathogenesis of ebola and marburg viruses inhibition of the nkp activating receptor by pp of human cytomegalovirus downregulation of the t-cell receptor complex and impairment of t-cell activation by human herpesvirus u protein role of amphipathic helix of a herpesviral protein in membrane deformation and t cell receptor downregulation herpesviral protein targets a cellular wd repeat endosomal protein to downregulate t lymphocyte receptor expression enhancement of human herpesvirus replication in adult human lymphocytes by monoclonal antibody to cd replication of human herpesvirus- in thymocytes activated by anti-cd antibody polar residue tagging of transmembrane peptides transmembrane interactions in the activation of the neu receptor tyrosine kinase optimizing synthesis and expression of transmembrane peptides and proteins computational design of peptides that target transmembrane helices designing transmembrane alpha-helices that insert spontaneously bioinformatic discovery of novel bioactive peptides visual detection of specific, native interactions between soluble and microbead-tethered alpha-helices from membrane proteins synthetic peptides as models for intrinsic membrane proteins discrepancy in cd -transmembrane peptide activity between in vitro and in vivo t-cell inhibition i thank walter m. kim for his help with writing the manuscript. key: cord- -s wmll q authors: shang, jian; zheng, yuan; yang, yang; liu, chang; geng, qibin; luo, chuming; zhang, wei; li, fang title: cryo-em structure of infectious bronchitis coronavirus spike protein reveals structural and functional evolution of coronavirus spike proteins date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: s wmll q as cell-invading molecular machinery, coronavirus spike proteins pose an evolutionary conundrum due to their high divergence. in this study, we determined the cryo-em structure of avian infectious bronchitis coronavirus (ibv) spike protein from the γ-genus. the trimeric ibv spike ectodomain contains three receptor-binding s heads and a trimeric membrane-fusion s stalk. while ibv s is structurally similar to those from the other genera, ibv s possesses structural features that are unique to different other genera, thereby bridging these diverse spikes into an evolutionary spectrum. specifically, among different genera, the two domains of s , the n-terminal domain (s -ntd) and c-terminal domain (s -ctd), diverge from simpler tertiary structures and quaternary packing to more complex ones, leading to different functions of the spikes in receptor usage and membrane fusion. based on the above structural and functional comparisons, we propose that the evolutionary spectrum of coronavirus spikes follows the order of α-, δ-, γ-, and β-genus. this study has provided insight into the evolutionary relationships among coronavirus spikes and deepened our understanding of their structural and functional diversity. a a a a a as large enveloped rna viruses, coronaviruses are capable of adapting to new hosts with relative ease through mutations and recombinations [ ] [ ] [ ] . as a result, coronaviruses infect a wide range of mammalian and avian species, and have genetically evolved into four major genera: α, β, γ, and δ [ ] . coronaviruses from the four genera all contain envelope-anchored spike proteins that mediate viral entry into host cells [ , ] . during viral entry, the spikes bind to host receptors through their s subunits and then fuse viral and host membranes through their s subunits. on the one hand, the spikes interact with host receptors and other host factors, hence needing to evolve for better adaptation to these host factors [ ] [ ] [ ] [ ] . on the other hand, they are exposed to the host immune system, thereby needing to evolve to evade the host immune surveillance [ ] [ ] [ ] [ ] . consequently, the spikes are the most divergent among all coronavirus proteins [ ] . the s subunits are particularly divergent, with little or low sequence similarities across different genera [ ] . how coronavirus spikes have evolved to their current diverse structures imposes a major evolutionary conundrum. traces of protein evolution can often be found more reliably in their tertiary structures and related functions than in their primary structures, because proteins generally need to evolve within certain structural and functional constraints [ , ] . to decipher the evolutionary puzzles surrounding coronavirus spikes, extensive structural studies have been carried out using both x-ray crystallography and cryo-electron microscopy (cryo-em) [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . these studies have resulted in structure determinations of s from the α-and β-genera and spike ectodomains from the α-, β-, and δ-genera. coronavirus spikes exist in two distinct conformations: the pre-fusion structures are present on mature virions and have a clove-like shape with three s heads sitting on top of a trimeric s stalk [ ] [ ] [ ] [ ] [ ] [ ] ; the post-fusion structures are the membrane-fusion state and have a dumbbell-like shape with three s subunits forming a six-helix bundle structure [ ] [ ] [ ] [ ] [ ] [ ] [ ] . whereas the structures of s from different genera are similar to each other in both the pre-and post-fusion states, the s subunits from different genera diverge structurally and they also recognize a variety of host receptors [ , ] . s contains two domains, n-terminal domain (s -ntd) and c-terminal domain (s -ctd), either or both of which can function as the receptor-binding domain. the s domains from different genera contain structural features that are unique to their genus. s -ctds are particularly diverse, with low or no structural similarity across different genera [ ] . overall, these previous studies have provided structural snapshots of coronavirus spikes from α-, β-, and δ-genera. however, because the structures of γ-coronavirus spikes were still missing, we lacked a clear picture of the evolutionary relationships among coronavirus spikes from different genera. in the current study, we determined the cryo-em structure of avian infectious bronchitis coronavirus (ibv) spike, the first such structure from the γ genus. the ibv spike possesses structural features that are unique to different other genera, suggesting that it falls in the middle of an evolutionary spectrum of coronavirus spikes. we also discuss how the structural evolution of coronavirus spikes has affected their functions as cell-invading molecular machinery. overall, our study has filled in a critical gap in the structural, functional and evolutionary studies of coronavirus spikes, and deepened our understanding of viral evolutions in general. expression and purification of ibv spike ectodomain ibv spike gene (virus strain m ; genbank number abi . ) was synthesized (genscript) with codons optimized for insect cell expression. its ectodomain (residues - ) was cloned into pfastbac vector (life technologies inc.) with a n-terminal honeybee melittin signal peptide and c-terminal gcn and his tags. it was expressed in sf insect cells using the bac-to-bac system (life technologies inc.) and purified as previously described [ ] . briefly, the protein was harvested from cell culture medium, and purified sequentially on ni-nta column and superdex gel filtration column (ge healthcare). ibv s -ctd (residues - ) was expressed and purified in the same way as the ibv spike ectodomain, although it only contains a c-terminal his tag and does not contain the gcn tag. for sample preparation, aliquots of ibv spike ectodomain ( μl, . mg/ml, in buffer containing mm tris ph . and mm nacl) were applied to glow-discharged cf- / - c c-flat grids (protochips). the grids were then plunge-frozen in liquid ethane using a fei markiii vitrobot system (fei company). for data collection, images were recorded using a gatan k summit direct electron detector in the direct electron counting mode (gatan), attached to a fei titan-krios tem, at arizona state university. the automated software serialem [ ] was used to collect~ , total movies at , x magnification and at a defocus range between and μm. each movie had a total accumulated exposure of . e/Å fractionated in frames of ms exposure. data collection statistics are summarized in s table. for data processing, whole frames in each movie were corrected for beam-induced motion and dose compensation using motioncor [ ] and~ , best images were manually selected (we manually discarded micrographs with only carbon field of view or thick ice after motion correction as well as micrographs with defocus parameter higher than . μm after ctf estimation). the final image was bin-averaged to lead to a pixel size of . Å. the parameters of the microscope contrast transfer function were estimated for each micrograph using gctf [ ] . particles were automatically picked and extracted using relion [ ] with a box size of pixels. initially,~ , particles were subjected to d alignment and clustering using relion, and the best classes were selected for an additional d alignment.~ , best particles were applied for creating the initial d model using relion.~ , particles selected from d alignment were then subjected to d classification and the best class with , particles were subjected to d refinement to generate the final density map. the final density map was sharpened with modulation transfer function of k operated at kv using relion post-processing. reported resolutions were based on the gold-standard fourier shell correlation (fsc) = . criterion, and fourier shell correction curves were corrected for the effects of soft masking by high-resolution noise substitution [ ] . data processing statistics are summarized in s table. the initial model of ibv spike ectodomain was obtained by fitting the seven parts (s -ntd, s -ctd, two parts of sd , two parts of sd , and s ) of the porcine delta coronavirus spike structure (pdb id: b n) individually into the cryo-em density map of ibv spike using ucsf chimera [ ] and coot [ ] . manual model rebuilding was carried out using coot based on the well-defined continuous density of the main chain; the side chain assignments were guided by the densities of n-linked glycans and bulky amino acid residues. the structural model of the ibv spike in the pre-fusion state was refined using phenix [ ] with geometry restrains and three-fold noncrystallographic symmetry constraints. refinement and model rebuilding in coot were carried out iteratively until there were no further improvements in geometry parameters and model-map correlation coefficient. the quality of the final model was analyzed with molprobity [ ] and emringer [ ] . the validation statistics of the structural models are summarized in s table. ibv pseudovirus entry assay ibv pseudovirus entry assay was carried out as previously described [ ] . briefly, full-length ibv spike gene was inserted into pcdna . (+) plasmid. retroviruses pseudotyped with ibv spike and expressing a luciferase reporter gene were prepared through co-transfecting hek t cells (source: american type culture collection) with a plasmid carrying env-defective, luciferaseexpressing hiv- genome (pnl - .luc.re) and the plasmid encoding ibv spike. the produced ibv pseudoviruses were harvested hours post transfection, and then used to enter df- cells (source: american type culture collection) and hek t cells. after incubation for hours at ˚c, the medium was changed and cells were incubated for an additional hours. cells were then washed with pbs and lysed. aliquots of cell lysates were transferred to optiplate- (perkinelmer), followed by addition of luciferase substrate. relative light units (rlus) were measured using enspire plate reader (perkinelmer). all the measurements were carried out in quadruplicates. recombinant ibv s -ctd was assayed for its cell-binding capability using flow cytometry as previously described [ ] . briefly, hek t and df- cells were incubated with recombinant ibv s -ctd containing a c-terminal his tag ( μg/ml) at room temperature for minutes, followed by incubation with phycoerythrin (pe)-labeled anti-his antibody for minutes. the cells were then analyzed for the binding of ibv s -ctd using flow cytometry. the total surface area and buried surface area of coronavirus s -ctds were calculated using the pisa server at the european bioinformatics institute (http://www.ebi.ac.uk/pdbe/prot_ int/pistart.html) [ ] . specifically, for each trimeric spike protein, a pdb file containing all of the six s domains (including three copies of s -ctds and three copies of s -ntds) was submitted to the pisa server, and the total surface area and buried surface area for each s -ctd were calculated. for the spike proteins used for the above analysis, all their s -ctds were in the "lysing down" state. the structures of mers-cov and hku spikes were not included in the above analysis because the former contain at least one s -ctd in the "standing up" state and the latter contains long stretches of missing residues in its s domains, both of which would interfere with the above analysis. we constructed the ibv spike ectodomain (from ibv strain m ) in the pre-fusion state by replacing its transmembrane anchor and intracellular domain with a c-terminal gcn trimerization tag, followed by a his tag ( fig a) . we expressed the protein in insect cells and purified the protein to homogeneity. we collected cryo-em data on ibv spike ectodomain, calculated a density map at . Å resolution (fig b; s fig) , built an atomic model of the structure and refined it (fig c and d) . the final structural model contains all of the residues from to (except residues - ) as well as glycans n-linked to sites. data collection and model statistics are shown in s table. the overall structure of ibv spike ectodomain resembles the pre-fusion structures of coronavirus spikes from the α-, β-, and δ-genera [ ] [ ] [ ] [ ] [ ] [ ] . it has a clove-like shape, with three s heads forming a crown-like structure and sitting on top of a trimeric s stalk. each overall structure of ibv spike ectodomain in the pre-fusion conformation. (a) schematic drawing of ibv spike ectodomain. s : receptor-binding subunit. s : membrane-fusion subunit. gcn -his : gcn trimerization tag followed by his tag. s -ntd: n-terminal domain of s . s -ctd: c-terminal domain of s . ch: central helix. fp: fusion peptide. hr and hr : heptad repeats and . residues in shaded regions (n-terminus, hr , gcn tag, and his tag) were not included in the structural model. question mark indicates that the exact location of fp is uncertain; the range of fp used for making figures is consistent with a previous structural study on β-genus mouse hepatitis coronavirus spike [ ] . monomeric subunit of s contains two major domains, s -ntd and s -ctd, and two subdomains, sd and sd (fig a and b ). the s -ctds from three different subunits sit on the top and center of the spike trimer, whereas the three s -ntds are located on the lower and sd ' and sd ": two parts of sd . sd ' and sd ": two parts of sd . (b) structure of monomeric s . s -ntd is colored in cyan. s -ctd is colored in green. sd is colored in magenta. sd is colored in orange. à indicates putative sugar-binding site. partial ceiling on top of the s -ntd core is labeled. putative receptor-binding motif loops (rbms) in s -ctd are also labeled. (c) structure of trimeric s . three s subunits are colored differently. (d) structure of monomeric s . the structural elements are colored in the same way as in fig a. (e) structure of trimeric s . dotted line indicates residues - that are missing in the structural model. the structural elements of subunit a are colored in the same way as in fig d. subunits b and c are colored in light purple and light pink, respectively. all structures are viewed from the side. https://doi.org/ . /journal.ppat. .g structure, function, and evolution of ibv spike protein outer side to s -ctds (fig c) . sd and sd connect s to s . the interface of trimeric s contains three central helices; each subunit of s contains one (fig d and e ). each subunit of s also contains two heptad repeat regions, hr and hr , and a fusion peptide (fp) ( fig d and e ). in the post-fusion structure of trimeric s , three copies of hr and three copies of hr would refold into a six-helix bundle structure, and fp would insert into the target membrane [ ] [ ] [ ] [ ] [ ] [ ] [ ] . as in the structures of other coronavirus spikes, the hr region (residues - ) in the pre-fusion ibv spike is disordered (figs a and d) . the exact residue range of coronavirus fp remains unknown, although biochemical studies have identified a region in coronavirus s that associates with membranes and likely corresponds to fp ( fig d) [ , ] . in the following sections of this paper, we will compare the structures and functions of ibv spike to those of the spikes from the other three genera, and discuss the evolution of coronavirus spikes. ibv s -ntd takes the same galectin fold as the s -ntds from the other three coronavirus genera, but it contains unique structural features (fig a- d ). its core structure is a twelvestranded β-sandwich, which consists of two six-stranded antiparallel β-sheet layers stacked together through hydrophobic interactions (figs b and c). the topology of the β-sandwich core is identical to that of human galectins (s fig). underneath the core structure is another β-sheet and an α-helix, which are also present in the s -ntds from the other three coronavirus genera. above the core structure are some loops that form a partial ceiling-like structure. this structure is not present in human galectins or s -ntds from α-or δ-genus, but becomes a more extensive ceiling-like structure in β-coronavirus s -ntds (fig a, b and d ). based on the structure and function of β-coronavirus s -ntds, we previously predicted that s -ntds from all of the genera have a galectin fold, and further correlated the galectin fold to their functions as viral lectins [ ] . recent structural studies, including the current one, have confirmed our previous structural predictions (s fig) . these studies also have unexpectedly revealed that the presence and extent of the ceiling-like structure on top of the core structure are unique structural features for s -ntds from different genera. it has been known that ibv spike binds sugar [ ] . a recent study further confirmed that the sugar-binding domain in ibv spike is its s -ntd [ ] . to date no structural information is available for the complexes of coronavirus s -ntds and their sugar ligand. mutagenesis study showed that in the s -ntd from β-genus bovine coronavirus (bcov), the sugar-binding site is located in the pocket formed between the core structure and the ceiling [ ] . in the structure of host galectins, despite no ceiling, the sugar-binding site is located in the same place [ ] . based on the structural similarity between the s -ntds from different coronavirus genera, the sugar-binding site in ibv s -ntd might also be located in the pocket formed between the core structure and the partial ceiling (figs b and c) . the structure determination of ibv s -ntd provides insight into the structural and functional evolution of coronavirus s -ntds. we hypothesized that coronavirus s -ntds originated from host galectins based on the structural similarities between coronavirus s -ntds and host galectins [ , ] . as host proteins, galectins are not recognized by the host immune system. in comparison, coronavirus s -ntds are under the host immune pressure to evolve. the gradual structural evolution of the ceiling on top of the core structure may have led to three functional outcomes. first, the ceiling could provide better protection to the sugar-binding site from host immune surveillance, which appears to be a common feature of viral lectins [ ] . this hypothesis on protected sugar-binding sites in viral lectins is also consistent with the "canyon hypothesis" which states that receptor-binding sites on viral surfaces are hidden from the host immune surveillance [ ] . second, the ceiling is also involved in the quaternary packing of s , which will be discussed later in this paper. third, in the structure of s -ntd from βgenus mouse hepatitis coronavirus (mhv), the outer surface of the ceiling has further evolved the capability to bind a protein receptor ceacam , while the presumed sugar-binding pocket has lost its capability to bind sugar [ ] . hence, the structural development of the ceiling is a possible indicator for the evolution of s -ntds in the direction of α-and δ-genera, then the γgenus, and finally the β-genus. furthermore, we performed quantitative structural comparisons of s -ntds from different genera by calculating the z-score and r.m.s.d. between each pair of the proteins (fig e) . the result confirmed that s -ntds are relatively conserved among different genera, as reflected by the generally high z-scores and low r.m.s.d. in terms of structural distances to α-coronavirus s -ntds, δ-coronavirus s -ntds are the closest, β- . although each subunit of nl s contains two copies of s -ntds (i.e., s -ntd and s -ntd ), s -ntd was used in structural comparisons with the s -ntds from the other genera because it occupies the same location as the s -ntds from the other genera in quaternary structures of the spikes (see fig a) . (b) structure of s -ntd from δ-genus porcine delta coronavirus (pdcov) (pdb id: b n). (c) structure of s -ntd from γ-genus ibv. (d) structure of s -ntd from β-genus sars coronavirus (pdb id: x ). à indicates sugar-binding site or putative sugar-binding site in sugarbinding s -ntds from each genus. core structure, partial ceiling, and extensive ceiling are labeled. arrows from panels (a) to (d) indicate evolutionary direction. (e) quantitative structural comparisons among s -ntds from different genera using software dali [ ] . both z-score and r.m.s.d. were calculated for each pair of the proteins. pdb ids for nl , pdcov and sars s -ntds are the same as in panels (a)-(d). pdb ids for mouse hepatitis coronavirus (mhv) and mers coronavirus are jcl and x f, respectively. ceacam b (pdb id: vst), whose β-sandwich fold is topologically different from that of coronavirus s -ntds [ ] , was used as a negative control. n.d.: no detectable structural similarity. https://doi.org/ . /journal.ppat. .g coronavirus s -ntds are the farthest, and γ-coronavirus s -ntds fall in the middle. moreover, the structural similarity between α-and δ-coronavirus s -ntds is slightly higher than that between two β-coronavirus s -ntds, suggesting that s -ntds within β-genus have diverged slightly more than those between the α-and δ-genera. taken together, s -ntds from the four genera form an evolutionary spectrum in the order of α-, δ-, γ-, and β-genus, with α-coronavirus s -ntds probably being the most ancestral (fig a- d ). the structure of ibv s -ctd is significantly different from the structures of s -ctds from the other genera (fig a- d; s fig) . its core structure is a β-sandwich containing two β-sheet layers: one is five-stranded and antiparallel, and the other is two-stranded and parallel (figs b and c; s fig) . the interactions between the two β-sheet layers are present but minimal. in contrast, the core structures of α-coronavirus and δ-coronavirus s -ntds are both standard β-sandwich folds with extensive interactions between the two β-sheet layers: one is threestranded and antiparallel, and the other is three-stranded and mixed (fig a and b; s fig) . even more drastically different are the β-coronavirus s -ctds, which contain only one fivestranded antiparallel β-sheet layer with the other layer turning into an α-helix and a coil ( fig d; s fig) . despite these dramatic structural differences, the s -ctds from all genera share the same structural topology (i.e., connectivity of secondary structural elements) (s fig) . moreover, the additional structural motifs on the edge of the core structure are also diverse among different genera (s fig). in the ibv s -ctd, two extended loops on the edge of the core structure function as putative receptor-binding motifs (rbms) by potentially binding to an unknown receptor (see below) (figs b and c) . in both the α-and δ-coronavirus s -ctds, three short discontinuous loops are located in the same spatial region; they function as the rbms in α-coronavirus s -ctds and putative rbms in δ-coronavirus s -ctds (fig a and b ). in β-coronavirus s -ctds, a long continuous subdomain is located in this spatial region and functions as the lone rbm (fig d) . structural variations of the rbms in the s -ctds within each of the genera further lead to different receptor specificities [ ] . in sum, ibv s -ctd contains a weakened β-sandwich core structure and two extended rbm loops; the former structural feature falls between the β-sandwich cores of α-and δ-genera and the βsheet core of β-genus, whereas the latter structural feature falls between the three short discontinuous rbm loops of α-and δ-genera and a single long continuous rbm subdomain of βgenus. to investigate the function of ibv s -ctd, we performed two assays to detect possible interactions between ibv s -ctd and its potential receptor on the host cell surface. first, we carried out an ibv-spike-mediated pseudovirus entry assay in the presence of recombinant ibv s -ctd (s a fig). to this end, retroviruses pseudotyped with ibv spike (i.e., ibv pseudoviruses) were used to enter host cells. in the absence of recombinant ibv s -ctd, ibv pseudoviruses entered df- cells (chicken fibroblast) efficiently, which was consistent with a previous report showing that df- cells are permissive to live ibv (strain m ) infections [ ] . as a negative control, their entry into hek t cells (human kidney) was inefficient. recombinant ibv s -ctd reduced the efficiency of ibv pseudovirus entry into df- cells in a dose-dependent manner, likely because it competed with ibv pseudoviruses for an unknown receptor on the host cell surface. second, we examined the binding of recombinant ibv s -ctd to the host cell surface using a flow cytometry assay (s b fig). to this end, recombinant ibv s -ctd was incubated with df- cells, and subsequently cell-bound s -ctd was detected using flow cytometry. recombinant ibv s -ctd bound to the surface of df- cells efficiently, but not the surface of hek t cells. taken together, ibv s -ctd binds to a yetto-be-identified receptor on the surface of chicken cells and hence functions as a receptorbinding domain (rbd). thus, the s -ntd and s -ctd of ibv spike may both function as rbds. because coronavirus s -ctds from the α-and β-genera all use the additional structural features on the edge of their core structure as their rbms, it is likely that the two extended loops in the same spatial region in ibv s -ctd function as the rbms. coronavirus s -ctds represent remarkable examples of divergent evolution of viral proteins. the core structures and the rbm regions of s -ctds are both divergent among different genera (fig a- d; s fig) . the core structures are β-sandwiches for α-and δ-coronavirus s -ctds, weakened β-sandwiches for γ-coronavirus s -ctds, and single β-sheet layer for βcoronavirus s -ctds. the rbms are three short discontinuous loops for α-and δ-coronavirus s -ctds, two reinforced loops for γ-coronavirus s -ctds, and a single continuous subdomain for β-coronavirus s -ctds. hence the s -ctds form an evolutionary spectrum, with αand δ-coronavirus s -ctds on one end, β-coronavirus s -ctds on the other end, and γcoronavirus s -ctds in between. we performed quantitative structural comparisons of s -ctds from all four genera (fig e) . the result confirmed that s -ctds are relatively poorly conserved among different genera, as reflected by the generally low z-scores and high r.m.s.d. in terms of structural distances to α-coronavirus s -ctds, δ-coronavirus s -ctds are the closest, β-coronavirus s -ctds are the farthest, and γ-coronavirus s -ctds fall in the middle. the functional outcomes of the core structure evolution are not clear, but the evolution of the rbms may have led to the following two functional outcomes. first, the diversity of the rbms from three short loops to two extended loops and then to a long subdomain may allow coronaviruses to explore a wider variety of receptors. second, the reinforced rbm regions in both β-and γ-coronavirus s -ntds facilitate quaternary packing of s , which will be discussed later in this paper. taken together, the s -ctds from different genera form an evolutionary spectrum in the order of α-, δ-, γ-, and β-genus, although the evolutionary direction could go either way (fig a- d ). curiously, coronavirus s from different genera take two types of quaternary packing modes (fig a- d ) [ ] [ ] [ ] [ ] [ ] [ ] . ibv s takes a cross-subunit quaternary packing mode where the s -ntd and s -ctd from different subunits pack together (fig c) . specifically, in the trimeric ibv spike, one s -ctd packs against two s -ctds from the other subunits as well as one s -ntd from another subunit. the putative rbms of ibv s -ctd and the partial ceiling structure, function, and evolution of ibv spike protein of ibv s -ntd are both involved in the cross-subunit packing. as a result, the putative rbms of ibv s -ctd are partially concealed, disallowing their full access to the host receptor. hence ibv s -ctd in the current structure was captured in a "lying down" state, and would need to "stand up" on the spike trimer for efficient receptor binding. this potential conformational change of ibv s can minimize the exposure of the putative rbms in its s -ctd to the immune system, thereby functioning as a possible strategy for viral immune evasion. β-coronavirus s also takes the cross-subunit packing mode, with the rbm of its s -ctd and the ceiling of its s -ntd both involved in the cross-subunit packing (fig d) [ ] [ ] [ ] . in contrast, α-and δ-coronavirus s both take an intra-subunit packing mode where the s -ntd and s -ctd from the same subunit pack together (fig a and b ) [ ] [ ] [ ] . the rbms of α-and δ-coronavirus s -ctds are involved in the intra-subunit packing. whether s packs in the intra-subunit or cross-subunit mode, the rbms of s -ctds are concealed or partially concealed in their "lying down" state, and would need to switch to the "standing up" state for receptor binding. overall, β-and γ-coronavirus s both take the cross-subunit quaternary packing mode, whereas α-and δ-coronavirus s both take the intra-subunit quaternary packing mode. we examined whether the quaternary structures of coronavirus s can lead to functional differences of coronavirus spikes. first, in both β-and γ-coronavirus spikes, the rbms of their s -ctds and the ceiling of their s -ntds have evolved to facilitate the cross-subunit packing. these additional structural features further evolved to gain other functions: the rbms of s -ctds recognize diverse protein receptors, whereas the ceiling of the s -ntds either protects the sugar-binding site or recognizes a new protein receptor [ ] . second, to investigate the structural restrain on s -ctds that may hinder their potential conformational change, we calculated the total and buried surface areas of the s -ctd on the spikes from different genera. the result did not reveal systematic difference between intra-subunit s packing and crosssubunit s packing in the buried surface area of s -ctds. however, it is worth noting that the s -ctd from β-genus sars-cov has the smallest buried surface area (in both absolute value and percentage) (s table) . the relative small buried surface area of sars-cov s -ctd indicates less structural restraint on the s -ctd from other parts of the spike s , possibly allowing the s -ctd to switch to the "standing up" and receptor-accessible conformation more easily. the "standing up" conformation of sars-cov s -ctd may also weaken the structural restraint of s on s (discussed in more detail later), potentially allowing membrane fusion to proceed more easily [ ] . indeed, frequent "standing up" of sars-cov s -ctd has been observed [ ] . overall, compared to the intra-subunit quaternary packing of α-and δ-coronavirus s , the cross-subunit quaternary packing of β-and γ-coronavirus s may have allowed their s to evolve additional functions in receptor recognition; moreover, the s -ctd from βgenus sars-cov spike has a relatively small buried surface area, which may be responsible for its dynamic receptor-binding conformation. the structure and function of ibv s are highly similar to those of s from the other coronavirus genera. in the pre-fusion structure of ibv s , hr is disordered, whereas hr and fp each consist of several α-helices and connecting loops (the exact residue range of fp is not clear) (fig d) . in the post-fusion structure, hr would refold into a long α-helix, hr would refold into a mixture of α-helices and coils, three copies of hr and hr would pack into a six-helix bundle structure, and fp would also refold and insert into the target membrane (s a fig) [ , ] . ibv s is locked in the pre-fusion state because of the structural restraint from s . specifically, because of the cross-subunit quaternary packing of trimeric ibv s , hr and fp of ibv s are structurally restrained by two s -ctds from the other subunits and sd from another subunit, respectively (s b fig). the structural restraints from s on s can be weakened by the standing up of s -ctds (which allows receptor binding) and can be lifted completely upon proteolysis removal of s . the packing between s and s in ibv spike is the same as those in β-coronavirus spikes [ ] [ ] [ ] . however, in α-and δ-coronavirus spikes, the packing between s and s becomes different due to the intra-subunit quaternary packing of their trimeric s : hr and fp are restrained by one s -ctd and one sd from another subunit, respectively (s c fig) [ - ] . other than the differences in s /s packing, the structural and functional similarities of coronavirus s from different genera suggest evolutionary conservation of coronavirus s . the fast evolutionary rates of viruses, particularly rna viruses, make it difficult to trace their evolutionary history [ ] [ ] [ ] . envelope-anchored coronavirus spike proteins guide viral entry into cells; they are the fastest evolving coronavirus proteins due to viral needs to engage diverse host receptors, maximize membrane-fusion efficiency, and evade host immune surveillance [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . coronavirus spikes from four different genera are divergent, and their evolutionary relationships pose a major puzzle in the virology field [ ] . because viral proteins need to function under certain structural and functional constraints, evolutionary information of viral proteins can be more reliably found in their tertiary structures and related functions than in their primary structures [ , ] . although extensive structural studies including both x-ray crystallography and cryo-em have been done on coronavirus spikes, a critical piece that was still missing is the structure of γ-coronavirus spikes, preventing a clear understanding of the evolutionary relationships among coronavirus spikes [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . in this study, we determined the cryo-em structure of ibv spike ectodomain, the first such structure from the γ-genus, which bridges the divergent structures of coronavirus spikes into an evolutionary spectrum and provides insight into the evolutionary relationships among coronavirus spikes. our study compares the structures and functions of coronavirus spikes from the four genera, and illustrates the structural and functional evolution of these proteins. first, coronavirus s -ntds from all genera share the same structural fold and possibly evolutionary origins with host galectins. from α-and δ-genera to γ genus and then to β genus, the s -ntds have evolved from simple galectin-fold core structure with an exposed sugar-binding site, to having a partial ceiling on top of the core structure, and to having an extensive ceiling to protect the sugar-binding site from host immune surveillance (the outer surface of the ceiling in one βcoronavirus can even bind to a novel protein receptor). the partial ceiling in γ-coronavirus s -ntds and the extensive ceiling in β-coronavirus s -ntds are also involved in the quaternary packing of s . second, coronavirus s -ctds from different genera are very diverse, but still form an evolutionary spectrum with α-and β-coronavirus s -ctds at two ends and δand γ-coronavirus s -ctds in the middle. the core structures of s -ctds have diverged from β-sandwich to weakened β-sandwich and then to β-sheet, whereas the rbms have diverged from short loops to extended loops and then to a long subdomain. the functional significance of the core structure evolution is not clear, but the rbm evolution may allow the viruses to expand receptor recognition and also contributes to the quaternary packing of s . third, from α-and δ-genera to β-and γ-genera, the quaternary packing of s has diverged from simple intra-subunit packing to more complex cross-subunit packing. the cross-subunit quaternary packing of β-and γ-coronavirus s may have allowed their s to evolve additional functions in receptor recognition. moreover, the relatively small buried surface area of the s -ctd from β-genus sars-cov may allow the s -ctd to be more dynamic for receptor binding. finally, the s from all four genera are structurally and functionally conserved, although there are some differences in their s /s packing. quantitative structural comparisons also demonstrate that in terms of structural distances to α-coronavirus s , δ-coronavirus s is the closest, β-coronavirus s is the farthest, and γ-coronavirus s is the intermediate. we also calculated the phylogenetic tree using the amino acid sequences of coronavirus spikes from different genera, and the result showed that in terms of amino acid sequence distances to αcoronavirus spikes, δ-coronavirus spike is the closest, β-coronavirus spike is the farthest, and γcoronavirus spike is the intermediate (s fig). taken together, coronavirus spikes from different genera form an evolutionary spectrum, with α-coronavirus spikes on one end, followed by δ-coronavirus spikes and γ-coronavirus spikes, and β-coronavirus spikes on the other end. because of their fast evolutionary rates, viruses are perfect model systems for studying evolution. our study has demonstrated that despite structural divergence among coronavirus spikes, particularly in their s where low or little structural similarities can be detected, we can still trace the evolutionary relationships among these viral proteins through detailed analyses of their structures and functions. our study also reveals that coronavirus spikes have evolved to remarkable diversity to expand their receptor recognition, facilitate membrane fusion, and evade host immune surveillance, while conserving basic membrane-fusion mechanisms. the evolutionary approaches that coronaviruses take and the evolutionary edges that they gain are good examples of viral evolution and deepen our understanding of evolution in general. supporting information s table. buried surface area of coronavirus spike s -ctds. (b) structural topology of the core structures of α-, γ-, and δ-coronavirus s -ntds. (c) structural topology of the core structures of β-coronavirus s -ntd. pdb ids of coronavirus s -ntds are the same as in fig . β-strands are shown as arrows. the two layers of the core structures are colored in green and magenta, respectively. n à and c à indicate n-and c-terminus, respectively. numbering of the secondary structures only counts secondary structural elements in the core region. (tif) s fig. structural topology of coronavirus s -ctds. (a) structural topology of the core structures of α-and δ-coronavirus s -ctds. (b) structural topology of the core structure of γcoronavirus s -ctd. (c) structural topology of the core structure of β-coronavirus s -ctd. pdb ids of coronavirus s -ctds are the same as in fig . β-strands are shown as arrows. αhelices are shown as cylinders. coil is shown as a curled line. the two layers of the core structures are colored in green and magenta, respectively. receptor-binding motifs (rbms) are colored in red and the relative lengths of the rbms are labeled in parentheses. in both γ-and δ-coronavirus s -ctds, the rbms have not been experimentally identified and thus their functions are putative. n à and c à indicate n-and c-terminus, respectively. numbering of the secondary structures only counts secondary structural elements in the core region. (tif) s fig. function of ibv s -ctd. (a) ibv pseudovirus entry into cells in the presence of recombinant ibv s -ctd. entry efficiency was characterized by luciferase activity accompanying entry. rlu: relative light units. mock: no ibv pseudoviruses were added. entry: ibv pseudovirus entry in the absence of any recombinant ibv s -ctd. (b) flow cytometry assay for the binding of recombinant ibv s -ctd to the surface of cells. cell-bound ibv s -ctd was detected using antibodies recognizing its c-terminal his tag. cells only or cells plus antibody without ibv s -ctd were used as negative controls. statistic analyses were performed using two-tailed t-test. arrow in the pre-fusion structure indicates the direction in which hr would need to extend to reach the post-fusion conformation. (b) packing between s and s in ibv spike. trimeric s and one monomeric s are shown. structural elements in monomeric s are colored in the same way as in panel (a). three s subunits are colored differently. (c) packing between s and s in porcine delta coronavirus spike (pdb id: b n). trimeric s and one monomeric s are shown. s and s are colored in the same way as in panel (b). all structures are viewed from the side. the phylogenetic tree was constructed using the neighbor-joining method as previously described [ ] . horizontal scale bars represent average numbers of substitutions per amino acid position. the genbank accession numbers of the selected spikes are marked before each virus name. (tif) recombination, reservoirs, and the modular spike: mechanisms of coronavirus cross-species transmission animal origins of the severe acute respiratory syndrome coronavirus: insight from ace -s-protein interactions receptor recognition and cross-species infections of sars coronavirus a comparative sequence analysis to revise the current taxonomy of the family coronaviridae coronaviruses post-sars: update on replication and pathogenesis structure, function, and evolution of coronavirus spike proteins. annual review of virology receptor recognition mechanisms of coronaviruses: a decade of structural studies mechanisms of coronavirus cell entry mediated by the viral spike protein host cell proteases: critical determinants of coronavirus tropism and pathogenesis ready, set, fuse! the coronavirus spike protein and acquisition of fusion competence evasion of antibody neutralization in emerging severe acute respiratory syndrome coronaviruses glycan shield and epitope masking of a coronavirus spike protein observed by cryo-electron microscopy cryo-em structure of porcine delta coronavirus spike protein in the pre-fusion state glycan shield and fusion activation of a deltacoronavirus spike glycoprotein fine-tuned for enteric infections evidence for a common evolutionary origin of coronavirus spike protein receptor-binding subunits the evolution of protein structures and structural ensembles under functional constraint the structure of protein evolution and the evolution of protein structure cryo-electron microscopy structure of a coronavirus spike glycoprotein trimer cryo-em structures of mers-cov and sars-cov spike glycoproteins reveal the dynamic receptor binding domains pre-fusion structure of a human coronavirus spike protein structure of sars coronavirus spike receptor-binding domain complexed with receptor crystal structure of nl respiratory coronavirus receptor-binding domain complexed with its human receptor crystal structure of mouse coronavirus receptor-binding domain complexed with its murine receptor crystal structure of bovine coronavirus spike protein lectin domain dipeptidyl peptidase is a functional receptor for the emerging human coronavirus-emc structural bases of coronavirus attachment to host aminopeptidase n and its inhibition by neutralizing antibodies crystal structure of the receptor-binding domain from newly emerged middle east respiratory syndrome coronavirus molecular basis of binding between novel human coronavirus mers-cov and its receptor cd tectonic conformational changes of a coronavirus spike glycoprotein promote membrane fusion conformational states of the severe acute respiratory syndrome coronavirus spike protein ectodomain structural basis for coronavirus-mediated membrane fusion. crystal structure of mouse hepatitis virus spike protein fusion core crystal structure of severe acute respiratory syndrome coronavirus spike protein fusion core core structure of s from the human coronavirus nl spike glycoprotein structure-based discovery of middle east respiratory syndrome coronavirus fusion inhibitor central ions and lateral asparagine/glutamine zippers stabilize the post-fusion hairpin conformation of the sars coronavirus spike glycoprotein. virology automated electron microscope tomography using robust prediction of specimen movements electron counting and beaminduced motion correction enable near-atomic-resolution single-particle cryo-em real-time ctf determination and correction relion: implementation of a bayesian approach to cryo-em structure determination high-resolution noise substitution to measure overfitting and validate resolution in d structure determination by single particle electron cryomicroscopy visualizing density maps with ucsf chimera features and development of coot phenix: a comprehensive python-based system for macromolecular structure solution molprobity: allatom structure validation for macromolecular crystallography emringer: side chain-directed model and map validation for d cryo-electron microscopy receptor usage and cell entry of bat coronavirus hku provide insight into bat-to-human transmission of mers coronavirus inference of macromolecular assemblies from crystalline state identification of the fusion peptide-containing region in betacoronavirus spike glycoproteins sars-cov fusion peptides induce membrane surface ordering and curvature binding of avian coronavirus spike proteins to host factors reflects virus tropism and pathogenicity mapping of the receptor-binding domain and amino acids critical for attachment in the spike protein of avian coronavirus infectious bronchitis virus x-ray crystal structure of the human galectin- carbohydrate recognition domain at . -angstrom resolution structural analysis of the evolutionary origins of influenza virus hemagglutinin and other viral lectins hiding the host cell receptor attachment site on a viral surface from immune surveillance activation of the chicken type i interferon response by infectious bronchitis coronavirus immunogenicity and structures of a rationally designed prefusion mers-cov spike antigen genetic characterization of betacoronavirus lineage c viruses in bats reveals marked sequence divergence in the spike protein of pipistrellus bat coronavirus hku in japanese pipistrelle: implications for the origin of the novel middle east respiratory syndrome coronavirus touring protein fold space with dali/fssp structural and molecular evidence suggesting coronavirus-driven evolution of mouse receptor initial cryo-em images were collected using fei tecnai tems maintained at the characterization facility of the university of minnesota. final cryo-em data were collected at the john m. cowley center for high resolution electron microscopy of arizona state university. we thank dr. dewight williams for helping us prepare grids and collect data. initial image analysis and computation work were performed using the workstations at the basic sciences computing laboratory of the university of minnesota supercomputing institute. key: cord- -ytxrrslz authors: züst, roland; dong, hongping; li, xiao-feng; chang, david c.; zhang, bo; balakrishnan, thavamalar; toh, ying-xiu; jiang, tao; li, shi-hua; deng, yong-qiang; ellis, brett r.; ellis, esther m.; poidinger, michael; zolezzi, francesca; qin, cheng-feng; shi, pei-yong; fink, katja title: rational design of a live attenuated dengue vaccine: ′-o-methyltransferase mutants are highly attenuated and immunogenic in mice and macaques date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: ytxrrslz dengue virus is transmitted by aedes mosquitoes and infects at least million people every year. progressive urbanization in asia and south-central america and the geographic expansion of aedes mosquito habitats have accelerated the global spread of dengue, resulting in a continuously increasing number of cases. a cost-effective, safe vaccine conferring protection with ideally a single injection could stop dengue transmission. current vaccine candidates require several booster injections or do not provide protection against all four serotypes. here we demonstrate that dengue virus mutants lacking ′-o-methyltransferase activity are highly sensitive to type i ifn inhibition. the mutant viruses are attenuated in mice and rhesus monkeys and elicit a strong adaptive immune response. monkeys immunized with a single dose of ′-o-methyltransferase mutant virus showed % sero-conversion even when a dose as low as , plaque forming units was administrated. animals were fully protected against a homologous challenge. furthermore, mosquitoes feeding on blood containing the mutant virus were not infected, whereas those feeding on blood containing wild-type virus were infected and thus able to transmit it. these results show the potential of ′-o-methyltransferase mutant virus as a safe, rationally designed dengue vaccine that restrains itself due to the increased susceptibility to the host's innate immune response. dengue virus (denv) is a member of the flaviviridae family. denv infection causes dengue fever (df) and the more severe forms of the disease, dengue hemorrhagic fever (dhf) and dengue shock syndrome (dss). denv has four serotypes (denv- to - ), each of which is capable of causing severe disease. the frequency, severity, and geographical spread of cases have increased over the past decades [ , ] . every year, one hundred million new cases of df and , dhf/dss are estimated by the who. at present, despite intensive global research efforts, no vaccine or antiviral treatment for dengue infection is available. vaccine development is complex due to multiple factors. (i) an effective vaccine must consist of a tetravalent formulation protecting against each of the four serotypes because more than one serotype typically circulates in a region. (ii) a sub-protective vaccine potentially increases the risk of vaccinees to develop the more severe forms of dengue during repeated infection because of a known association of pre-existing immunity with severity [ , ] . (iii) since most infections occur in developing countries, an ideal vaccine should be affordable and fully protective [ ] . taken together, a vaccine inducing a robust level of immunity ideally with only one inoculation is required. live-attenuated vaccines are replication-competent viruses, which can induce an immune response and an immune memory that are comparable to those induced by the wild-type virus. live-attenuated viruses do not cause disease because of the low level of replication and hence low levels of inflammation. prominent examples of successful live-attenuated vaccines providing long-term immunity are those against vaccinia virus, poliovirus (sabin), and two members of the flaviviridae family, yellow fever virus (yf- d) and japanese encephalitis virus (jev sa - - ) [ ] . liveattenuated denv vaccines have been shown to induce protective neutralizing antibody titers in mice, monkeys, and humans [ ] [ ] [ ] . in addition, evidence that a balanced t cell response contributes to protection is accumulating, emphasizing the importance of t cell epitopes in a vaccine [ ] . flaviviruses are positive-sense, single-stranded rna viruses. the flavivirus genome encodes for structural (c, prm, and e) and non-structural proteins (ns , ns a, ns b, ns , ns a, ns b, and ns ). ns is a multifunctional protein, consisting of the rna-dependent rna polymerase [ ] and methyltransferase (mtase) activities responsible for rna cap formation [ , ] as well as internal rna methylation [ ] . while n- -methylation is essential for rna translation and stability, the function of -omethylation has remained elusive until recently. we and others demonstrated that while -o-mtase is not essential for viral replication in vitro, viruses bearing mutations in the highly conserved mtase catalytic k-d-k-e tetrad are severely attenuated in the host due to the inability of the virus to shield viral rna from recognition by host innate immune factors [ , ] . denv rna binds to rig-i and mda [ , ] , which activates interferon (ifn)-b production via a cascade involving ifn-b promoter stimulator (ips- ) [ ] . ifns in turn activate ifn stimulated genes (isgs), which induce antiviral responses in infected and neighbouring cells. ifn-induced proteins with tetratricopeptide repeats (ifits) are critical for the inhibition of viral infections, although their functions are only partially understood [ , ] . the human ifit gene family comprises four members: ifit , ifit , ifit ( = ifit ), and ifit ; whereas mice only express ifit , and [ ] . interestingly, ifit homologs are conserved from amphibians to mammals, suggesting that they play a central role in the innate immune response [ ] . ifit and bind to eukaryotic initiation factor (eif ) and inhibit translation [ ] , whereas ifit amplifies the antiviral signal by connecting ips- and tbk , resulting in more ifn production [ ] . the role of human ifit is less well understood. here we demonstrate that denv strains bearing a mutation in the catalytic site of the -o-mtase replicated to high titres in cell culture whereas these mutant viruses were highly attenuated in mice and rhesus monkeys. the mutation was stable over several passages and reversion to wild-type (wt) was not observed. for further safety improvement, a second mutation in the -o-mtase catalytic tetrad was introduced without affecting the viability of the virus in vitro. a single dose administration to rhesus macaques (rm) conferred protection to homologous denv challenge. mice immunized with a single dose of a divalent (denv- / ) formulation of the mutant viruses and mice immunized with the monovalent formulation showed comparable antibody responses, demonstrating that there was no interference between two serotypes of the denv mtase mutants. moreover, no enhanced infection and increased tnf-a levels were observed in immunized mice upon challenge with heterologous virus. overexpression of ifits in hek-dc-sign cells suggested a role for ifit in the attenuation of mtase mutant in human cells. taken together, these results demonstrate the potential of -o-mtase mutants as a denv vaccine. to our knowledge, this is the first live-attenuated rational vaccine approach, tailored to optimally activate the innate and adaptive immune response while being severely attenuated due to its susceptibility to the ifn response. flaviviruses replicate in the cytoplasm. the cytoplasm-replicating viruses have evolved n -and -o-methyltransferases (mtase) to methylate their viral mrna cap structures [ ] . we have previously shown for west nile virus (wnv) and denv- that mutation of the asp of the tetrad k-d-k-e completely abolished both n -and -o-mtase activities and was lethal for viral replication; mutations of the other three residues of the tetrad abolished -o-methylation (with a decrease in n -methylation), and led to attenuated viruses [ , ] . since there are four serotypes of denv, we introduced the same mtase mutations into denv- to examine whether the same approach was feasible with more than one serotype. a wt recombinant mtase, representing the n-terminal amino acids of the denv- ns (strain tsv ), was cloned and expressed. two mutant mtases containing ala-substitutions at the k-d-k-e tetrad ( fig. a) were prepared: one with a single e a mutation and another with double k a+e a mutations. the mutant enzymes retained % and % of the wt n -methylation activity, respectively; neither mutant exhibited any -o-methylation activity (fig. b) . bhk- cells transfected with equal amounts of wt and mutant (e a and k a+e a) genomelength rnas of denv- generated equivalent numbers of viral e protein-expressing cells (fig. c) . both wt and mutant rnas produced infectious viruses (passage ) with similar plaque morphologies (fig. d ). the replication of mutant viruses was attenuated in mammalian vero and mosquito c / cells (fig. e ). continuous culturing of the mutant viruses on vero cells or hek- cells expressing dc-sign (hek-dc-sign) for ten rounds ( - days per round) did not change their plaque morphologies ( fig. d and data not shown). the expression of dc-sign facilitates denv infection [ ] . sequencing of the passage and viruses from both vero and hek-dc-sign cells showed that the engineered mutations were retained (supplementary fig. s a and s b). similar results were obtained for denv- containing the e a (e in denv- mtase is equivalent to e in denv- mtase) or k a+e a mutation in mtase ( supplementary fig. s ). collectively, the results demonstrate that the -o-mtase mutant denv- and - are slightly attenuated, but stable in cell culture. the four serotypes of dengue virus cause severe outbreaks globally in tropical countries with thousands of patients requiring hospitalization. the health care and indirect economic cost of dengue in endemic countries is huge. despite this, no clinically approved vaccine or antiviral treatment is currently available. dengue transmission could be stopped with a vaccine that provides full protection to all serotypes. dengue afflicts many developing countries and a vaccine should therefore be cost-effective and should provide protection with ideally a single injection. here we present a novel dengue vaccine approach that harbours mutation(s) in the -o-methyltransferase (mtase), a viral enzyme that methylates viral rna as a strategy to escape the host immune response. nonmethylated rna is recognized as ''foreign'' and triggers an interferon response in the cell. the mtase mutant virus is immediately recognized by the host's immune response and hardly has a chance to spread in the organism while an immune response is efficiently triggered by the initially infected cells. mice and monkeys infected with the mutant virus developed an immune response that fully protected them from a challenge with wild-type virus. furthermore, we show that mtase mutant dengue virus cannot infect aedes mosquitoes. collectively, the results suggest -o-mtase mutant dengue virus as a safe, highly immunogenic vaccine approach. denv -o-mtase mutants are attenuated in mice and induce a protective immune response we infected ag mice with the wt and -o-mtase mutants (called ''e a'' for denv- and ''e a'' for denv- from this point) to assess viral replication and immunogenicity in vivo. ag mice lack the receptors for type i and type ii ifns, and have been used widely for antiviral and vaccine testing [ ] [ ] [ ] [ ] . mice were intraperitoneally (i.p.) infected with . plaque-forming unit (pfu) of wt or mutant viruses. the viremia result showed that mutating k a or e a in denv- and mutating e a in denv- attenuated the virus compared to the wt virus ( fig. a and b) . next, we examined a combination of two mtase mutants (e a and e a) representing denv- and denv- to address a potential competition effect that has been described previously with attenuated strains in humans [ ] and in mice [ ] . to this end, mice were injected i.p. with . pfu of e a or e a or a combination of both (a total of . pfu viruses). at days post immunization, mice were challenged i.p. with pfu of wt denv- or wt denv- . denv specific igg titers and viremia were observed. all mice immunized with e a and/or e a were protected against homologous challenge (fig. c) , demonstrating that the immune response was protective even though the igg titers in e a and/or e ainfected mice were to times lower than those in the wt virusinfected mice ( fig. d and e) . a general concern for live attenuated vaccines is their theoretical potential to mutate back to wt under pressure of the immune system. to address this in our system, virus from mice infected with mutant denv or denv was isolated at day after infection and the mutations were found to be stable ( supplementary fig. s c ). to rule out that compensatory mutations were introduced into the viral genome the input and output (day after infection) virus was sequenced using illumina deep sequencing technology. as summarized in supplementary table s , only the single nucleotide polymorphisms (snps) responsible for the e a or e a mutation were found when comparing the sequences to wild-type denv- or - , respectively. we next compared the neutralization and infection enhancing capacity of serum collected days post immunization (table and supplementary fig. s ) [ ] . mutant viruses cause the same or less antibody-dependent enhancement (ade) than the respective wild-type viruses in the heterologous setting ( . . vs. . . for denv- immunization and ade tested against denv- and . . vs. . . for denv- immunization and ade tested against denv- ) ( table ). more importantly, we did not observe enhanced infection in vivo ( fig. c and see later challenge experiments with a virulent denv- strain). these data suggest that vaccination with the e a/e a mutants does not cause ade during heterologous challenge even though lower neutralizing ab titers are generated by the mutant strains compared to the wild-type virus. recombinant mtases were assayed for gpppa-rnarm gpppa-rna and m gpppa-rnarm gpppam-rna conversions to indicate n -and -omethylation activities, respectively. the reactions were analyzed on thin-layer chromatography (tlc) plates [ ] . relative methylation activities were indicated below the tlc images with wt activity set as %. (c) immunofluorescence analysis (ifa). bhk- cells were electroporated with equal amounts of wt and mutant genome-length rnas of denv- . at indicated days post transfection, the cells were subjected to ifa using mouse antibody g against denv e protein and anti-mouse igg conjugated with fitc as primary and secondary antibodies, respectively [ ] . (d) plaque morphology. wt and mutant viruses recovered from genome-length rna-transfected cells (passage ), as well as the viruses after culturing on vero cells for rounds (passage ) were analyzed by plaque assays [ ] . vaccinated mice generate a non-structural proteinspecific cd t cell response while antibodies are crucial to reduce the viral load by binding and neutralizing virus particles, t cells are necessary for efficient viral clearance [ , ] . ag mice are not suitable to study t cell responses because of their lack of ifn-c signaling, which is critical to activate t cells. we therefore used ifnar mice lacking the receptor for ifn-a/b [ ] . ifnar mice were immunized with . pfu denv- e a or denv- wt and spleens were harvested at day for restimulation in vitro and detection of ifn-c production (fig. a ). mutant and wt virus elicited a strong cd and cd t cell response after re-stimulation with denv- . the cd response was weaker in e a-immunized mice, likely due to the lower total viral load in e a-immunized mice compared to mice immunized with the wt virus (fig. b ). to test for targeted denv t cell response splenocytes were re-stimulated with a pool of ns b and ns cd peptides described by yauch et al [ ] . no significant difference in the ns b and ns -specific t cell response was seen between mice immunized with e a or wt denv- ( fig. b ). taken together, denv -o-mtase mutants induce a t cell response and epitope presentation that is similar to wt infection. nevertheless, additional studies in mice and monkeys are necessary to assess the t cell response in greater detail and to test its functional contribution to protection. vaccinated mice are protected against challenge with the virulent denv- strain denv- strain k and denv- strain tsv do not cause pathology in mice. to test for protection against a more virulent strain we immunized mice with denv- e a, denv- e a, a mixture of e a and e a, wt denv- (westpac) or wt denv- (tsv ) or pbs and challenged them with the virulent denv- strain d y p [ ] days later (fig. ) . denv- e a protected against the homologous challenge (fig. a ). immunization with denv- e a protected % of the mice, showing limited cross-protection after infection with d y p ( fig. a and b ). no enhanced disease was detected after heterologous challenge. increased tnf-a levels were associated with pathology in the ag mouse model in the context of ade [ ] . to further assess the possibility of ade-associated pathology, we measured tnf-a levels in plasma three days after challenge. high levels of tnf-a were only detected in unimmunized (pbs) mice, showing that tnf-a as a marker of pathology was independent of ade, and that immunization with e a did not cause ade after heterologous challenge. these data demonstrate that immunization with e a protects mice against challenge with an aggressive, virulent denv- strain that causes % mortality in unimmunized mice. to assess the safety (viremia profile) and efficacy (neutralizing antibody response and protection against challenge) of the -o-mtase mutant denv vaccine approach in an immunologically competent host, three groups of rhesus monkeys (rm) were immunized with different doses of e a. one group received a low dose ( pfu), one group a medium dose ( pfu), and one group a high dose ( pfu) of e a virus. viremia was monitored during days after inoculation. the e a virus was severely attenuated, and no viremia was detected except for one animal (r ) that had received a high dose ( pfu) and developed a low viremia (table ) . virus was extracted for sequencing, and it was confirmed that the e a mutation was retained in the virus extracted at days , and from this animal. importantly, full virus genome sequencing of the viral rna recovered at day showed that no compensatory mutations were introduced (data not shown). all immunized monkeys developed neutralizing antibodies to denv- on day after immunization (table ) . ade was analyzed in a k assay and a similar enhancement pattern was fig. s ). this argues against a physiologically relevant infection enhancement, which would only be expected after heterologous infection. by day after immunization, all monkeys including the ones with low dose immunization developed high titers (gmt$ ) of neutralizing antibodies ( table ). the monkeys were then challenged with pfu of wt denv- on day post-immunization. no viremia was detected in all immunized monkey, whereas all four pbs-immunized controls had a mean peak virus titer of . log pfu/ml and mean viremia duration of . days (table ). in all animals except one (r ), anamnestic antibody responses were observed after challenge (table ). these data demonstrate that live, attenuated denv mtase mutant virus, even when administrated at low dose ( pfu), can induce protective immunity in non-human primates. the -o-methylation of the cap of wnv and coronavirus rna functions to subvert innate host antiviral response through escape of ifit-mediated suppression [ , ] . to assess whether this is true for denv as well, we pretreated hek e a was lost in the presence of ifn, whereas wild-type virus resisted the ifn pressure in both cell lines. e a isolated from passage three in hek-dc-sign and from passage one in u -dc-sign was isolated for sequencing. the e a mutation was retained and no compensatory mutations were introduced (data not shown). to elucidate the molecular mechanism of attenuation, we overexpressed human ifit , , , or in hek-dc-sign cells. the cells were infected with wt or mutant denv- and assessed for the number of infected cells by flow cytometry (fig. c ). the wt virus infection was not affected, whereas e a mutants were significantly inhibited by ifit , but not ifit , , or . however, ifit over-expression did not completely block e a infection nor did it affect virus output from the infected cells (fig. d) , suggesting that other ifn-mediated signals are involved in the response against denv. both mutant and wt virus show similar growth kinetics in untreated cells (fig. e) . we currently don't know why the mutant virus is attenuated in vero cells but not in hek-dc-sign since both lines are deficient in ifn production. it should be noted that the maximum antiviral effect of ifits table . viremia in e a-immunized rms after challenge with wild-type denv- *. we compared the effect of -o-mtase mutation on viral fitness in mosquito ae. aegypti, the natural transmission vector for denv. the mosquitoes were fed with blood containing wt or e a. after the mosquitoes were fed at a titer of pfu/ml, significant differences in oral infection and dissemination between the wt and mutant viruses were observed days post-infection ( table ). the wt virus infected % of mosquitoes at the highest titer ( pfu/ml), but only - % of mosquitoes at lower titers ( and pfu/ml). when orally fed with pfu/ ml wt virus, approximately % of mosquitoes were infected after ; the wt virus disseminated in % of the mosquitoes (table ). when fed with and pfu/ml wt virus, the dissemination rates reached - %. in contrast, the mutant virus was unable to infect the ae. aegypti and, subsequently, no dissemination was observed for all titers (table ) . to examine whether the e a mutant could replicate in vivo, we intra-thoracically inoculated the wt and mutant viruses into ae. aegypti mosquitoes. intra-thoracic inoculation bypasses the mosquito midgut, which is the key barrier to establish infection during natural feeding route. both wt and mutant viruses reached % infection rate upon intra-thoracic inoculation. the mean genome copy number reached . and . , respectively ( supplementary fig. s ). the genome copy number of the wt virus was approximately % higher than that of the mutant virus (p = . ). overall, the results demonstrate that the -o-mtase mutant virus is compromised in vector fitness. various dengue vaccine strategies are currently under development, including live attenuated virus, subunit vaccines, chimeric viruses, and dna vaccines [ , ] . the yfv d-based chimeric dengue vaccine developed by sanofi-pasteur is the most advanced in clinical testing [ , ] . the establishment of reverse genetic manipulation of denv has greatly facilitated the generation of promising vaccine candidates [ , ] . the recent progress in understanding the mechanism of attenuation of -o-mtase mutant flaviviruses has provided a novel approach for vaccine and antiviral development [ ] . here we show in a proof-of-concept study that mtase mutant e a denv- and e a denv- strains are stable in vitro, and safe and immunogenic in vivo. importantly, enhancement of infection was not observed after heterologous infection of immunized mice. the fear in a clinical setting is that sub-neutralizing titers of antibodies could enhance infections, even though this has so far not happened in the context of vaccine trials in humans [ ] . a commonly used approach to address ade in vitro is to infect k cells in the presence of antibodies. virus alone is not able to infect k cells efficiently, whereas virus-antibody immune complexes bind to k cells via fc-c receptors (fccr's), assisting the internalization of the virus and infection of the cells. we found that k cells could be infected in the presence of serum from immunized mice and monkeys at dilutions that were approximately % neutralizing in the u -dc-sign system (supplementary fig. s and s ). this is in line with a previous report, which found that even strongly neutralizing antibodies are enhancing at concentrations that are close to the % neutralizing titer [ ] . clinically relevant ade would be expected at sub-neutralizing titers and only after heterologous infection, and this was not observed in our experiments. a caveat of the k system is that the cells do not express inhibitory fccriib, which is present on human target cells (dendritic cells and macrophages) and which negatively regulates ade [ , ] . physiological amounts of complement, another negative regulator of ade, are also not taken into account [ ] . in summary, while the k assays done here did not show more ade for heterologous infections, we cannot exclude ade because of the limitations of the assay. potential ade will have to be addressed in further monkey studies. live attenuated dengue vaccine candidates have several advantages. importantly, they can induce long lasting humoral and cellular immune responses to both structural and non-structural viral proteins. in this study we show a cd response to ns b and ns peptides that is similar in mice immunized with mutant or wt virus, suggesting that the response is qualitatively equivalent. chimeric viruses using the same backbone for all four denv serotype glycoproteins would induce a type-specific response restricted to the structural proteins of one denv serotype [ ] . the interdependence of the t and b cell response for the efficient generation of immune memory has been demonstrated in a number of human studies [ , ] . we speculate that the advantage of an attenuated non-chimeric denv that includes all naturally occurring t and b cell epitopes could be that only one vaccination is required to confer long-term immunity to reinfection, as seen for natural denv infections [ , ] . a singledose vaccine would facilitate the logistics of a vaccination program and would significantly reduce its cost compared to candidates requiring several booster immunizations. the -o-mtase mutant denv vaccine approach, with a known mechanism of attenuation, can be readily generated using a reverse genetic system. this is in contrast to the method to develop live attenuated vaccines by passaging of wt viruses in cell lines, leading to the introduction of random mutations. the reverse genetic system-based rational vaccine ensures that the vaccine maintains the attenuated genotype. additionally, a tetravalent formulation would contain the same attenuating mutation in all four serotype recombinant vaccine strains, making the generation of a more pathogenic virus by intra vaccine-strain recombination impossible. moreover, recombination in cell culture is hardly observed in flaviviruses, suggesting that flaviviruses are not prone to evolution by recombination [ ] . by introducing additional mutations in the k-d-k-e tetrad of -o-mtase, further safety and attenuation can be achieved. only a virus that has at least two mutations will be acceptable in the clinical setting. our data demonstrate that the -o-mtase e a virus is attenuated in mice and monkeys. we cannot explain fully why the -o-mtase mutant virus was attenuated ( -fold lower virus titer compared to wt virus) in ag mice, which are unable to respond to ifn-signals. it is likely that pattern recognition receptors and downstream pathways activated by the mutant virus trigger antiviral defense mechanisms in an ifn-dependent and ifnindependent manner. whether the balance between low virulence and high immunogenicity is achieved in humans by -o-mtase mutants remains to be elucidated. our studies in human hek cells show increased susceptibility of denv e a mutant to ifn-b in vitro, suggesting that denv e a mutants will be attenuated in humans as well. in the monkey immunization experiments, one monkey out of four in the high dose group experienced peak viremia of about pfu, which is comparable to other live attenuated vaccine candidates [ ] . indeed, weak replication of the vaccine approach is desirable in order to induce a strong protective cellular immune response. replication should be restricted enough to preclude onset of illness, whereas sub-clinical symptoms such as mild rash, transient leukopenia, and mildly elevated liver enzyme values are generally accepted [ ] [ ] [ ] . furthermore, studies with murine hepatitis virus have shown that mtase mutants are highly attenuated in its natural host, induce ifn, which could further induce the immunogenicity of a vaccine, and are genetically stable in vivo [ ] . moreover, the replication level of wnv -o-mtase mutant in mice was largely decreased in the spleen, serum, or brain in comparison with the wt wnv infection. intracranial inoculation of pfu of -o-mtase mutant wnv did not cause any mortality and morbidity in mice, demonstrating the safety of this vaccine approach [ ] . taken together, these evidences demonstrate the safety and immunogenicity of the mtase-mutant vaccine approach. we are currently working on the tetravalent formulation to develop the strategy towards a clinical application. all experimental procedures involving rhesus monkeys were approved by and carried out in strict accordance with the guidelines of the animal experiment committee of state key laboratory of pathogen and biosecurity, beijing, china. all procedures were performed under sodium pentobarbital anesthesia by trained technicians and all efforts were made to ameliorate the welfare and to minimize animal suffering in accordance with the ''weatherall report for the use of non-human primates'' recommendations. the mouse experiments were conducted according to the rules and guidelines of the agri-food and veterinary authority (ava) and the national advisory committee for laboratory animal research (naclar), singapore. the experiments were reviewed and approved by the institutional review board of biological resource center, singapore (iacuc protocols and ). bhk- , c / , and hek- were purchased from the american type culture collection (http://www.atcc.org). hek- and u cells expressing dc-sign were obtained by lentiviral transfection and subsequent cell sorting. all cells were maintained in minimal essential medium supplemented with fetal bovine serum ( %- %). wt mtases representing the n-terminal and amino acids of denv- and - ns , respectively, were cloned, expressed, and purified as reported previously [ ] . mutagenesis of mtase was performed using quikchange ii xl site-directed mutagenesis kit (stratagene). the complete sequence of each mutant mtase was verified by dna sequencing. n -and -omethylation assays were performed as described before [ ] . full-length infectious cdna clones of denv- (western pacific strain) and denv- (tsv strain) [ , ] were used to generate wt and mutant viruses. a standard mutagenesis protocol was used to engineer mutations into the mtase region as reported previously [ ] . the protocols for in vitro transcription, rna transfection, ifa, plaque assay, and growth kinetics were reported previously [ ] . strain d y p was described previously [ ] . female or male - week old ifn a/b/c receptor deficient mice (ag ) were purchased from b&k universal limited with permission from dr. m. aguet (isrec, school of life sciences ecole polytechnique fédérale (epfl)). ifn a/b receptor deficient mice (ifnar) on a c bl/ background were provided by prof. ulrich kalinke [ ] . all mice were bred and kept under specific pathogen-free conditions at the biomedical resource centre, singapore. for immunization, bhk- derived mutant and wt viruses were used. for challenge experiments denv produced in c / cells was used. fourteen rms, weighing from . to . kg, were pre-screened negative for igg antibodies against denv and jev by indirect immunofluorescence assay. animals were randomly divided into four groups and inoculated subcutaneously (s.c.) in the deltoid region of left arm with . ml of denv- e a dilutions containing . , . , . log pfu, respectively. animals in the control group received pbs. blood was collected from each rm daily post immunization for days to detect viremia. for neutralizing antibody tests, blood was taken before immunization (day ) and on days and post-immunization. on day post-immunization, all immunized animals including the pbstreated control animals were challenged by s.c. inoculation of . ml containing log pfu of denv- (tsv- ). for the following days, blood was collected for determination of viremia. neutralizing antibody levels in serum were measured by the standard % plaque reduction neutralization test (prnt ) on days and post-challenge, respectively. viremia in serum samples was determined by plaque assay in bhk- cell monolayers in -well plates. undiluted serum or serial -fold dilutions of serum were inoculated onto bhk cells. after h of adsorption at uc, wells were overlaid with ml of dmem supplemented with % fbs and % agarose. plates were incubated for days at uc in % co . monolayers were fixed by addition of ml of % formalin solution to the overlay medium. after h of fixation at room temperature, the fixative was removed, wells were washed with water, and monolayers were stained with % crystal violet in % methanol. plaques were counted, and titers were expressed as pfu/ml. for determination of dengue virus-neutralizing antibody titers, serial twofold dilutions of serum (starting at a dilution of : ) were mixed with equal volumes of a suspension of , pfu of denv- tsv /ml. the serum-virus mixtures were incubated at uc for h and tested ( . ml/well) for concentration of infectious virus using the plaque assay described above. the neutralization titer was defined as the lowest serum dilution at which the infectious virus concentration was reduced by % from the concentration found when virus was incubated with culture medium. cells were seeded at per well in a -well plate and treated hours prior to infection with medium or varying concentrations of human recombinant ifn-b (immunotools). cells were then infected at an moi of with wt or mtase mutant virus (tsv ), respectively, incubated for hours and harvested and processed for flow cytometry as described. supernatants were collected for plaque assay. ifit expression plasmids were a kind gift from a. pichlmair ( ) . for ifit overexpression cells were seeded at per well in a -well plate. hours later cells were transfected using fectin according to manufacturer's protocol. one day post transfection cells were trypsinized and seeded in a -well plate at per well. after hours of incubation cells were infected and analysed as described previously. transfection rate was - % judged by parallel experiments with gfp expression plasmid. to determine the percentage of infected cells, cells were harvested, washed in pbs and fixed and permeabilized with cytofix/ cytoperm (bd). intracellular dengue e protein was stained with antibody g conjugated to alexa and fluorescent cells were measured by flow cytometry. for the assessment of ade, g or serum/plasma was serially diluted and a constant amount of virus was added. the antibodyvirus mixture was incubated at uc for min and then ml of the mixture was added to ' k cells per -plate well (moi . - ). after h of infection ml rpmi medium containing fcs was added. after . days of incubation the infected cells were fixed and stained intracellularly with g -alexa . the percentage of infected cells was quantified by flow cytometry. for the measurement of neutralization, g or heat-inactivated serum/plasma was serially diluted and a constant amount of virus was added. the antibody-virus mixture was incubated at uc for min and then ml of the mixture was added to , u cells (atcc) stably transfected with human dc-sign (moi . - ). after h of infection ml rpmi medium containing fcs was added. after incubation over night the infected cells were fixed and stained intracellularly with g -alexa . the percentage of infected cells was quantified by flow cytometry and data were analyzed with graphpad prism software for the calculation of the nt . spleens were harvested at day after infection and single cell suspensions were incubated with live virus or a pool of the following peptides: ns b - , ns b - and ns - [ ] overnight. brefeldin (biolegend) was added for h before cells were washed and stained with antibodies cd -apc, cd -percpcy . and ifn-c-pe-cy (biolegend). cells were acquired on a facscantoii (bectondickinson) and data were analyzed with flowjo (treestar ltd.) -well polystyrene plates were coated with concentrated, heat inactivated dengue virus. plates were incubated overnight at u. before use, plates were washed three times in pbs (ph . ) containing . % tween- (pbs-t). non-specific binding was blocked with % non-fat dry milk diluted in pbs (pbs-m) for h at room temperature (rt). after washing, sera were diluted : in pbs-m, heat inactivated for hour at uc and threefold serial dilutions were added to the wells. plates were incubated for h at rt, followed by three washes with pbs-t. peroxidase-conjugated rabbit anti-mouse igg, in pbs-m was added, followed by h of incubation at rt and three additional washes with pbs-t. tmb was used as the enzyme substrate. the reaction was stopped with m hcl and the optical densities were read at nm using an automatic elisa plate reader. endpoint titers were defined as the lowest dilution of plasma in which binding was twofold greater than the mean binding observed with the negative controls. vector competence experiments were performed using a colony of ae. aegypti mosquitoes in which % of the population is derived from field obtained eggs each month. batches of - female mosquitoes, aged - days were fed with pig blood containing wt or mtase mutant denv- at titers of , , and log pfu/ml. fully engorged mosquitoes were held at uc, % relative humidity, and h photoperiod for days, after which the abdomen was separated from the thorax and homogenized. homogenates were inoculated into vero cell culture. after culturing the inoculated cells for days, viral infection was assayed using an indirect fluorescent antibody test (ifa). antibody b c- against flavivirus group e protein (at : dilution; provided by the usa cdc as a mouse hybridoma) and an anti-mouse antibody conjugated with fitc were used as a primary and secondary antibody, respectively. positive fluorescence determinations were performed manually using an inverted fluorescent microscope (olympus ix ). chi-square and contingency table statistical tests were performed to detail heterogeneity in vector competence within/between wt and mutant viruses. intra-thoracic inoculation of . ml of wt denv- and e a at a titer of pfu/ml was performed using female mosquitoes each. following inoculation, mosquitoes were held for seven days under the same conditions as described above. mosquitoes were then killed by freezing and homogenized. viral rna was quantified by real-time qrt-pcr using primers and methods reported previously [ ] . briefly, whole mosquito homogenate viral rna was extracted using qiaamp viral rna mini kit (qiagen). qrt-pcr was completed using invitrogen super-script iii platinum one-step qrt-pcr mix (without rox) and cfx real-time pcr detection system (biorad). cycling parameters performed were uc for min, uc for min, followed by cycles of uc for sec, uc for sec. a two-tailed unpaired t-test was performed to determine the statistic difference between the mean genomic equivalents calculated for wt and mutant viruses. virus was isolated from mouse serum with qiagen viral rna extraction kit. fifty ng of viral rna were used to prepare cdna libraries using the illumina truseq rna sample preparation kit according to manufacturer's protocol. the only protocol modification was the removal of the mrna enrichment step. the cdna libraries were sequenced as a multiplex in a single lane of an illumina hiseq (next generation sequencing core facility, genomic institute of singapore). one to million bp paired-end reads were generated for each virus. wild-type and mutant virus samples were mapped to their respective reference genomes using bowtie [ ] . mapping statistics and genotype calls were made with samtools [ ] . data analysis was performed in pipeline pilot (http://www.accelrys.com). at least two reads with an alternate base at a given position were defined as a snp. statisitical tests were performed with graphpad prism software, using students t test, two-way anova or chi-square and contingency table statistical tests as indicated in the figure legends. figure s neutralization and ade assay with ag mouse plasma. plasma from ag mice was analyzed days after immunization with mutant or wild-type denv. upper graphs in panels a, b and c show ade assays using k cells and lower graphs show the corresponding neutralization assay using u -dc-sign as target cells. groups of mice were immunized with a) denv- e a, denv- wt, denv- e a and denv- e a combined or pbs; b) denv- e a or denv- wt. c) antibody g was used as a technical control. symbols in panels a) and b) are the means sem of three mice per group, tested in duplicate. the shown experiment is representative for one of two. the mean sd from the two independent experiments (n = - per group) are shown in table . (tif) figure s neutralization and ade assay with nhp serum. the serum of three monkeys per group was analyzed for ade activity. sera from day after challenge in pbs animals (day post infection) or days after challenge in animals which had been immunized with e a days earlier (day post challenge). a) k cells were infected with denv- or denv- in the presence of serum diluted as indicated in the x axes. symbols are means sem of three sera per group from two independent ade assays testing the sera in duplicate each. b) the same sera were tested for neutralization by using u -dc-sign as target cells. symbols are means sd of three sera per group, tested in duplicate each. c and d) g antibody was used as a technical control for the infection of k cells (c) or u -dc-sign cells (d). symbols are means sd of duplicate values. (tif) figure s e a does not mutate and escape ifn pressure in human cell lines hek -dc-sign and u -dc-sign. hek -dc-sign cells (a) and u -dc-sign cells (b) were seeded in a well plate, incubated for hours with , or iu/ml of ifn-b and infected at moi of with e a or wt denv- . hours post infection the percentage of infected cells was determined by facs. ml of the supernatant (passage p ) was transferred to newly seeded ifnb pre-treated cells. the remaining supernatant was kept for isolation of viral rna and sequencing. this procedure was repeated two more times (p and p ). p was collected after instead of hours to allow any potential mutants to have enough time to grow to high titers. (tif) figure s comparison of genome copy numbers of mutant (mt) and wild-type (wt) virus after intrathoracic infection of ae. aegypti. ten female mosquitoes were inoculated intra-thoracic with . ml of wt denv- or e a at a titer of pfu/ml. seven days later mosquitoes were killed by freezing and homogenized. viral rna was quantified by real-time qrt-pcr. mean and % ci intervals are indicated by horizontal bars, each point represents a single female mosquito. p = . , unpaired t test. (tif) towards a global dengue research agenda the global emergence/resurgence of arboviral diseases as public health problems dengue hemorrhagic fever caused by sequential dengue - virus infections over a long time interval: havana epidemic health economics of dengue: a systematic literature review and expert panel's assessment immunological mechanisms of vaccination recombinant, liveattenuated tetravalent dengue virus vaccine formulations induce a balanced, broad, and protective neutralizing antibody response against each of the four serotypes in rhesus monkeys a human challenge model for dengue infection reveals a possible protective role for sustained interferon gamma levels during the acute phase of illness introduction of mutations into the non-structural genes or untranslated region of an attenuated dengue virus type vaccine candidate further decreases replication in rhesus monkeys while retaining protective immunity recombinant dengue type virus ns protein expressed in escherichia coli exhibits rnadependent rna polymerase activity west nile virus -cap structure is formed by sequential guanine n- and ribose -o methylations by nonstructural protein an rna cap (nucleoside- -o-)-methyltransferase in the flavivirus rna polymerase ns : crystal structure and functional characterization -o methylation of internal adenosine by flavivirus ns methyltransferase -o methylation of the viral mrna cap evades host restriction by ifit family members ribose -o-methylation provides a molecular signature for the distinction of self and non-self mrna dependent on the rna sensor mda flavivirus induces interferon-beta gene expression through a pathway involving rig-i-dependent irf- and pi k-dependent nf-kappab activation distinct rig-i and mda signaling by rna viruses in innate immunity ifit is an antiviral protein that recognizes -triphosphate rna the isg /ifit gene family a new pathway of translational regulation mediated by eukaryotic initiation factor ifn-induced tpr protein ifit potentiates antiviral signaling by bridging mavs and tbk flavivirus methyltransferase: a novel antiviral target biochemical and genetic characterization of dengue virus methyltransferase dc-sign (cd ) mediates dengue virus infection of human dendritic cells immunogenicity and efficacy of chimeric dengue vaccine (denvax) formulations in interferon-deficient ag mice immune response in mice that lack the interferon-gamma receptor new mouse model for dengue virus vaccine testing a dengue fever viremia model in mice shows reduction in viral replication and suppression of the inflammatory response after treatment with antiviral drugs interference and facilitation between dengue serotypes in a tetravalent live dengue virus vaccine candidate antibodies targeting dengue virus envelope domain iii are not required for serotype-specific protection or prevention of enhancement in vivo cd + t cells are not required for the induction of dengue virus-specific cd + t cell or antibody responses but contribute to protection after vaccination a protective role for dengue virus-specific cd + t cells type i interferons directly regulate lymphocyte recirculation and cause transient blood lymphopenia a single amino acid in nonstructural protein ns b confers virulence to dengue virus in ag mice through enhancement of viral rna synthesis lethal antibody enhancement of dengue disease in mice is prevented by fc modification dengue vaccines: progress and challenges progress towards a dengue vaccine immune response to dengue virus and prospects for a vaccine protective efficacy of the recombinant, live-attenuated, cyd tetravalent dengue vaccine in thai schoolchildren: a randomised, controlled phase b trial small molecule inhibitors that selectively block dengue virus methyltransferase long-term safety assessment of live attenuated tetravalent dengue vaccines: deliberations from a who technical consultation the stoichiometry of antibody-mediated neutralization and enhancement of west nile virus infection human fcgammarii cytoplasmic domains differentially influence antibody-mediated dengue virus infection ligation of fc gamma receptor iib inhibits antibody-dependent enhancement of dengue virus infection infection-enhancing and -neutralizing activities of mouse monoclonal antibodies against dengue type and viruses are controlled by complement levels mechanistic insights into the impairment of memory b cells and antibody production in the elderly adjuvanted h n vaccine induces early cd + t cell response that predicts long-term persistence of protective antibody levels neutralizing antibodies after infection with dengue virus etiologies of the experimental dengues of siler and simmons recombination and flavivirus vaccines: a commentary recombinant chimeric yellow fever-dengue type virus is immunogenic and protective in nonhuman primates liveattenuated, tetravalent dengue vaccine in children, adolescents and adults in a dengue endemic country: randomized controlled phase i trial in the philippines phase clinical trial of three formulations of tetravalent live-attenuated dengue vaccine in flavivirus-naive adults recent progress on sanofi pasteur's dengue vaccine candidate relationship of a dengue isolate from townsville, , to international isolates functional analysis of two cavities in flavivirus ns polymerase serotype-specific detection of dengue viruses in a fourplex real-time reverse transcriptase pcr assay fast gapped-read alignment with bowtie the sequence alignment/map format and samtools we thank duane j. gubler for helpful discussions during the course of this study. we are grateful to prof. ulrich kalinke for providing ifnar mice. key: cord- - anybxnk authors: ireland, derek d. c.; stohlman, stephen a.; hinton, david r.; kapil, parul; silverman, robert h.; atkinson, roscoe a.; bergmann, cornelia c. title: rnase l mediated protection from virus induced demyelination date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: anybxnk ifn-α/β plays a critical role in limiting viral spread, restricting viral tropism and protecting mice from neurotropic coronavirus infection. however, the ifn-α/β dependent mechanisms underlying innate anti-viral functions within the cns are poorly understood. the role of rnase l in viral encephalomyelitis was explored based on its functions in inhibiting translation, inducing apoptosis, and propagating the ifn-α/β pathway through rna degradation intermediates. infection of rnase l deficient (rl(−/−)) mice with a sub-lethal, demyelinating mouse hepatitis virus variant revealed that the majority of mice succumbed to infection by day p.i. however, rnase l deficiency did not affect overall control of infectious virus, or diminish ifn-α/β expression in the cns. furthermore, increased morbidity and mortality could not be attributed to altered proinflammatory signals or composition of cells infiltrating the cns. the unique phenotype of infected rl(−/−) mice was rather manifested in earlier onset and increased severity of demyelination and axonal damage in brain stem and spinal cord without evidence for enhanced neuronal infection. increased tissue damage coincided with sustained brain stem infection, foci of microglia infection in grey matter, and increased apoptotic cells. these data demonstrate a novel protective role for rnase l in viral induced cns encephalomyelitis, which is not reflected in overall viral control or propagation of ifn-α/β mediated signals. protective function is rather associated with cell type specific and regional restriction of viral replication in grey matter and ameliorated neurodegeneration and demyelination. type i interferon (ifn-a/b) induced anti-viral responses are critical for the initial control of most virus infections. although anti-viral responses are initiated in infected cells, they act in an autocrine and paracrine fashion, antagonizing virus replication in host cells and inducing a protective anti-viral state in neighboring cells. the anti-viral state is distinguished by the expression of numerous ifn stimulated genes (isg), which directly or indirectly interfere with both viral and host rna transcription and translation [ ] . many of these pathways can also result in the apoptosis of infected cells [ ] . nevertheless, the relative contributions of these various anti-viral effector mechanisms to overall virus control differ with the virus as well as the tissue and cell type infected. the best characterized anti-viral mechanisms are mediated by activation of double-stranded rna-dependent protein kinase (pkr) and the - -oligoadenylatesynthase (oas)/ribonuclease l (rnase l) pathways [ ] [ ] [ ] . upon recognition of double-stranded rna, the oas family of proteins convert atp into unique, unstable - -a oligomers, which are bound by latent rnase l monomers, driving the dimerization and activation of rnase l. activated rnase l cleaves single-stranded rna, of u-a and u-u sites found in both viral and cellular rna. rnase l endoribonuclease activity is thus not limited to viral rna, but also degrades host cell rna, including ribosomal rna. as oas genes are stimulated by ifn-a/b, elevated oas levels enhance the antiviral state in adjacent, non-infected cells responding to ifn-a/b. nevertheless, inappropriate activation of rnase l is counterbalanced by the unstable nature of - a oligomers and the endogenous rnase l inhibitor, rl [ ] . a novel aspect of the oas/rnase l pathway is the cleavage of host rna releasing free -monophosphates that can activate the cytoplasmic rna helicases rig-i and mda- [ ] . recognition by these pattern recognition receptors initiates ifn-b transcription, similar to the activation mediated by recognition of specific viral rna structures. rnase l thus not only exerts anti-viral effects via rna degradation and apoptosis, but also has the capacity to amplify and prolong expression of anti-viral genes and other isgs. the anti-viral activity of the oas/rnase l system has been studied in several virus infections in vitro and in vivo [ , ] . protective mechanisms of the oas/rnase l pathway are generally more evident for rna relative to dna virus infections. however, the contribution of rnase l to disease severity and viral control varies significantly depending on the virus and even virus strain studied, presumably reflecting the diverse mediators and targets of these anti-viral mediators. for example, encephalomyocarditis virus (emcv) replication was only modestly increased in rnase l deficient (rl / ) mouse embryonic fibroblasts, consistent with enhanced susceptibility to infection in vivo. although rnase l prolonged survival, mortality rates of infected rl / and control mice were similar. furthermore, ifn-b treatment increased the survival time of infected wild-type (wt) and to a lesser extent rl / mice, indicating that alternate ifn-dependent anti-viral mechanisms act in the absence of rnase l [ ] . in contrast, the requirement for rnase l activity to protect from another picornavirus, coxsackievirus b , was evident by significantly increased mortality rates of infected rl / compared to wt mice. however, increased mortality was not the result of uncontrolled virus replication, as virus titers were only slightly elevated within pancreatic islet cells in vivo. by contrast, pancreatic islet cells derived from rl / mice, treated with ifn-a and infected in vitro exhibited increased virus replication and loss of cellular integrity, compared to ifn-a treated rnase l sufficient cells [ ] . these results revealed distinct differences in direct antiviral functions of rnase l in vitro and in vivo. cell type specific effects of anti-viral rnase l activity are also clearly evident from studies with west nile virus (wnv). while mouse embryonic fibroblasts display rnase l dependent anti-viral activity [ ] , ifn-b treatment revealed no affect of rnase l in reducing wnv replication in either cortical or peripheral motor neurons [ ] . nevertheless, a modest effect was observed in bone marrow derived macrophages, consistent with increased viral burden in cd b + splenocytes derived from rl / compared to wt mice [ ] . furthermore, increased mortality of rl / mice in response to peripheral wnv infection correlated with increased replication in lymphoid tissue, but not enhanced viral dissemination into the cns. a minor role for rnase l in control of wnv in the cns was supported by similar viral titers in brains as well as kinetics and rates of mortality following intracranial infection of rl / and wt mice. although viral replication was only transiently increased in spinal cords of intracranially infected rl / mice, the most prominent effect was increased spread to the spleen and liver [ ] . the more critical role of rnase l in limiting wnv spread in peripheral tissues rather than the cns highlights the complexities of oas/rnase l mediated anti-viral mechanisms in vivo. ifn-a/b mediated innate responses are also essential to control virus replication and survival following coronavirus infections [ ] [ ] [ ] [ ] . infections in vitro and in vivo induce upregulation of ifn-a/b and anti-viral mediators, including pkr and oas [ , , ] ; however, the participation of these pathways in the anti-viral response to cns infection has not been elucidated. in vitro, the dual liver and neurotropic mhv-a strain does not elicit degradation of s and s ribosomal rna, suggesting the absence of rnase l activation in hela cells [ ] . however, administration of an rnase l agonist inhibited replication of the hepatotropic mhv- strain in peritoneal macrophages in vitro and in liver in vivo [ ] . nevertheless, reduced viral replication via rnase l function in vivo was insufficient to prevent liver necrosis and death. the nonlethal gliotropic jhm strain of mouse hepatitis virus (mhv-jhm) replicates exclusively in the cns following intracranial infection and is controlled by both innate and adaptive responses [ , ] . cns infection results in a non-lethal encephalitis with immune-mediated demyelination resulting in hind limb paralysis [ ] . based on expanded tropism to hippocampal neurons and widespread dissemination within the cns of mice deficient in ifn-a/b signalling (ifnar / ) [ ] , the gliotropic mhv-jhm variant was used to probe an anti-viral role of rnase l within the cns. while the vast majority of wt mice survived, rl / mice succumbed to infection, albeit delayed compared to ifnar / mice. although enhanced morbidity suggested a prominent role of rnase l in anti-viral protection, the absence of rnase l neither diminished overall virus control in the cns, nor enhanced neuronal infection. furthermore, rl / mice did not reveal any impairment in ifn-a/b induction, alterations in proinflammatory cytokines or inflammation compared to wt mice. these data rather reveal a novel role of rnase l in specifically counteracting focal microglia/macrophage infection, thus potentially sustaining microglia mediated neuroprotective effects and ameliorating demyelination. mice deficient in ifn-a/b signalling succumb to an otherwise sub-lethal gliotropic mhv-jhm within days of infection despite functional cd t cell responses [ ] . uncontrolled viral replication in the absence of ifn-a/b implicated a crucial role of anti-viral innate mechanisms in stemming viral spread within the cns. to elucidate the role of rnase l in cns pathogenesis, gliotropic mhv-jhm infection was examined in rl / mice [ ] . increased susceptibility of rl / mice was evident by more severe clinical symptoms of acute encephalitis at - days post infection (p.i.) (fig. , top panel) . with the exception of increased severity, onset and progression of clinical symptoms in infected rl / were similar to those exhibited by infected wt mice. these included initial symptoms of acute viral encephalitis, including: lethargy, dehydration and weight loss, which progressed to diminished hind limb function. furthermore, mortality rates were significantly higher in rl / mice, with over % succumbing to infection by days p.i. (fig. , middle panel) , approximately - days delayed relative to ifnar / mice [ ] . assessment of direct anti-viral functions of rnase l [ , ] revealed no initial spread of viruses is controlled by type i interferon induced antiviral molecules. early intervention with viral replication is especially critical in central nervous system infections to reduce loss of non-renewable cells and mitigate immune pathology. one of the best characterized anti-viral mechanisms is mediated by ribonuclease l (rnase l). rnase l exerts activity at multiple levels, including degradation of viral and host rna, induction of apoptosis, and propagation of the ifn-a/b pathway. recent studies suggest that rnase l antiviral activity is dependent on the virus, as well as the cell type and tissue infected. this study demonstrates that rnase l protects mice infected with a sub-lethal, demyelinating neurotropic coronavirus by ameliorating encephalitis and morbidity, albeit without affecting control of infectious virus or ifn-a/ b expression. rnase l specifically protected the brain stem from sustained infection and prevented spread of virus to microglia/macrophages located in spinal cord grey matter. the subtle regional alteration in tropism in the absence of rnase l coincided with increased apoptotic cells and earlier onset as well as increased severity of axonal damage and demyelination. the results demonstrate how subtle regional alterations in viral tropism within the cns may severely affect the balance between neuroprotection and neurotoxicity mediated by microglia/macrophages. significant changes of infectious virus in brains of rl / , compared to wt mice. virus titers were increased less than . log in rl / mice (fig. , bottom panel) . very modest anti-viral rnase l activity in the cns was confirmed by analysis of viral rna from cns tissue. levels of viral mrna encoding the viral nucleocapsid protein were increased less than . -fold in spinal cords of rl / compared to wt mice at any time point (table ) . as liver may constitute an extraneural infection site in immunocompromised mice, viral replication in this tissue was also assessed by real-time pcr. the abundant viral mrna encoding the nucleocapsid protein was detected in liver at very low levels compared to the cns in both groups, albeit only at early time points (table ) . although viral rna was elevated in livers of rl / mice at day p.i., clearance by day p.i. suggested hepatitis did not contribute to mortality. necrotic foci were not evident in either mouse group by gross examination. a minor role of rnase l in viral control in vivo was reminiscent of the previously described picornavirus models [ , , ] . in addition to rna degradation, rnase l also induces a multitude of genes in the ifn-a/b pathways during infections with sendai virus and emcv [ ] . thus, both self and virus small rna products released by rnase l activate cytoplasmic rna helicases rig-i and mda- to optimize and perpetuate anti-viral responses. however, ifn-a and ifn-b expression levels were slightly increased, rather than decreased in brains of mhv-jhm infected rl / mice relative to wt mice (fig. ) . furthermore, oas- , ifit- , ifit- , and irf- , representing prominently activated isgs, were induced to similar, or modestly increased levels in the cns of infected rl / mice. slightly enhanced ifn-a/b and isg expression was also observed in spinal cords from infected rl / relative to wt mice (data not shown). consistent with similar virus replication and control in the cns, neither ifn-a/b nor the expression of downstream isgs were impaired at the tissue level. these data suggest that a positive rnase l-dependent feedback loop in propagating ifn-a/b signalling is not activated during mhv-jhm infection in the cns. an unanticipated feature of rl / mice is delayed tissue rejection in transplant studies [ ] , suggesting a role of rnase l in modifying antigen presentation, lymphocyte trafficking or function. to assure that altered pathogenesis was not due to differential inflammatory responses, the cns was examined for extent, composition and localization of infiltrating cells. characterization of cells recruited to the cns using flow cytometry revealed similar total numbers of infiltrating cells and no alterations in their composition (fig. ). unlike ifnar / mice [ ] , neutrophil infiltration was not affected in rl / mice and macrophages comprised the most prominent population early p.i. in both strains. cd + and cd + t cells peaked to nearly identical numbers at day p.i. in both groups. furthermore, cns infiltrating cd + t cells in both groups were comprised of % and % virus specific cells at day and pi, respectively, as monitored by d b /s class i tetramer staining [ ] . similarly, no significant differences in the extent of inflammatory cells were observed by histochemical analysis (data not shown). these data confirm that priming and trafficking of virus specific t cells was unaffected by the loss of rnase l activity, consistent with effective clearance of infectious virus. rnase l did not overtly affect overall cns viral replication; however, it may act in a tissue or cell type specific manner [ ] . mhv-jhm initially infects ependymal cells and spreads to astrocytes, microglia/macrophages and predominantly oligodendrocytes, but rarely infects neurons [ ] . however, mhv-jhm induced mortality in the absence of ifn-a/b signalling is associated with a dramatically expanded virus tropism to hippocampal neurons [ ] . to assess the possibility that rnase l exerts cell type dependent anti-viral effects not apparent from whole tissue homogenates, the distribution of virus infected cells was analysed by immunohistochemistry. sequential analysis demonstrated a limited infection of neurons within the brain with a predominance in the brain stem at day p.i. in both rl / and wt mice (fig. ) . glia tropism prevailed in both groups and overall distribution of virus infected cells was similar with occasional neurons still infected at day p.i. (data not shown). by day p.i. virus infected cells declined in cortex and brain stem of both wt and rl / mice. however, in contrast to wt mice which had cleared virus from brain stem, virus infected cells were sustained in the brain stem of rl / mice and were predominantly microglial in appearance (fig. ) . the overall extent and distribution of viral infected cells was thus similar during peak virus replication in brains of rl / and wt mice, with the exception of sustained infection in brain stem in the absence of rnase l. preliminary experiments indicate that these cells are microglia or macrophage/ monocytes (see below). these data contrast with the infection of ifnar / mice [ ] which show predominant neuronal infection. therefore, preferential infection of neurons could not account for the increased mortality in rl / mice. the data also indicated that protective rnase l functions are not overtly reflected in early anti-viral activity but are manifested in region specific protection. rnase l activation leads to apoptosis and elimination of infected cells, a process requiring activated caspase [ , ] . furthermore, ifna/b mediated activation of rnase l also induces apoptosis in non-infected cells [ ] . during mhv-jhm infection of wt mice the majority of apoptotic cells are lymphocytes [ ] , which are not targets of infection. although oligodendrocyte apoptosis has been linked to the demyelinating process, the demonstration of apoptotic oligodendrocytes during mhv-jhm infection has been elusive [ , ] . the retention of virus infected cells in brain stem may result in enhanced local t cell activation and apoptosis of either t cells, infected cells, or both. therefore, the frequency of apoptotic cells was examined at day p.i. in contrast to the brain stems of wt mice which contained few apoptotic cells, apoptotic cells were prominent in brain stems of rl / mice (fig. ) coincident with retention of virus infected cells. although increased focal virus infection associated with substantially increased apoptotic cells presumably dysregulates neuronal function and potentiates tissue damage, there was no evidence that apoptotic cells are preferentially located adjacent to neurons. sustained brain stem infection and apoptosis in rl / mice coincided with increased morbidity and mortality, despite similar overall control of viral replication. the morbidity in mhv-jhm infected rl / mice also coincided temporally with the onset of acute primary demyelination in wt mice. rl / mice were thus examined for enhanced tissue pathology and demyelination. brain stems of infected wt mice revealed minimal demyelination by day p.i. (fig. ). by contrast, demyelination was more severe in brain stems of rl / mice, correlating with increased numbers of virus infected as well as apoptotic cells. demyelination is associated with a variable degree of axonal damage [ , ] . primary immune mediated demyelination in the spinal cord during mhv-jhm infection coincides with early axonal degeneration [ , ] . while axons were largely intact in brain stems of wt mice, axonal integrity within demyelinated lesions was severely compromised in rl / mice as indicated by a striking decrease in neurofilament and an increase in dystrophic axons within lesions (fig. ) . in spinal cords of wt mice, demyelination was also minimal at day p.i. and increased by day p.i. (fig. ) , when control of virus replication is clearly evident (table ) . by contrast, large focal areas of severe demyelination were already apparent in rl / mice at day p.i., resembling those in wt mice at day p.i. in addition to the accelerated kinetics of demyelination, the severity of myelin loss was more pronounced in the absence of rnase l at both days and p.i. consistent with the more rapid loss of myelin in infected rl / mice, quantification of demyelinated areas at day p.i. revealed . . % demyelination in spinal cords of wt mice and . . % in rl / mice. increased demyelination in both brain stem and spinal cord are thus a hallmark of infected rl / mice, irrespective of overall viral control. apoptotic cell numbers and axonal damage in the spinal cords of rl / mice were also evaluated to determine an association with increased demyelination. in wt mice, apoptotic cells were undetectable in spinal cord at day p.i. (data not shown), but were prominent in white matter by day p.i. (fig. ) . by contrast, spinal cords from rl / mice already exhibited small areas of apoptotic cells at days p.i., albeit only in white matter. however, by day p.i. numerous apoptotic cells were evident in both white matter and grey matter of spinal cords from infected rl / mice. furthermore, in contrast to brain stems, apoptotic cells were often detected in close proximity to neurons (inset fig. ) ; however, there . rnase l does not alter the extent of neuronal infection but delays viral clearance from brain stem. viral nucleocapsid antigen detected by immunoperoxidase staining using mab j. . in brains from infected wt and rl / mice at days and p.i. (top panels: brown chromogen; hematoxylin counterstain). note neuronal infection (arrows) and similar foci of viral antigen in glial cells in brain stem at day p.i. by day p.i. infection is controlled in wt mice, but foci of infected non neuronal cells with glia morphology are sustained in rl / mice. scale bar = mm. immunohistochemical staining for activated caspase- (bottom panels) at day p.i. indicates increased numbers of cells undergoing apoptosis in brain stem of rl / relative to wt mice. scale bar = mm. doi: . /journal.ppat. .g was no evidence for neuronal apoptosis. these data suggest that the absence, rather than presence, of rnase l contributes to enhanced apoptosis in white matter, and subsequently also in grey matter. analysis of axonal integrity revealed that all axons were intact in spinal cords of wt mice consistent with minimal demyelination at day p.i. (fig. ) . by contrast, a striking decrease in neurofilament and an increase in dystrophic axons within demyelinated lesions demonstrated axonal integrity was already compromised in white matter of rl / mice by day p.i. at day p.i. the amount of axonal degeneration in wt mice resembled that of rl / mice at day p.i., consistent with the severity of demyelination. however, by day p.i. spinal cords of rl / mice showed marked axonal loss located in areas of myelin loss (fig. ) , similar to results in brain stem. the increased pathological changes in both brain stem and spinal cord thus correlated with the high mortality rates of rl / mice starting at day p.i. the inability to directly link increased axonal damage with enhanced neuronal infection remains puzzling and supports dysregulation of oligodendrocyte function and/or neuroprotective functions of microglia as contributing factors to the severe pathological phenotype and clinical outcome. increased demyelination in spinal cords has been correlated with the extent of white and grey matter infection in mice infected with heterologous mhv strains [ ] . furthermore increased apoptosis, specifically in spinal cord grey matter of rl / mice supported grey matter infection. increased demyelination as well as apoptosis in grey matter in rl / mice thus prompted a more detailed investigation of viral antigen distribution in spinal cords, where the demarcation between grey and white matter is linear as opposed to the more complex organization of brain stem. neuronal infection was not observed in spinal cords of either rl / or wt mice. furthermore, the number of infected cells with oligodendrocyte morphology in the white matter of rl / mice was similar to wt mice at day p.i. (fig. ) . however, a prominent difference in viral antigen positive cells was noted in spinal cord grey matter. rl / mice harboured several foci of infected cells with microglia morphology, whereas viral antigen positive cells were rarely observed in spinal cord grey matter of wt mice (fig. ) . furthermore, a large number of infected cells in the grey matter of rl / mice were identified as microglia and/or infiltrating monocytes based on co-localization with iba- positive cells (fig. ). foci of iba- positive cells co-expressing viral antigen in the grey matter were only observed in rl / but not wt mice, confirming results obtained by immunohistochemistry (fig. ) . nevertheless, infected iba- positive cells were dispersed throughout the white matter of wt and rl / mice. similar characterization of the brain stem at day p.i. also identified iba - positive cells as prominent cells harbouring sustained viral infection (data not shown). co-stains with the astrocyte specific marker gfap showed no evidence of infected astrocytes in spinal cord grey matter; astrocytes were also only rarely infected in white matter of wt and rl / mice (data not shown). these data indicate that rnase l mediated antiviral function acts specifically in microglia and/or monocytes. moreover the focal nature of infection in grey matter suggests microglia localization is associated with enhanced susceptibility to infection in the absence of rnase l. to determine whether infection of iba- positive cells was biased to microglia or infiltrating monocytes, both cell populations were purified from spinal cords of infected mice by fluorescence activated cell sorting. measurement of viral mrna encoding the nucleocapsid protein revealed that microglia derived from rl / mice harbored . -fold higher levels of viral rna at day p.i., while the relative levels in monocytes was only increased . -fold relative to infected wt mice (table ) . however, viral mrna levels decreased in both cell types reaching comparable levels by day p.i. while rna levels for tnf were elevated in rnase l deficient microglia and monocytes by -fold and . fold, respectively, inos levels remained similar relative to wt derived cells (data not shown). ifnc relative to gapdh mrna levels in spinal cord were also modestly increased from . . in wt mice to . . in rl / mice at the peak (day p.i.) and declined to . . and . . , respectively, by day p.i., confirming a modest contribution of proinflammatory cytokines to pathogenesis. moderately increased viral rna levels in microglia are thus consistent with foci of microglia infection in spinal cord grey matter not present in wt mice. overall these data demonstrate that accelerated and enhanced demyelination in the absence of rnase l coincided with significant neuronal damage in the absence of expanded neuronal infection. furthermore, the limited extent of increased focal glial infection appeared insufficient to mediate the pathological effect. to assess potential differences in the overall activation state of microglia, the distribution and morphology of iba- positive cells was assessed in spinal cord grey matter. microglia in wt mice were activated as demonstrated by their intense staining and ramified phenotype and localized in close proximity to neuronal cell bodies (fig. ) . by contrast, in infected rl / mice, fewer microglia were activated based on their iba- staining and morphology and did not show preferential association with neurons. whether the absence of rnase l itself or infection of microglia mitigates a neuroprotective function of microglia remains to be explored. the notion that inflammatory insults disrupt neuroprotective functions by microglia is based on microglia mediated neuroprotection in models of injury and lps preconditioning [ ] [ ] [ ] and loss of protection following viral infection [ ] . ifn-a/b dependent innate immunity is essential to contain viral spread during most viral infections prior to control by adaptive responses. nevertheless, the in vivo anti-viral effector mechanisms contributing to innate viral control remain poorly understood. the best characterized pathways are mediated by activation of pkr and rnase l [ ] [ ] [ ] . however, in contrast to the conclusive effects of pkr and rnase l deficiency on viral replication in vitro, in vivo studies demonstrate more subtle anti-viral effects [ , , ] . during wnv infection, rnase l deficiency is manifested in profoundly altered morbidity, despite similar viral control after footpad inoculation [ ] . ifn-a/b-mediated anti-viral responses are also critical for controlling spread of neurotropic coronavirus within glial cell populations and preventing infection of neurons [ ] . based on its activation by numerous rna viruses, rnase l was investigated as a prototypical anti-viral effector molecule in controlling mhv-jhm virus replication in the cns. rnase l deficiency was not sufficient to duplicate uncontrolled replication and the rapid uniform lethality observed in ifnar / mice [ ] . nevertheless, a critical contribution to virus susceptibility was demonstrated by a significant increase in both clinical disease and mortality. mortality was delayed by - days in rl / relative to ifnar / mice, but could not be attributed to uncontrolled viral replication or spread to neurons. in fact, overall virus replication in brains and spinal cords of rl / mice was only modestly increased and declined with kinetics similar to wt mice. these latter findings were reminiscent of transiently increased viral replication in the cns of rl / mice following intracerebral wnv inoculation [ ] . in addition to direct anti-viral activities, activated rnase l degrades viral and host rna [ , ] . the cleavage products may in turn activate rig-i/mda- cytoplasmic pattern recognition receptors, propagating the expression of ifn-a/b and isgs [ ] . while this function of rnase l is indeed evident by reduced ifn-b production in emcv infected rl / mice [ ] , mhv-jhm infection of the cns presented no evidence for this pathway. the negligible affects of rnase l deficiency on overall viral replication [ ] . nevertheless, the preferential susceptibility of rnase l deficient microglia/ monocytes to mhv-jhm infection demonstrates that viral rna does activate cellular rna sensors, albeit in a cell type specific manner. this is supported by mda- triggered activation of the ifn-a/b pathway in microglia and macrophages infected with mhv-a in vitro [ ] . whether the apparent inability of gliotropic mhv-jhm to activate rnase l in other cell types in vivo resides in distinct basal oas/rnase l expression levels, activation of distinct oas enzymes, or other rna sensing receptors such as mda- has not been elucidated. numerous functions of rnase l, not directly associated with anti-viral activity, may contribute to the increased susceptibility of rl / mice to mhv-jhm infection. rnase l plays a role in translational inhibition, regulation of mrna stability, apoptosis, proliferation and tumor suppression [ , ] . for example, rnase l contributes to ifn-a/b mediated apoptosis, as well as homeostasis of thymocytes and splenocytes in young naïve mice [ ] . rnase l deficiency also delays tissue graft rejection [ ] implicating a defect in t cell function or trafficking. nevertheless, both histochemical and flow cytometric analysis revealed a similar extent and composition of inflammatory cells in the cns of infected rl / and wt mice. the lack of rnase l mediated alterations in t cells was supported by similar peripheral expansion of virus-specific cd t cells and kinetics of viral control. increased morbidity could thus not readily be attributed to altered inflammation or pro-inflammatory signals at the tissue level. neuronal infection by gliotropic mhv-jhm is sparse and only evident early p.i. in wt mice. no evidence for enhanced neuronal infection in rl / mice thus suggested that the ifn-a/b mediated anti-viral mechanisms in neurons are rnase l independent. this contrasts with a protective role for rnase l in inhibiting hsv- replication in neurons of ifn-b treated trigeminal ganglia [ ] , and supports virus specific susceptibilities to innate anti-viral immunity. distinguishing features in mhv-jhm infected rl / mice are the sustained areas of microglia infection in brain stem and focal areas of infected microglia within the spinal cord grey matter. infected cells in spinal cord grey matter are also evident in scid [ ] and ifnar / mice (unpublished observation) following infection with the gliotropic mhv-jhm. in rl / mice the prominent infected cells in brain stem and spinal cord grey matter were identified as iba- positive microglia or infiltrating monocytes, which cannot readily be distinguished by immunohistochemistry. nevertheless the morphology of infected cells in the grey matter was consistent with activated microglia, rather than the monocyte/macrophages with dense cytoplasm found prominently in demyelinating lesions. furthermore, preferential microglia infection was supported by a relatively greater increase of viral rna in microglia relative to monocytes, when comparing rl / versus wt mice. the prominent location of iba- positive cells in grey matter of rl / mice could not be attributed to the absence of activated microglia in grey matter of wt mice. in fact, microglia in wt mice exhibited a more ramified phenotype surrounding neurons compared to microglia in rl / infected mice. it remains unclear whether abrogation of the proximal localization of microglia to neurons in rl / spinal cords reflects an unknown function of an rnase l enhanced neuroprotective effect, or altered function due to infection. surprisingly, both prolonged brain stem infection and enhanced spinal cord grey matter infection was associated with more severe demyelination and axonal damage. overall, the cns pathology characteristic of mhv-jhm infection was accelerated by - days in rl / relative to wt mice. although infection of microglia correlated with a subsequent increase in apoptotic cells, apoptotic cells surrounding neurons were only evident in spinal cord, not in brain stem. the inability to detect increased numbers of infected or apoptotic cells in spinal cord white matter is consistent with both an apparent lack of rnase l activation in oligodendrocytes during mhv-jhm infection and paucity of apoptotic oligodendrocytes in wt mice [ ] . whether apoptotic cells originated from infected myeloid cells themselves or from lymphocytes migrating to the infected areas and exerting effector function is unclear. preliminary analysis showed no co-localization of activated caspase and iba- (data not shown). overall, the neurologic disability and morbidity of the infected rl / mice appears to result from axonal or neural degeneration, independent of neuronal infection in both brain stem and spinal cord. the enhanced demyelination phenotype is thus distinct from demyelination attributed to enhanced white matter infection by recombinant neuronotropic mhv variants [ ] . rnase l deficiency and infection of microglia may contribute to this pathology in several ways. infection in the absence of rnase l may impair neuroprotective effects exerted by microglia under inflammatory conditions [ ] [ ] [ ] . alternatively, enhanced infection of microglia may increase proinflammatory responses resulting not only in enhanced recruitment of t cells and monocytes, but also increased local production of neurotoxic factors such as tnf, nitric oxide, oxidative radicals, and matrix metalloproteases. however, only modest increases in ifnc mrna in spinal cord, as well as minor differences in tnf and inos mrna in microglia/monocytes suggest these effects may only be apparent at a focal level. increased localized t cell effector function, manifested in release of perforin and granzymes, has also been shown to contribute to axonal injury without affecting demyelination [ ] . lastly, rnase l deficient and/or infected microglia may perturb normal microglia functions in maintaining neuronal health as indicated by disruption of neuronal autophagy by siv infected microglia [ ] . it thus remains to be determined to what extent accelerated and more severe pathology is due to disruption of the neuroprotective functions of microglia and/or from overt activation due to infected cells [ ] [ ] [ ] [ ] [ ] . independent of a pathogenic effect, it is interesting to note that the absence of rnase l also enhanced macrophage susceptibility to wnv infection [ ] . whether this reflects differential activation of oas enzymes or participation of other pattern recognition receptors such as mda- in susceptible cell types in vivo remains to be elucidated. in summary, these data highlight a novel role of rnase l in protection from virus induced demyelination. although rnase l did not play an overt anti-viral role as measured by viral replication, it did provide specific protection from focal infection of microglia/macrophages in the cns. rnase l was not crucial in protecting neurons from infection and played no obvious role in protecting oligodendrocytes or astrocytes. furthermore, rnase l deficiency did not alter proinflammatory responses, diminish ifna/b mediated signals, or dampen adaptive immune mediators. the results rather uncover how subtle local alterations in viral tropism may affect the balance between neuroprotection and neurotoxicity mediated by microglia/macrophages. animals, viruses, and clinical scores c bl/ mice were purchased from the national cancer institute (nci, fredrick, md). c bl/ -rl / mice were bred and housed under pathogen-free conditions in the biological resources unit of the cleveland clinic. all procedures were performed in compliance with protocols approved by the institutional animal care and use committee. mice were infected at weeks of age by intracranial injection with pfu of the gliotropic mhv-jhm variant v . - [ ] in ml endotoxinfree dulbecco's phosphate buffered saline (dpbs). the severity of clinical disease was graded as previously described [ ] : = ruffled fur, = slow righting reflex, = loss of righting reflex, = moribund. following intraperitoneal administration of ketamin/xylaxine ( mg/kg/ mg/kg), mice were perfused intracardially with ml dpbs. brains, spinal cords, spleens, cervical lymph nodes (cln) and livers were collected and processed as described below. brains were bisected sagittally. one half-brain and whole spinal cord from each mouse were homogenized in ice cold tenbroeck glass grinders in ml or ml of dpbs, respectively. homogenates were clarified by centrifugation for min at g. supernatants were stored at uc. virus in supernatants was measured by plaque assay on monolayers of delayed brain tumor (dbt) astrocytoma cells as previously described [ ] . cns cells from homogenate pellets were resuspended in rpmi containing mm hepes, adjusted to % percoll (pharmacia, upsalla, sweden), underlayed with ml % percoll, centrifuged at g for minutes and collected from the %/ % percoll interface as previous described [ ] . purified cns cells were washed and resuspended in rpmi. cell populations isolated from the brain and spinal cord were phenotyped using four-color flow cytometry. prior to staining, cells were incubated with % mouse serum and % rat anti-mouse fcciii/iir monoclonal antibody (mab) in fluorescent antibody cell sorting (facs) buffer ( . % bovine serum albumin in dpbs) for minutes at uc to block non-specific binding. cell types were identified using fluorescein isothiocyanate-, phycoerythrin-, peridinin-chlorophyll-protein complex-or allophycocyanin-conjugated anti-mouse mab: ly- g ( a ), cd (gk . ), cd ( . ) (all from bd biosciences, san diego, ca) and f / (ci:a - ; serotec, raleigh, nc). virus specific cd t cells were identified using h- d b /s mhc class i tetramers as described previously [ ] . cells were incubated with antibodies for minutes on ice, washed twice with facs buffer and fixed with % paraformaldehyde for minutes on ice. at least , events were acquired on a facscalibur flow cytometer (bd biosciences, san jose, ca) for subsequent data analysis using flow-jo software (tree star, inc. ashland, or). for fluorescence activated cell sorting of microglia and monocyte populations, spinal cords from eight mice per group were finely minced using a razor blade and dissociated in a . % trypsin solution as described [ , ] at uc for minutes with periodic tituration. trypsin was quenched by addition of rpmi supplemented with mm hepes and % new born calf serum. the dissociated cells were washed in rpmi containing mm hepes, % fcs, then isolated from the interphase of a %/ % percoll gradient as described above. cells were incubated with % mouse serum and cd / prior to staining with allophycocyanin-conjugated mab specific for cd ( -f ), peridinin-chlorophyll protein-conjugated cd b (m / ) (bd biosciences, san diego, ca) and phycoerythrin-conjugated mab specific for f / (ci:a - ; serotec, raleigh, nc). monocyte/ macrophages and microglia were purified on a facsaria cell sorter (bd biosciences, san diego, ca) based on their respective cd hi cd b + f / + and cd lo cd b + f / + phenotypes. one half-brain and whole spinal cords were fixed with formalin and embedded in paraffin. sections were stained with either hematoxylin and eosin or luxol fast blue as described [ ] . distribution of viral antigen was determined by immunoperoxidase staining (vectastain-abc kit; vector laboratory, burlingame, ca) using mab j. . , specific for the carboxyl terminus of the viral nucleocapsid protein as primary antibody, biotinylated horse antimouse as secondary antibody, streptavidin-conjugated horse radish peroxidise and , -diaminobenzidine substrate (vector laboratory) [ ] . similarly, immunoperoxidase staining for neurofilament used mouse anti-phosphorylated and anti-non-phosphorylated neurofilament mab (smi- and smi- , covance, princeton, nj) and immunoperoxidase staining for microglia/macrophages used anti-ionized calcium-binding adapter molecule- antibody (iba- , abcam, cambridge, ma). apoptotic cells were detected by rabbit anti-activated caspase- ab (asp , cell signalling technology, beverly, ma). sections were scored for inflammation, viral antigen, apoptotic cells, axonal damage and demyelination in a blinded fashion. representative fields-of-view were identified based on average scores of all sections in each experimental group. for calculation of the percentage area of demyelination, sections were digitized and analysed as previously described [ ] . to identify infected cell types, spinal cords were embedded in tissuetek o.c.t. compound (andwin scientific, tryon, nc), flash frozen in liquid nitrogen and stored at uc. blocks were warmed to uc prior to cutting mm sections by cryostat at this temperature. following fixation with % paraformaldehyde for minutes at room temperature, non-specific antibody binding was blocked using % bovine serum albumin, % normal goat serum. infected cells were identified using anti-mhv-jhm j . mab, rabbit anti-glial fibrillary acidic protein antibody (gfap, abcam, cambridge, ma) and rabbit anti-ionized calcium-binding adapter molecule- antibody (iba- , wako, richmond, va) in combination with goat anti-mouse alexa-fluor and goat anti-rabbit alexa-fluor -conjugated igg (invitrogen, carlsbad, ca) as secondary antibodies, respectively. sections were mounted with prolong gold antifade mounting media containing , -diamidino- -phenylindole (invitrogen, carlsbad, ca). imaging of immunofluorescent sections was performed with a leica sp- confocal microscope (leica microsystems, bannockburn, il). z-projections of sections were processed by imagej software (national institutes of health, bethesda, md) one-half brains and whole spinal cords were snap frozen in liquid nitrogen and stored at uc. frozen brain or spinal cord tissue was homogenized in ml and ml trizol reagent (invitrogen, carlsbad, ca), respectively, in rnase-free tenbroeck glass grinders. rna was purified according to the manufacturer's protocol (invitrogen, carlsbad, ca). briefly, . ml chloroform/ ml trizol (sigma-aldrich, st. louis, mo) was added to homogenate, mixed and centrifuged at , g for minutes at uc. rna was precipitated from the aqueous phase by addition of isopropyl alcohol and centrifugation at , g for min at uc washed in rnase-free % ethanol and resuspended in ultrapure dnase/rnase-free water (invitrogen, carlsbad, ca). cells isolated by facs were immediately resuspended in ml of trizol reagent (invitrogen, carlsbad, ca) and treated as above. isolated total rna was treated with dnase , using the dna-free kit (applied biosystems, foster city, ca) following the manufacturer's protocol. the concentration and purity of rna was measured by spectrophotometry at / nm. rna integrity was confirmed by . % formaldehyde-agarose gel electrophoresis. reverse transcription was performed on mg total rna isolated from brain and spinal cord or all total rna isolated from facs sorted cells, primed with ng random hexamers, (invitrogen, carlsbad, ca) using mmlv reverse transcriptase (invitrogen, carlsbad, ca) for minutes at uc. real-time pcr was performed using sybr green master mix (applied biosystems, foster city, ca) for the following primer sets: interferon-induced protein with tetratricopeptide repeats ( the reaction conditions were: uc for minutes, followed by cycles of: denaturation at uc for seconds, elongation at uc for seconds and annealing at uc for seconds. ifn-b , ifn-a and ifn-c levels were determined by real time pcr using abi gene expression assays with universal taqman fast master mix (applied biosystems, foster city, ca), using manufacturer default cycling conditions and gapdh expression as an endogenous control. all real-time reactions were run using an abi fast real-time cycler and analysed with fast software (applied biosystems, foster city, ca). data presented are expressed as fold-induction relative to gapdh based on the following formula: ( (ct(gapdh)-ct(target)) )* . students' t-test with equal variance was used to compare rl / and wt c bl/ mice. significant differences between groups are noted by: *, = p# . . interferons at age : past, current and future impact on biomedicine type interferons and the virus-host relationship: a lesson in detente a scientific journey through the - a/rnase l system viral encounters with , -oligoadenylate synthetase and rnase l during the interferon antiviral response the dsrna protein kinase pkr: virus and cell control cloning and characterization of a rnase l inhibitor. a new component of the interferonregulated - a pathway small self-rna generated by rnase l amplifies antiviral innate immunity interferon action and apoptosis are defective in mice devoid of , -oligoadenylate-dependent rnase l rnase l and double-stranded rna-dependent protein kinase exert complementary roles in islet cell defense during coxsackievirus infection rnase l plays a role in the antiviral response to west nile virus pkr and rnase l contribute to protection against lethal west nile virus infection by controlling early viral spread in the periphery and replication in neurons type i interferons are essential in controlling neurotropic coronavirus infection irrespective of functional cd t cells interferon and cytokine responses to sars-coronavirus infection control of coronavirus infection through plasmacytoid dendritic cell-derived type i interferon type i ifn-mediated protection of macrophages and dendritic cells secures control of murine coronavirus infection viral induction of central nervous system innate immune responses murine coronavirus mouse hepatitis virus is recognized by mda and induces type i interferon in brain macrophages/microglia mouse hepatitis coronavirus a nucleocapsid protein is a type i interferon antagonist a , -oligoadenylate analogue inhibits murine hepatitis virus strain (mhv- ) replication in vitro but does not reduce mhv- -related mortality or induction of procoagulant activity in susceptible mice coronavirus infection of the central nervous system: host-virus stand-off murine coronavirus infection: a paradigm for virus-induced demyelinating disease skin allograft rejection is suppressed in mice lacking the antiviral enzyme, , -oligoadenylate-dependent rnase l inverted immunodominance and impaired cytolytic function of cd + t cells during viral persistence in the central nervous system sequential infection of glial cells by the murine hepatitis virus jhm strain (mhv- ) leads to a characteristic distribution of demyelination diverse functions of rnase l and implications in pathology ctl effector function within the central nervous system requires cd + t cells inhibition of interferon-gamma signaling in oligodendroglia delays coronavirus clearance without altering demyelination differential induction of apoptosis in demyelinating and nondemyelinating infection by mouse hepatitis virus contrasting roles for axonal degeneration in an autoimmune versus viral model of multiple sclerosis: when can axonal injury be beneficial? absence of perforin expression confers axonal protection despite demyelination role of ifn-gamma responsiveness in cd tcell-mediated viral clearance and demyelination in coronavirus-infected mice axonal damage is t cell mediated and occurs concomitantly with demyelination in mice infected with a neurotropic coronavirus demyelinating and nondemyelinating strains of mouse hepatitis virus differ in their neural cell tropism evidence for synaptic stripping by cortical microglia microglia overexpressing the macrophage colony-stimulating factor receptor are neuroprotective in a microglial-hippocampal organotypic coculture system enhanced cell-to-cell contacts between activated microglia and pyramidal cell dendrites following kainic acid-induced neurotoxicity in the hippocampus disruption of neuronal autophagy by infected microglia results in neurodegeneration interferon action: rna cleavage pattern of a ( - )oligoadenylate-dependent endonuclease interferon actionsequence specificity of the ppp(a p)na-dependent ribonuclease rnase l: its biological roles and regulation interferon-beta suppresses herpes simplex virus type replication in trigeminal ganglion cells through an rnase l-dependent pathway memory cd + t-cell-mediated protection from lethal coronavirus encephalomyelitis microglia as neuroprotective, immunocompetent cells of the cns molecular and cellular immune mediators of neuroprotection multiple sclerosis: novel perspectives on newly forming lesions experimental models of neuroprotection relevant to multiple sclerosis the role of macrophage/microglia and astrocytes in the pathogenesis of three neurologic disorders: hiv-associated dementia, alzheimer disease, and multiple sclerosis pathogenicity of antigenic variants of murine coronavirus jhm selected with monoclonal antibodies induction of class i antigen processing components in oligodendroglia and microglia during viral encephalomyelitis perforin-mediated effector function within the central nervous system requires ifn-gamma-mediated mhc up-regulation the authors thank dr. bruce trapp for helpful discussions and comments on the manuscript. we also thank wen wei and ernesto barron for excellent assistance with the histopathology and jennifer powers for cell purification by facs. conceived and designed the experiments: ddci sas ccb. performed the experiments: ddci pk. analyzed the data: ddci sas drh pk rhs raa ccb. contributed reagents/materials/analysis tools: ccb. wrote the paper: ddci sas drh rhs ccb. key: cord- - bbqz o authors: beitzel, brett f.; bakken, russell r.; smith, jeffrey m.; schmaljohn, connie s. title: high-resolution functional mapping of the venezuelan equine encephalitis virus genome by insertional mutagenesis and massively parallel sequencing date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: bbqz o we have developed a high-resolution genomic mapping technique that combines transposon-mediated insertional mutagenesis with either capillary electrophoresis or massively parallel sequencing to identify functionally important regions of the venezuelan equine encephalitis virus (veev) genome. we initially used a capillary electrophoresis method to gain insight into the role of the veev nonstructural protein (nsp ) in viral replication. we identified several regions in nsp that are intolerant to small ( bp) insertions, and thus are presumably functionally important. we also identified nine separate regions in nsp that will tolerate small insertions at low temperatures ( °c), but not at higher temperatures ( °c, and °c). because we found this method to be extremely effective at identifying temperature sensitive (ts) mutations, but limited by capillary electrophoresis capacity, we replaced the capillary electrophoresis with massively parallel sequencing and used the improved method to generate a functional map of the entire veev genome. we identified several hundred potential ts mutations throughout the genome and we validated several of the mutations in nsp , nsp , e , e , e and capsid using single-cycle growth curve experiments with virus generated through reverse genetics. we further demonstrated that two of the nsp ts mutants were attenuated for virulence in mice but could elicit protective immunity against challenge with wild-type veev. the recombinant ts mutants will be valuable tools for further studies of veev replication and virulence. moreover, the method that we developed is applicable for generating such tools for any virus with a robust reverse genetics system. alphavirus endemic to regions of south america. normally maintained in a rodent reservoir, veev can be transmitted by mosquitoes to horses and humans where it can cause debilitating and potentially fatal encephalitis. there are currently no vaccines for vee licensed for use in humans. alphaviruses contain an approximately - kb single-strand, capped and polyadenylated positive-sense rna genome. the two-thirds of the genome encode the non-structural proteins; nsp , nsp , nsp , and nsp , which are involved in genome replication and transcription. the one-third of the genome encodes the structural proteins; capsid, e , e , k, and e . much of what is known about the functions of alphavirus nonstructural proteins has been elucidated through molecular and classical genetics studies of two prototypical alphaviruses: sindbis virus (sinv), and semliki forest virus (sfv) (reviewed in [ , , , ] ). the non-structural proteins are initially translated as two polyproteins, p and p . in sinv, and several other alphaviruses including veev, the major non-structural polyprotein, p , is produced by translation termination at an opal codon at the end of nsp . occasional read-through of the opal termination codon produces p . cleavage of p in cis by a protease activity that resides in nsp generates a complex of p and nsp that can initiate minus-strand rna synthesis. p is cleaved into nsp , nsp , and nsp , and these fully cleaved forms generate a complex with cellular proteins, replicate the full-length viral genome from minus-strand templates, and transcribe the subgenomic rna encoding the viral structural proteins. the enzymatic activities and functional roles of nsp , nsp , and nsp have been partially characterized. nsp has methyltransferase and guanylyltransferase activity [ , , , , ] , required for capping rna, and is necessary for synthesis of minus-strand rna [ ] . nsp has multiple functions in viral replication. it has rna helicase activity [ ] and ntpase activity [ , ] , and the c-terminus of nsp functions as a cysteine protease that cleaves the non-structural polyproteins p and p [ , , , ] . nsp has been found to enter cell nuclei and to be an inhibitor of transcription of cellular messenger and ribosomal rnas including those involved in innate immune responses [ , , ] nsp is the rna dependent rna polymerase [ , , , ] . nsp is the least characterized of the alphavirus nonstructural proteins. studies with sinv showed that nsp is essential for both minus-strand and plus-strand synthesis, but the precise role that it plays in these activities is unknown [ , , ] . the protein consists of two domains, an n-terminal macrodomain that is highly conserved among alphaviruses and a hypervariable, phosphorylated c-terminal domain. macro domains (also called x domains) are found in proteins from bacteria, archea, and eukaryotes, as well as several viruses including alphaviruses, coronaviruses, hepatitis e virus, and rubella virus [ , , , ] . they bind adp-ribose and poly(adp-ribose) and exhibit adpribose -phosphatase activity. the crystal structure of the veev macrodomain was recently solved, revealing a conserved adenosine binding pocket [ ] . nsp is the only alphavirus non-structural protein that is phosphorylated [ ] , with most or all of the phosphorylation on serine and threonine residues in the cterminus [ , ] . the studies that we report here were initiated to gain insight into the function of veev nsp . toward this goal, we used transposon mutagenesis, reverse genetics, and fragment analysis by capillary electrophoresis to identify regions of the nsp gene that are important for replication and that result in temperature sensitive (ts) mutations. although this work demonstrated the utility of using insertional mutagenesis for identifying such regions, the method was limited by the inherently low capacity of fragment analysis by capillary electrophoresis. consequently, we developed a novel high-resolution functional mapping technique that couples transposon insertional mutagenesis with tag sequencing on a next-generation sequencing platform. we used transposon mutagenesis to construct a cdna library with small dna fragments randomly inserted throughout the veev genome and then produced replication-competent virus through reverse genetics. comparing transposon insertion sites in the resultant viruses to those in the starting library, we were able to produce a functional map of the entire genome of veev, and to identify several hundred potential ts mutations, including those we originally identified with the capillary electrophoresis method. we further validated the mutations in cell culture assays of recombinant viruses generated through reverse genetics and tested two of the nsp ts mutants in mice to see if they could act as attenuated vaccines. these studies provide both new information about the association of nsp gene regions with virulence, as well as a rapid and effective method for identifying and creating new ts mutants for replication studies. we used a multistep transposon mutagenesis process to generate a library of full-length veev clones with single insertions in nsp ( figure ). our final library consisted of . full-length clones of the veev genome with bp insertions in nsp and had an average complexity of approximately -fold coverage at every nucleotide position. the bp insertions consist of bp derived from a modified mua transposon and bp derived from duplication of the transposition target site. the sequence derived from the transposon contains a unique noti restriction site that allows for mapping the insert location in the veev genome. we produced virus pools from the insert library by using standard alphavirus reverse genetics, as depicted in figure s . infectious rna was produced by in vitro transcription of the insert library, and was transfected into bhk cells. the transfected bhk cells were then incubated at uc to generate virus particles. virus was collected from the supernatant of the transfected cells and used to infect fresh vero cells at an moi of . . the low moi infection was intended to prevent trans-complementation caused by coinfecting mutant viruses, which could confound our downstream analyses. infected cell cultures were incubated at either uc, uc, or uc to determine if there would be a temperaturedependent difference in the functional maps of viruses produced at these temperatures. rna from our starting unselected pool, and rnas isolated from the uc, uc, and uc infection supernatants were reverse transcribed and used as templates for pcr amplification using primer combinations (tables s and s ) that generated amplicons of approximately bp long with a fluorescent fam label on the end, and a biotin tag on the end. the amplicons were bound to streptavidin-coated magnetic beads, and then digested with noti to release any fragments containing a noti site derived from a transposon insertion. the sizes of the released fragments were analyzed by capillary electrophoresis on a prism xl genetic analyzer. we included a fam-labeled sequencing ladder of each amplicon to accurately size fragments. to analyze results, we generated electropherograms ( figure ) in which the x-axis indicates the size of the dna fragment (the transposon insertion position), and the y-axis indicates the fluorescence intensity (the number of transposon insertions at that position). examination of the electropherograms from the starting, unselected library showed that it contained insertions after approximately - % of the nucleotides in nsp . the observation that less than half of the nucleotide positions in nsp had an insertion appeared to be due to a bias in mua transposition, and not inadequate coverage in the library, as a second, independent insertion library gave a virtually identical insertion pattern (data not shown). comparison of the library electropherograms to those derived from viral genomes clearly revealed regions in nsp that would and would not tolerate bp insertions. as expected, most regions of the highly conserved end of nsp would not tolerate insertions. only one region in the end, from nucleotides to (relative to the genome of the trinidad donkey strain of veev, genbank accession number l ), would tolerate insertions at all temperatures (region , figures a and b) . in contrast, the hypervariable end of nsp tolerated insertions at most positions. from nt (near the start of the hypervariable region indicated in figure c ) to nt , there was no discernable difference in the vrna electropherograms compared to the unselected library rna electropherogram. however, we did find two regions in the end, one from venezuelan equine encephalitis virus (veev) is a new world alphavirus that was first identified in venezuela in . veev normally circulates in rodent populations, but during outbreaks it can jump to horses and humans where it can cause debilitating and potentially fatal disease. there are currently no vaccines or antiviral agents against veev licensed for use in humans. in this study, we describe a technique that we have developed that allows for the rapid identification of viral mutants that can be useful for studying the basic biology of viral replication. these mutants can also be used to generate vaccines that protect against infection with wild-type virus. we demonstrate the utility of this technique by identifying over mutations spread throughout veev genome that make the virus unable to replicate efficiently at higher temperatures ( uc or uc.) furthermore, we show that two of the mutant viruses work as vaccines, and protect mice against lethal infection with veev. this technique can be applied to studying other viruses, and may allow for the rapid identification of numerous vaccine candidates. nucleotide positions to (region ), and the other from to (region ) that were intolerant of insertions. we were also able to detect different sites in the end of the nsp gene that would tolerate insertions at uc, but not at uc or uc (summarized in figure f ). the degree of impairment of viral replication varied from site to site. some of the sites allowed reduced replication at uc (i.e., detectable but reduced electropherogram peaks relative to the uc peaks), while others had no detectable replication at uc (e.g., compare figure for whole-genome analysis by massively parallel tag sequencing, it was necessary to develop a modified sequencing library preparation protocol based on the roche gs-flx protocol such that the libraries had the sequencing adapter (adapter a) ligated onto the not i sites that were present in the transposon insertions. a schematic of the protocol used for preparing the sequencing libraries is shown in figure s . each prepared sequencing library ( uc virus, uc virus, and unselected in vitro transcribed rna) was assessed on a single large region of a gs-flx picotiter plate. we obtained , sequencing reads from the uc library, , reads from the uc library, and , reads from the unselected control rna library. all analyses were normalized to account for the different number of sequences obtained from each sample. sequencing reads arising from a transposon insertion should have a characteristic sequence tag at the end; thus, we analyzed the bulk sequences obtained for the presence of the correct tag, and those sequence reads lacking the tag were excluded from further analysis. we used the first nucleotides of each sequencing read to map the insert location on the veev genome. because the veev genome is relatively small (, . kb), bp was sufficient to uniquely identify the insert locations. the total number of insertions recorded at each nucleotide position in the genome was tallied and used to build a histogram of insertion frequencies across the entire genome (data set s , and figure - ) . as expected, the histogram of the unselected rna sample showed insert locations spread throughout the entire genome ( figure ). in contrast, the histogram of insert locations isolated from virus produced at uc showed several regions of the genome that did not tolerate insertions. these regions were presumably functionally important, and many of them mapped to domains with functions known to be required for viral replication, such as the nsp protease active site and substrate binding pocket [ ] , and the nsp / protease cleavage site. although the histograms of the uc virus and the uc virus appeared quite similar, there were many locations within the genome where we detected insertions at uc, but not at uc (figure ), including those we identified in nsp using capillary electrophoresis analysis of the insertional library ( figure s ). in total, we found approximately nucleotide positions within the genome where the ratio of insertions detected at uc was greater than or equal to times the number of insertions detected at uc (data set s ). using the data obtained by comparing the histograms of insert locations in the uc and uc vrnas, we chose several locations in which insertions would be predicted to cause a ts phenotype. we constructed mutants that contained bp insertions that would mimic the insertions generated by a transposon insertion (table . ) we examined the single-cycle replication kinetics of each of these mutants at uc and uc, as compared to wild-type v . all of the mutants, with the exception of ts , replicated at rates similar to wild type at uc ( figure ). two of the mutants, ts and ts , replicated to higher titers than wild type at both h and h post infection. however, at uc, all of the mutants had titers that were reduced between -and -fold relative to v at h post infection (excluding mutant ts which had undetectable titers at h). at h post infection, most of the mutants still had titers greater than -fold lower than wild-type v , but three mutants (ts , ts - , ts - ) had titers similar to wild type. mutant ts had an extremely slow growth phenotype that made it difficult to measure. plaques were barely visible at days post infection, and took to days before accurate plaque counts could be obtained. plaques from the other ts mutants were counted on day post infection. temperature-sensitive mutants have been used extensively to elucidate replication and virulence properties of alphaviruses. to demonstrate the utility of our functional mapping method for identifying useful ts veev mutants, we further studied two nsp ts mutants, ts - and ts - for replication, virulence, and immunogenicity in mice. the insert locations in these two mutants are shown in figure . in addition to these two mutants, we constructed a mutant with both the ts - and ts - insertions (double ts). we infected groups of balb/c mice subcutaneously (s.c.) with either a low dose ( pfu) or a high dose ( pfu) of ts - , ts - , double ts, or wild-type v . a negative control group was inoculated with pbs. all of the mice receiving the high-dose v had to be euthanized when moribund on day , while of mice receiving the low-dose v inoculation were euthanized when moribund by day . all of the mice that received either high doses or low doses of any of the three ts mutants survived without signs of disease, as did the negative control group. as a verification that the ts mutants had actually infected the mice, we assayed their sera for the presence of neutralizing antibodies days after infection. one mouse each in the low dose v , ts - , and ts - groups had no detectable neutralizing antibodies, but all others did ( table and table s ). neutralizing antibodies are a correlate of protective immunity to veev; thus we were interested in determining if the ts mutants were able to serve as protective, live-attenuated vaccines. consequently, we challenged all of the surviving mice with pfu of veev, strain trinidad donkey, days after their initial infection with the ts mutants ( figure ). all of the negative control mice that had received pbs in the initial inoculation were euthanized when moribund on day post challenge. one of the two mice that had survived the low-dose v inoculation, as well as one mouse each in the low-dose ts - and ts - groups were euthanized when moribund on day post challenge. all of the remaining mice survived for an additional days post challenge without displaying disease symptoms. the analysis of ts mutants has been used extensively to study alphavirus replication, and has helped to identify the activities and interactions of many viral proteins [ , , , , , , , , , ] . we originally planned to use functional mapping to gain insight into the role(s) that nsp plays during viral replication simply by mapping domains that would and would not tolerate short insertions. however, we found that by performing functional mapping on virus pools that had been produced at different temperatures, we could identify large numbers of ts mutants, which could be used for more in depth studies of viral replication. our mapping of nsp identified several interesting features. not surprisingly, most of the conserved n-terminus of the protein was intolerant to short insertions while most of the variable c-terminus would tolerate insertions. two regions in the c-terminus that would not tolerate insertions spanned nucleotides - and nucleotides - . the latter region might be expected to be intolerant to insertions, as that region encodes the extreme c terminus of nsp , including the cleavage site between nsp and nsp . disruptions in this region could prevent cleavage of nsp from nsp , which is an essential step in viral replication [ ] . the other site is more enigmatic. the region from - has no obvious homology to known regions of importance, and will require additional study to determine why insertions are not tolerated. one recent study with sfv indicated that the extreme c-terminus of nsp contains a degradation signal and a signal important for its' cellular localization [ ] . the regions we have identified here might play a similar role in veev replication. in addition to these two intolerant regions, we also identified nine separate regions in the end of nsp that are ts for insertions. several of these regions mapped near ts regions identified in sinv [ , , , ] , but ts regions , , , and were in regions that were not previously identified. construction of these ts mutants through reverse genetics will be required to more fully characterize their roles in viral replication. to generate a functional map of nsp , we used fragment analysis by capillary electrophoresis to generate functional maps in a manner similar to one reported earlier [ , ] . while this technique worked relatively well for analyzing short stretches of dna or rna (i.e., up to kb), the technique had limitations that made it cumbersome for analysis of larger gene regions. for example, we could only analyze individual amplicons of approximately - bp due to the inherent loss of size resolution in larger fragments. we also needed to include a sequencing ladder in each electrophoretic analysis to obtain accurate fragment size information. because our mapping fragments had short transposon-derived sequences appended to the end, fragment sizing with the sequencing ladder was not exact. we theorized that functional mapping by using massively parallel sequencing might overcome some of these issues. in principle, the large number of sequencing reads obtained would allow us to measure the relative frequencies of insertions at each nucleotide position in the genome. massively parallel sequencing would also provide an exact sequence readout of the locations of inserts and generate highly accurate maps. lastly, mapping by for our studies, we used a roche gs-flx system, which was adequate for mapping the approximately . kb veev genome (, k- k reads per sample.) viruses with larger genomes could be mapped by using other sequencing platforms such as the illumina genome analyzer or applied biosystems solid sequencer. these platforms produce hundreds of millions of sequencing reads, and would permit the analysis of a much larger number of samples per sequencing run. therefore, they are probably the optimal systems for this type of analysis. although the length of sequencing reads obtained on these systems are shorter than those obtained on the gs-flx, the + bp reads that they generate should be enough to map insertion sites onto the relatively simple genomes of viruses. in our analysis of veev, we found that the first nts of sequence was sufficient for mapping each read back to the genome. functional mapping of the veev genome by using the massively parallel sequencing method that we developed revealed several hundred sites, spread throughout the genome in every gene, where the number of insertions detected at uc was greater than times higher than the number of sequences detected at uc. all sites that we chose for further analysis gave rise to a virus with a ts phenotype, thus it is likely that most or all of the other sites could also be reverse engineered to generate novel ts mutant viruses that could be used for functional studies of veev proteins. many of the sites fell outside of the known functional domains of veev proteins, such as the helicase and peptidase domains of nsp and the rna-dependent rna polymerase domain of nsp , and may represent additional domains with unknown functions in these proteins. elucidating the mechanisms of attenuation for these mutants will be important for identifying additional functions required for viral replication. in addition to providing new ts mutants for studying viral replication, this method can also be used to map virulence properties. for example, we demonstrated one use of functional mapping data by constructing ts mutants that acted as attenuated vaccines in a mouse model of lethal veev infection. none of the mice vaccinated with these viruses showed any signs of disease, and most were protected from challenge with wild-type veev. of the three mice that did succumb to challenge, all were in groups that were inoculated with only pfu of ts mutant virus; thus, it is possible that these mice were not infected in the first place. this is supported by the observation that one mouse in each group did not develop neutralizing antibodies after infection with the ts mutants. the utility of this technique for designing ts vaccines lies not only in the ability to detect large numbers of ts mutants, but also in the ability to multiplex those mutations to generate viruses with a potentially more stable ts phenotype. in our study, we demonstrated that we could combine two mutants in our double ts mutant and still recover viable ts virus. it might be possible to combine more than two mutations, with each additional mutation reducing the reversion potential of the final attenuated virus. finally, although we only presented functional mapping data relating to ts phenotypes, functional mapping of other phenotypes is also possible using this technique. for example, comparison of functional maps generated from rna isolated from infected cells to vrnas isolated from the supernatant of infected cells might identify mutants defective for particle formation. as an additional example, we have performed preliminary functional mapping of veev propagated in mosquito cells (c / ) to identify insertions that confer a species-specific replication phenotype, and have identified several insertions in the veev genome that appear to allow virus to replicate in vero cells but not in c / cells, and vice versa. similar studies could be performed to examine animal-, organ-, or cell-specific replication characteristics of viruses. this method should provide a powerful and new means to generate tools for studying a myriad of characteristics of any virus with a robust reverse genetics system. bhk and vero cells were cultured in eagle's minimal essential medium (emem) supplemented with % fetal bovine serum (fbs). veev strain v is derived from a molecular clone of the wild-type trinidad donkey strain of veev. for studies using only the nsp gene we used a veev reverse genetics system derived from plasmid pvee replicon . [ ] , which contains a t rna promoter driving expression of the veev utr and nsp through nsp . pv s contains the end of the veev genome, including the subgenomic promoter, the structural proteins and utr. to prevent interference with restriction digestions required for genetic analysis, noti sites in both pvee replicon . and pv s were changed to asci sites by digestion with noti followed by ligation of a linker with the sequence -ggccggcgcgcc- . this change did not interfere with virus production from the full-length genome. the modified pvee replicon . was named pbb , and the modified pv s was named pbb . a mua transposon, entranceposon m -kan r ( bp, part of the mutation generation kit from finnzymes, espoo, finland), was transposed into pbb . briefly, target dna, entranceposon m -kan r , and mua transposase were combined in transposition buffer, and the transposition was allowed to proceed for h at uc. the transposase was then inactivated by heating to uc for min. the transposition reaction was desalted on a sephadex g- spin column and transformed into dh a e. coli. a primary insertion library with full-length transposon insertions in pbb was isolated by plating the transformed cells on lb containing ampicillin and kanamycin. the mutation generation kit has been optimized so that greater than % of the clones in the library will contain a single transposon insertion. a fragment containing transposon insertions in nsp (and small fragments of nsp and nsp ) was isolated from the primary insertion library by digestion with avrii and bsiwi. this fragment was cloned into avrii-bsiwi cut pbb , yielding a secondary library that only contained fulllength transposon insertions in nsp . digestion of the secondary library with noti, followed by intramolecular ligation, generated a tertiary library consisting of clones with bp inserts spread throughout nsp . finally, the s apai-asci fragment from pbb was cloned into the apai-asci cut tertiary library, generating the final library of full-length veev genomes with bp insertions in nsp . for studies using the entire veev genome, we constructed a plasmid containing cdna representing the full-length genome of veev, strain trinidad donkey (genbank accession number l ), named pbb . pbb was generated by cloning the apai-asci fragment from pbb (containing the veev structural proteins) into apai-asci cut pbb . pbb has a t promoter immediately upstream, and a unique asci site immediately downstream of the veev genome. after linearization of the plasmid with asci, infectious viral rnas (vrna) were produced by in vitro transcription with t rna polymerase. massively parallel sequencing identified nucleotide differences from the published trinidad donkey (accession #l ) sequence as follows: nucleotide (nt) a to g, nt t to c, nt t to c, nt g to a, nt a to t, nt t to c, nt c to t, nt a to g, nt c to t, and deletion of nt t in the utr. entranceposon m -kan r was transposed into pbb and a primary insertion library generated as described above. a secondary library was prepared by digesting the primary library with asci and sbfi to release the veev genome from the plasmid backbone. size fractionation of this digest on an agarose gel allowed isolation of veev genomes containing transposon insertions, and removal of wild-type genomes lacking transposon insertions. the transposon-containing genomes were re-cloned into pbb . this secondary library was digested with noti, followed by intramolecular ligation, to produce a tertiary library consisting of clones with bp inserts spread throughout the veev genome. at least -fold coverage of the veev genome was maintained in the library throughout the cloning process. recombinant veev was produced essentially as described previously [ , ] . briefly, the nsp only or full genome insertion libraries were linearized by asci digestion, and transcribed in vitro using t rna polymerase (ribomax, promega, madison, wi). this rna was introduced into bhk cells by electroporation and the cells were then propagated in emem + % fbs at uc until noticeable cytopathic effects (cpe) were seen (approximately days). cell culture supernatant was centrifuged to remove cellular debris, aliquoted, and frozen. the titer of the bhk-produced virus was determined by plaque assay on vero cells. in a second round of infection, vero cells were infected at a multiplicity of infection (moi) of . (low moi) with bhk-produced virus. cells were incubated at uc, uc or uc (for the nsp library), or uc or uc (for the full genome library) and supernatant was collected after extensive cpe was observed. virus from ml of supernatant from the low moi infections was concentrated by ultracentrifugation. vrna was isolated from the viral pellets by trizol-ls extraction (invitrogen, calsbad, ca). in vitro-transcribed rna that had not been used to produce virus was used as the unselected pool, and was processed in parallel with vrnas. for capillary electrophoresis fragment analysis, rnas were reverse transcribed with random hexamer primers, and the resulting cdna was amplified by pcr in ml reactions (platinum pcr supermix high fidelity, invitrogen) using the following combinations of primers (table s ) : bbu + bbu , bbu + bbu , bbu + bbu , bbu + bbu , bbu + bbu . in all cases, the forward primer was labeled with -fam to facilitate detection and the reverse primer was labeled with biotin for subsequent purification steps. the amplicons were designed to overlap so that regions that would be obscured near the ends of one amplicon could be analyzed in the overlapping amplicons. for massively parallel tag sequencing, rna was processed with the firstchoice rlm-race kit (applied biosystems/ambion, austin, tx) to add a -race adapter to the vrnas, and reverse transcription was performed using a mixture of veev-specific primers and a -race adapter. the resulting cdnas were amplified by pcr, generating six overlapping amplicons (table s ). the amplicons spanned the entire genome, and were designed to overlap so that primer binding sites that would be obscured at the ends of each amplicon could be analyzed in the overlapping amplicons. the primers used for pcr added asci restriction sites to both ends of each amplicon to facilitate sequencing library preparation. products from the rt-pcr reactions were processed with a pcr purification kit (edge biosystems, gaithersburg, md), to remove remaining primers, enzymes, and free nucleotides. samples were resuspended in restriction endonuclease buffer (neb, beverly, ma) and bound to streptavidin-coated magnetic beads (invitrogen) for h at uc. after binding, beads were washed three times with ml of restriction endonuclease buffer, and were resuspended in ml of restriction buffer containing units of noti restriction enzyme (neb). digests were incubated at uc for h. after digestion, bead eluates were desalted on a sepharose filtration spin column and dried. dried samples were resuspended in ml of deionized formamide with a rox-labeled size standard (geneflo , chimerx, milwaukee, wi). samples were denatured at uc for min, and then loaded onto a prism xl genetic analyzer (applied biosystems, foster city, ca) for capillary electrophoresis. fam-labeled dideoxy sequencing ladders of each amplicon were run in parallel to allow for accurate fragment sizing. settings for the run were the same as the default settings for fragment analysis on a cm pop- array, except that the run time was changed from s to s. data from the capillary electrophoresis runs were analyzed with genemapper software version . (applied biosystems) using the local southern setting for peak sizing. amplicons that had been generated by rt-pcr were mixed in an equimolar ratio to a final mass of mg. the mixture was digested with asci to generate ligatable ends on each amplicon. the asci was heat inactivated, and the digest mixture was purified over sepharose to remove the small end fragments generated by asci digestion. the purified amplicons were ethanol precipitated and resuspended in a ml ligation mix including t dna ligase and t polynucleotide kinase, and the ligation was incubated overnight at uc. the product of this ligation was a collection of high molecular weight dnas (hmwdna) with sizes greater than approximately kb as visualized on an ethidium bromide stained agarose gel. the hmwdnas were processed with a gs-flx library preparation kit (roche) with a few modifications to the kit protocol. as per the protocol, hmwdnas were nebulized to generate small fragments of double stranded dna approximately - bp in size. the ends of these fragments were polished with t dna polymerase and t polynucleotide kinase to produce blunt, phosphorylated ends. in a departure from the kit protocol, sequencing adapter ''b'' was ligated to both ends of the dna fragments. the fragments were then digested with not i (which cuts in the transposon insertions), and a modified sequencing adapter ''a'' was ligated onto the exposed not i ends. at this point, we returned to the library preparation kit protocol to finish preparing the sequencing libraries for each sample. the final product for each was approximately ng of sequencing library. sequencing out from the ''a'' adapter present on each dna fragment allowed us to identify the locations of the transposon insertions in each sample. the sequencing libraries were amplified onto sequencing beads using the roche empcr kit ii. this kit produces templates for sequencing reactions starting from the ''a'' adapters. after cleanup of the empcr reactions, sequencing beads were loaded onto a picotiter plate and sequenced on the gs-flx. sequences from one large region of a picotiter plate were obtained for each sample. the sequences obtained (between , and , per sample) were processed by a short perl script to identify those that contained a sequence tag indicative of a ''true'' sequence from a transposon insertion event. the sequence of these tags was a(r)*cggccgca. the first a was from the modified ''a'' sequencing adapter. the (r)* came from a multiplex identifier (mid) that was included at the end of the modified ''a'' adapter. the mid consisted of either four a or four g residues. the cggccgc was the residual not i site, and the final a came from the end of the transposon insertion. approximately % of the sequences from each sample contained this sequence tag. the sequence of the first bases after the tag was blasted to the veev genome to identify the location of the insertion [ ] . to be included in the final analysis, each blast result had to match the veev genome % over the base sequence. approximately % of the tag-containing sequences were successfully mapped to the veev genome using these criteria. the insertion sites identified by blast were tallied to build a histogram of insertion frequencies across the veev genome. construction of temperature sensitive mutants veev genomes carrying bp insertions that mimicked a transposon insertion were constructed by using a quikchange xl kit (stratagene, la jolla, ca.) mutations were first constructed in plasmids containing subgenomic fragments. the mutations were confirmed by sequencing, and then transferred into a full-length genomic plasmid (pbb ) by restriction digestion and cloning. mutant viruses were prepared from these full-length clones as described above. vero cells plated in -well dishes were infected in duplicate with wild-type and mutant viruses at an moi of . after binding the virus for h at the appropriate temperature ( uc or uc,) the wells were washed once with pbs, and fresh medium was added. a sample was taken immediately after adding the fresh medium, and represented the h time point. infected cells were then cultured at either uc or uc, and additional samples were withdrawn from the supernatant at h and h post infection. all samples were frozen at uc prior to plaque assay. groups of -to -week-old balb/c mice were inoculated s.c. with either pfu or pfu of ts mutant viruses. control groups received or pfu of v or pbs. mice were weighed and examined daily for signs of disease, and euthanized when moribund. twenty-eight days after the initial inoculation with the veev ts mutants, surviving mice were challenged s.c. with pfu of veev, strain trinidad donkey. mice were weighed and examined daily for signs of disease, and euthanized when moribund. sera from vaccinated mice were serially diluted in hank's balanced salt solution (hbss) containing % fbs. serum dilutions were incubated overnight at uc with pfu of veev trinidad donkey. after incubation, the mixtures were assayed for plaque formation on vero cells. the % plaque reduction neutralization titer (prnt ) was calculated as the serum dilution at which plaque formation was reduced by % relative to a control that was incubated in the absence of serum. figure s transposon mutagenesis and alphavirus reverse genetics. a graphical representation of the protocol described in the text. a plasmid containing the entire veev genome (pbb ) was subjected to insertional mutagenesis by transposition of a modified mua transposon (entranceposon m -kan r shown in the inset.) removal of the bulk of the transposon by noti digestion followed by intramolecular ligation leaves a library of clones, each containing a bp insertion at an essentially random location in the genome (blue xs shown in the bp insert library). each insertion contains a unique noti site that can be used to map the insert location. the library is transcribed in vitro to produce infectious virus-like rnas. these rnas are transfected into cells, yielding recombinant viruses after - hours. found at: doi: . /journal.ppat. .s ( . mb pdf) figure s processing of vrnas for massively parallel sequencing. a graphical representation of the protocol described in the text. viral genomic rnas (vrnas) are isolated from the supernatant of infected cell cultures. race and rt-pcr are used to generate amplicons that span the entire viral genome. the amplicons are mixed in an equimolar ratio, and digested with asci (added by the primer during pcr) to generate phosphorylated ends. this mix is then ligated into a random jumble of high molecular weight dna (hmwdna) to generate a starting material for sequencing library preparation. the hmwdna is nebulized into fragments - bp in size, and the ends are polished to generate blunt, phosphorylated ends. sequencing adapter ''b'' (shown in green) from the roche library preparation kit is ligated onto both ends of the polished dnas. this mixture is then digested with noti to expose the ends of the transposon insertions, and a biotinylated modified adapter ''a'' (shown in blue) is ligated onto the exposed noti ends. the mix is bound to streptavidin coated magnetic beads and washed to remove any fragments lacking the biotin tag. templates for gs-flx sequencing are eluted from the magnetic beads as single stranded dna. table s serum neutralizing titers for individual mice vaccinated with veev ts strains. % plaque reduction neutralization titers (prnt ) of individual animals after inoculation with veev ts mutants. titers registered as , indicate that no neutralization activity was seen at the lowest serum dilution ( : ) . titers registered as . indicate that plaque numbers were reduced . % (compared to a no-serum control) at the highest dilution used in the assay ( : ). found at: doi: . /journal.ppat. .s ( . mb pdf) data set s compiled sequencing data. sequence reads obtained from gs-flx sequencing were processed as described in the text. columns f-h indicate the raw number of sequence reads detected at each nucleotide position in the veev genome. the alphaviruses: gene expression, replication, and evolution functions of alphavirus nonstructural proteins in rna replication alphavirus positive and negative strand rna synthesis and the role of polyproteins in formation of viral replication complexes fields virology putative rna capping activities encoded by brome mosaic virus: methylation and covalent binding of guanylate by replicase protein a critical residues of semliki forest virus rna capping enzyme involved in methyltransferase and guanylyltransferase-like activities reaction in alphavirus mrna capping: formation of a covalent complex of nonstructural protein nsp with -methyl-gmp association of the sindbis virus rna methyltransferase activity with the nonstructural protein nsp expression of sindbis virus nsp and methyltransferase activity in escherichia coli functional analysis of the a complementation group mutants of sindbis hr virus rna helicase activity of semliki forest virus replicase protein nsp identification of a novel function of the alphavirus capping apparatus. rna -triphosphatase activity of nsp atpase and gtpase activities associated with semliki forest virus nonstructural protein nsp characterization of the cysteine protease domain of semliki forest virus replicase protein nsp by in vitro mutagenesis evidence that sindbis virus nsp is an autoprotease which processes the virus nonstructural polyprotein processing the nonstructural polyproteins of sindbis virus: nonstructural proteinase is in the c-terminal half of nsp and functions both in cis and in trans identification of the active site residues in the nsp proteinase of sindbis virus roles of nonstructural protein nsp and alpha/beta interferons in determining the outcome of sindbis virus infection sindbis virus nonstructural protein nsp is cytotoxic and inhibits cellular transcription nuclear localization of semliki forest virus-specific nonstructural protein nsp mapping of rna-temperature-sensitive mutants of sindbis virus: complementation group f mutants have lesions in nsp evolution and taxonomy of positive-strand rna viruses: implications of comparative analysis of amino acid sequences analysis of rna-dependent rna polymerase structure and function as guided by known polymerase structures and computer predictions of secondary structure primary structural comparison of rna-dependent polymerases from plant, animal and bacterial viruses genetic analysis of the nsp region of sindbis virus: evidence for roles in minus-strand and subgenomic rna synthesis alphavirus nsp functions to form replication complexes transcribing negative-strand rna regulation of sindbis virus rna replication: uncleaved p and nsp function in minus-strand rna synthesis, whereas cleaved products from p are required for efficient plus-strand rna synthesis structural basis of severe acute respiratory syndrome coronavirus adp-ribose- -phosphate dephosphorylation by a conserved domain of nsp structural and functional basis for adp-ribose and poly(adp-ribose) binding by viral macro domains expression, purification and crystallization of the sars-cov macro domain adp-ribose- -monophosphatase: a conserved coronavirus enzyme that is dispensable for viral replication in tissue culture the crystal structures of chikungunya and venezuelan equine encephalitis virus nsp macro domains define a conserved adenosine binding pocket phosphorylation of sindbis virus nsp in vivo and in vitro phosphorylation site analysis of semliki forest virus nonstructural protein deletion and duplication mutations in the c-terminal nonconserved region of sindbis virus nsp : effects on phosphorylation and on virus replication in vertebrate and invertebrate cells the crystal structure of the venezuelan equine encephalitis alphavirus nsp protease demonstration in vitro of temperature-sensitive elongation of rna in sindbis virus mutant ts sequence analysis of three sindbis virus mutants temperature-sensitive in the capsid protein autoprotease mapping of rna-temperaturesensitive mutants of sindbis virus: assignment of complementation groups a, b, and g to nonstructural proteins synthesis and processing of the nonstructural polyproteins of several temperature-sensitive mutants of sindbis virus functional defects of rna-negative temperature-sensitive mutants of sindbis and semliki forest viruses temperature sensitive shut-off of alphavirus minus strand rna synthesis maps to a nonstructural protein a second nonstructural protein functions in the regulation of alphavirus negative-strand rna synthesis mutants of sindbis virus. i. isolation and partial characterization of new temperature-sensitive mutants roles of nonstructural polyproteins and cleavage products in regulating sindbis virus rna replication and transcription novel functions of the alphavirus nonstructural protein nsp c-terminal region functional analysis of nsp phosphoprotein mutants of sindbis virus high-resolution functional profiling of hepatitis c virus genome highresolution functional profiling of a gammaherpesvirus rta locus in the context of the viral genome replicon-helper systems from attenuated venezuelan equine encephalitis virus: expression of heterologous genes in vitro and immunization against heterologous pathogens in vivo specific restrictions in the progression of venezuelan equine encephalitis virus-induced disease resulting from single amino acid changes in the glycoproteins gapped blast and psi-blast: a new generation of protein database search programs the mouse research protocol was approved by the us army medical research institute of infectious disease institutional animal care and use committee in compliance with the animal welfare act and other federal statutes and regulations relating to animals and experiments involving animals and adheres to principles stated in the guide for the care and use of laboratory animals, national research council, . the facility where the research was conducted is fully accredited by the association for assessment and accreditation of laboratory animal care, international. opinions, interpretations, conclusions, and recommendations are those of the author and are not necessarily endorsed by the u.s. army.we wish to thank tim read, kim bishop-lilly, and kristin willner for their assistance with gs-flx sequencing. we also wish to thank mike parker and catherine luke for their helpful comments. conceived and designed the experiments: bfb css. performed the experiments: bfb rrb jms. analyzed the data: bfb. contributed reagents/materials/analysis tools: bfb. wrote the paper: bfb css. key: cord- - lyt gfq authors: griffiths, samantha j.; koegl, manfred; boutell, chris; zenner, helen l.; crump, colin m.; pica, francesca; gonzalez, orland; friedel, caroline c.; barry, gerald; martin, kim; craigon, marie h.; chen, rui; kaza, lakshmi n.; fossum, even; fazakerley, john k.; efstathiou, stacey; volpi, antonio; zimmer, ralf; ghazal, peter; haas, jürgen title: a systematic analysis of host factors reveals a med -interferon-λ regulatory axis against herpes simplex virus type replication date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: lyt gfq herpes simplex virus type (hsv- ) is a neurotropic virus causing vesicular oral or genital skin lesions, meningitis and other diseases particularly harmful in immunocompromised individuals. to comprehensively investigate the complex interaction between hsv- and its host we combined two genome-scale screens for host factors (hfs) involved in virus replication. a yeast two-hybrid screen for protein interactions and a rna interference (rnai) screen with a druggable genome small interfering rna (sirna) library confirmed existing and identified novel hfs which functionally influence hsv- infection. bioinformatic analyses found the hfs were enriched for several pathways and multi-protein complexes. of particular interest was the identification of med as a strongly anti-viral component of the largely pro-viral mediator complex, which links specific transcription factors to rna polymerase ii. the anti-viral effect of med on hsv- replication was confirmed in gain-of-function gene overexpression experiments, and this inhibitory effect was specific to hsv- , as a range of other viruses including vaccinia virus and semliki forest virus were unaffected by med depletion. we found med significantly upregulated expression of the type iii interferon family (ifn-λ) at the mrna and protein level by directly interacting with the transcription factor irf . the synergistic effect of med and irf on ifn-λ induction suggests this is the major transcription factor for ifn-λ expression. genotypic analysis of patients suffering recurrent orofacial hsv- outbreaks, previously shown to be deficient in ifn-λ secretion, found a significant correlation with a single nucleotide polymorphism in the ifn-λ (il b) promoter strongly linked to hepatitis c disease and treatment outcome. this paper describes a link between med and ifn-λ, provides evidence for the crucial role of ifn-λ in hsv- immune control, and highlights the power of integrative genome-scale approaches to identify hfs critical for disease progression and outcome. up to % of the global population is infected with the aherpesvirus herpes simplex virus type i (hsv- ). whilst hsv- is largely responsible for outbreaks of vesicular oral skin lesions (fever blisters, or cold sores), it can also cause a variety of more severe diseases including encephalitis, meningitis and keratitis [ , ] . furthermore, the frequency of association with genital lesions (previously associated mainly with hsv- infection) is increasing. as co-infection with hsv is a significant contributing factor to transmission of the human immunodeficiency virus (hiv), our understanding of hsv disease, and herpesviruses in general, has wide implications for global healthcare. like all herpesviruses, hsv- establishes lytic (epithelial cells) and asymptomatic latent infection (sensory neurons in trigeminal and sacral ganglia) which undergoes periodic reactivation [ ] . the equilibrium between these two infection states requires a fine balance between innate and adaptive immune responses, and viral immune evasion mechanisms [ ] . whilst aspects of the hsv- replication cycle have been intensively investigated, there remain gaps in our understanding of the complexity of virus:host interactions. for example, a proteomics study identified over changes in the cellular proteome within the first h of infection with hsv- [ ] , and a recent analysis of virionincorporated cellular proteins found that about % of these directly affected virus growth [ ] . to systematically identify host factors (hfs) required for viral replication, rnai screens have been performed with a range of different rna and dna viruses including hiv- [ , , ] , influenza a virus [ , , ] , hepatitis c virus [ ] , west nile virus [ ] , dengue virus [ ] , enterovirus [ ] and vaccinia virus [ , ] . the overlap between the results of these studies is generally very low [ ] , reflecting either differences in biology, or different experimental set-ups, cutoff and selection criteria. in addition, microenvironmental effects might also play a role for the differences of the results [ ] . whilst loss-of-function sirna screens provide functional information on specific genes, protein interaction studies can provide insight into the mechanism of action by identifying physical interaction partners between pathogen and host. genome-scale virus-host protein interaction screens using the yeast-two-hybrid system have been performed for hcv [ ] , influenza a virus [ ] , epstein barr virus (ebv) [ ] , vaccinia virus [ , ] , sars coronavirus [ ] and several non-human viruses [ ] . based on these genome-scale studies and individual interactions found by literature curation, several virus-host interaction databases have been created including the hiv- , human protein interaction database at ncbi [ ] , virhostnet [ ] , virusmint [ ] , pig [ ] and hpidb [ ] . although there is little overlap between individual cellular interactors of different viruses, targeting of a number of cellular processes such as cell cycle regulation, nuclear transport and immune response appears to be conserved [ ] . understanding the complex interplay between viral and host components is critical to the definition of herpesvirus infection and pathogenesis. as herpesviruses encode a large number of proteins, in contrast to small rna viruses such as hiv and influenza, many cellular processes may be directly affected by viral proteins, and whilst there exists a wealth of information on individual viral proteins, there remain large gaps in our understanding of the hsv- life cycle and its interaction with its host. here, we present data from the first integrative and systematic screening approach to characterise the role of cellular proteins in the hsv- life cycle. a genome-scale rnai knockdown screen to identify hfs functionally influencing hsv- replication was performed in parallel with a yeast two-hybrid (y h) protein interaction screen to simultaneously gain insight into potential mechanisms of action. combined analyses confirmed the importance of known cellular proteins involved in processes such as cell cycle, proteins transport and gene expression important for virus replication. furthermore, we identified a subunit of the mediator multi-protein complex, med , as a key regulator of ifn-l induction, which appears to be of crucial significance for the control of hsv- both in vitro and in vivo. these data demonstrate the power of a combined screening strategy to investigate pathogen:host interactions and identify novel host factors and cellular pathway targets for the development of essential clinical interventions. host factors (hfs) which positively or negatively regulate hsv- replication were identified by screening a druggable genome sirna library ( sirnas per gene) targeting , human genes against a hsv- reporter virus expressing the enhanced green fluorescent protein (egfp; hsv- strain c ) in the epithelial hela cell line, due to their ease of transfection and susceptibility to hsv- infection [ ] . to generate a robust and reliable dataset the screen was carried out three times in triplicate, with one replicate used in a cell viability assay to determine any cytotoxic effects of gene depletion and duplicates infected for the virus infection assay. the sirna library was reversetransfected into hela cells before infecting with hsv- and monitoring virus growth kinetics as a measure of gfp-fluorescence (figure b). by following virus growth over multiple rounds of replication, host proteins involved in all stages of the virus life cycle can be identified. replication slopes during linear growth were normalized to controls (mock-transfected cells, and cells transfected with a sirna unable to be processed by the rna silencing complex, rscf) and the mean of six replicates was calculated. sirnas found to be cytotoxic ( in total) were excluded from further analyses, and a hitlist of containing the top . % inhibitory and the top . % enhancing hfs was generated ( table s in text s ). the identified hsv- hfs were compared to datasets from published sirna depletion screens aimed at identifying cellular factors affecting hiv- [ , , ] , west nile virus (wnv) [ ] , hepatitis c virus (hcv) [ ] , dengue virus [ ] and influenza a virus [ , , ] . of our hfs, cellular proteins ( . %) overlapped with these other virus screens (influenza a, ; hiv- , ; hcv, ; wnv, ; dengue virus, ) ( figure c ; table s in text s ). a genome-scale yeast two-hybrid protein interaction screen identifies novel viral protein interaction partners hsv- is currently known to encode at least proteins, expressed sequentially under strict temporal regulation during herpes simplex virus type (hsv- ) infects the vast majority of the global population. whilst most people experience the relatively mild symptoms of cold sores, some individuals suffer more serious diseases like viral meningitis and encephalitis. hsv- is also becoming more common as a cause of genital herpes, traditionally associated with hsv- infection. co-infection with hsv- is a major contributor to hiv transmission, so a better understanding of hsv- /hsv- disease has wide implications for global healthcare. after initial infection, all herpesviruses have the ability to remain dormant, and can awaken to cause a symptomatic infection at any stage. whether the virus remains dormant or active is the result of a finely tuned balance between our immune system and evasion techniques developed by the virus. in this study we have found a new method by which the replication of the virus is counteracted. the cellular protein med was found to actively induce an innate anti-viral immune response in the form of the type iii interferons (ifnlambda), by binding irf , a key regulator of interferons, and modulating its activity. interferon lambda is well known to be important in the control of hepatitis c infection, and a genetic mutation correlating to an increase in interferon lambda levels is strongly linked to clearance of infection. here we find the same association between this genetic mutation and the clinical severity of recurrent cases of hsv- infection (coldsores). these data identify a med -interferon lambda regulatory axis of innate immunity, show that interferon lambda plays a significant role in hsv- infection, and contribute to the expanding evidence for interferon lambda in disease control. infection. to gain further mechanistic insight into host factors involved in hsv- infection, in parallel to the sirna depletion screen we carried out a yeast two-hybrid protein interaction screen to identify cellular interaction partners of viral proteins. we generated a collection of partial and full-length hsv- cdna constructs and tested them for interactions with proteins encoded by a library of , human cdna clones [ ] . hsv- human protein interactions were detected once (low confidence), and more than once (high-confidence)( table s in text s ) . using these high-confidence interactions, the previously reported hsv- interactome [ ] was connected into a human interactome ( , published protein interactions) to generate a combined pathogen-host interactome ( figure s a ). both degree centrality (which indicates the number of interactions a protein has, where high values represent highly interactive 'hubs') and betweenness centrality (which indicates the number of shortest paths between ( replicates) or the capacity to influence replication of the hsv- gfp reporter virus c ( replicates) from to h post-infection. virus replication slopes during the linear phase were calculated and normalized to mock-transfected cells. replication slopes were then compared to replication upon knockdown of essential (icp , vp ) or non-essential (vp / ) viral genes, a cellular receptor for hsv- (hvem) or control risc-free sirna (rscf). (c) overlap between the hsv- hfs identified in this study with those published in hiv- [ , , ] , hepatitis c virus (hcv) [ ] and influenza a virus [ , , ] . doi: . /journal.ppat. .g any pair of proteins passing through the protein considered) were significantly increased for hsv- interactors, particularly in the high-confidence network (figure s b-e). these data suggest hsv- proteins preferentially target highly connected central human proteins in the cellular interaction network, similar to other viruses [ ] . analysis of this interactome for hfs identified by rnai found they were enriched in the fraction of cellular proteins that directly interact with viral proteins or that interact via one intermediate, in comparison to proteins that only interact via or more intermediates (p = . , fisher's exact test)( figure s f) . a direct comparison of hsv- protein interaction partners and the sirna screen hfs found genes in common. of those, ten ( . %) were identified as a hit in both screens ( table s in text s ), suggesting that these technologies identify complimentary yet not necessarily overlapping hfs. an extended literature and database search identified cellular proteins that interact with or are involved in infection with human herpesviruses. the overlap between the high-confidence y h cellular interactors ( ) and hfs ( ) with this set was statistically significant (p = . ) (figure a; figure s h ; table s in text s ). from this combined analysis, a subset of hfs was chosen for further validation. protein interactions were tested in a mammalian cell system by lumier pull-down assay [ ] . of the interactions tested, ( . %) were confirmed, with strongly positive (z-score . ) and weakly positive (zscore - ) ( figure s g ). sirna deconvolution ( sirnas per gene tested individually) was used to further validate hfs (figure b ; figure s ). the replication phenotype could be confirmed ($ or more sirnas gave the same or better replication slope than observed in the primary screen) in a high proportion ( . %) of candidates, highlighting the reliability of the primary screen dataset. quantitative rt-pcr analysis of mrna expression levels found a minimum depletion of % (mean %) in a subset of genes (data not shown; table s in text s ) confirming the observed effects on hsv- replication are genuine and not due to 'off-target' effects or insufficient gene knockdown. to further investigate the virus-specificity of our identified hfs, we tested this subset for their effect on the replication of an additional a-herpesvirus (varicella-zoster virus, vzv), the bherpesvirus cytomegalovirus (cmv), and a completely unrelated rna virus, semliki forest virus (sfv). none of the three proteins which enhanced hsv- replication upon knockdown had an effect on either vzv or cmv, and one (nr c ) was even inhibitory for sfv ( figure c ). of the sirnas which inhibited hsv- , ( . %) were also inhibitory for vzv, for cmv ( . %) and ( . %) for sfv replication ( table s in text s ) . some functional groups (transcriptional regulators) were required by most viruses, but there were notable differences between other proteins. for example ifitm- , previously identified as an inhibitor of influenza a, dengue virus and wnv [ ] , inhibited vzv yet had a positive effect on hsv- replication. these data suggest that whilst there are some hfs which are broad in their effects on virus replication, a large proportion are species-specific. table s in text s ). pathways included those involved in gene expression, transcription, splicing and translational regulation (rnai screen), and protein transport, cell cycle, and transcriptional repressor activity (y h screen). a combined analysis of hfs from both screens found dominant functional categories centred on the regulation of transcription (rna polymerase ii-associated genes, splicing factors, transcription activation and the mediator complex) ( figure s c, d) . the physiological relevance of some hfs and pathways was confirmed by further biological validation. protein transport pathways (in the form of dynein microtubule networks) are exploited by hsv- early after infection to shuttle viral capsids to the nucleus. these screens confirmed known interactions between dynein subunits and viral proteins, and identified additional previously unknown interactions (text s and figure s a ). several dynein chain subunits were found to be essential for virus replication, whilst the moderate effect of depletion of other subunits demonstrated a level of functional redundancy in hsv- capsid transport ( figure s b -e) [ , ] . intrinsic anti-viral host defense mechanisms, in the context of cellular e ubiquitin ligases, were also investigated. the immediate-early viral protein icp , an e ubiquitin ligase, is crucial for blocking anti-viral defense mechanisms by degrading promyeloctic leukemia (pml) nuclear bodies (nd domains) in the presence of cellular e -ubiquitin-conjugating enzymes (e s). our sirna screen found multiple e s were required for this, and suggests that hsv- icp is promiscuous in its exploitation of e s to mediate pml degradation and ensure successful infection (text s and figure s ). combined bioinformatic analyses of protein interaction and sirna depletion screens found a significant functional enrichment for proteins involved in transcription, and identified multi-protein complexes enriched for pro-viral hfs which strongly inhibited hsv- upon depletion, including the rna-polymerase ii, eif and mediator complexes ( figure a) . the mediator complex links the cellular transcription machinery (rna polymerase ii) to specific transcription factors, and the identification of many mediator subunits as hfs in other viral sirna depletion screens highlights its significant role in viral genome transcription [ , , , ] (table s in text s ) . further, several mediator subunits (med , , and ) are known to interact with the hsv- transactivator vp (ul ) and other herpesviral proteins [ ] ( figure s a) . consistently, the mediator complex was found to be strongly required for hsv- replication, with depletion of the majority of subunits (med , , , , , , , , , , and ) leading to a severe reduction in virus replication in the primary screen ( figure s b ) or in confirmatory deconvolution assays ( figure b) . however, depletion of the med subunit was striking in that it led to a significant enhancement of virus growth ( figure b ). flow cytometry quantification found that removal of med not only increased the total number of infected cells (combination of gfp lo and gfp hi cells; . % in comparison to . % in mock-transfected cells) but also the copy number of virus genomes (gfp hi cells; . % in comparison to . % in mocktransfected cells) (figure c med could exert anti-viral effects either by having an inhibitory effect on viral transactivators or by interacting with and having a positive effect on an existing anti-viral factor. we first tested whether med directly affects viral gene expression using luciferase reporters with hsv- promoters, however observed no inhibitory effect (data not shown). since the mediator complex and med in particular is known to be involved in jak/statmediated interferon signaling [ ] , we used the lung epithelial cell line a and its stat- -deficient derivative a -v [ ] to determine if med influences hsv- replication by modulating innate immunity. in the parental a cells the phenotype of hsv- replication was the same as that observed in hela cells, where depletion of med enhanced replication and overexpression inhibited virus growth. however, in the stat -deficient a -v cells hsv- replication was unaffected by both depletion and over-expression of med (fig. a) , indicating that med requires an intact jak/stat signalling pathway to exert its anti-viral effects. to determine which interferon may be responsible for the antiviral effects of med , a cells were depleted for med and infected with hsv- following pre-stimulation with type i (ifn-a or ifn-b), type ii (ifn-c) or type iii (the distinct ifn-l or the almost identical ifn-l and -l , termed ifn-l / ) interferons. whilst treatment with ifn-a, -b and -c significantly decreased hsv- replication levels, the observed , -fold enhancement of hsv- replication following med depletion was still seen. however, pre-treatment with the both ifn-l and ifn-l / blocked the enhancing effect of med depletion (figure b) . investigation into the effect of med on interferon induction by qrt-pcr found that whilst med over-expression induced ifnb (, -fold increase), induction of ifn-l and l / was considerably and statistically significantly higher (, -fold induction; p = . and . , respectively) ( figure c ). this induction was specific, as levels of other cytokines and interferon-regulatory factors (irfs) were unaffected by med overexpression ( figure s a ). secretion of ifn-l / protein was also increased in all cell lines tested, but most significantly to , -fold in a cells (figure d) , which is consistent with a recent report showing that type iii interferons are the dominant type of ifns expressed by primary airway epithelial cells [ ] . furthermore, qpcr analysis found depletion of med inhibited the induction of ifn-l expression following hsv- infection of a cells in comparison to cells transfected with the rscf sirna control ( figure e ). together, these data suggest that ifn-l is responsible for the observed inhibitory effect of med on hsv- replication. as ifn-l expression is induced following activation of pathogen recognition receptors (prrs) by virus infection [ , , , ] , we tested whether med induced ifn-l by directly interacting with an interferon-responsive transcription factor (irf). y h and confirmatory co-immunoprecipitation experiments in mammalian cells with a panel of irfs found that med interacted with irf and irf (figure a ; figure s b ). we also observed a weak interaction with irf , which may explain the previously observed effect of med on jak/stat signalling [ ] . to determine if this interaction had a functional effect, we looked at whether med influenced irf-mediated induction of ifn-l. in a luciferase reporter assay, neither irf nor irf led to a significant induction of the ifn-l promoter, either alone or in conjunction with med (data not shown). irf induced expression from the ifn-b and ifn-l promoters to similar levels (, -fold and -fold higher than background, respectively), whilst the isre, induced by irf and also present in the irf promoter, was induced , -fold ( figure b ). whilst co-expression of med with irf had no further effect on ifn-b expression, a synergistic induction of the ifn-l promoter and, to a lesser extent, the isre, was observed (ifn-l doubled to , -fold, p = . ) ( figure b ). interestingly, a med mutant unable to induce immediate early gene expression via jun/fos (r q, or r q in med transcript variant used here) synergistically induced isre expression with irf , yet was unable to further enhance irf mediated induction of ifn-l (data not shown). a similar synergistic effect of med and irf was seen at the protein level, where co-expression increased supernatant levels of ifn-l more than -fold those seen with med or irf alone ( figure s c , d). successful disease and treatment outcome in hepatitis c virus infection (demonstration of a sustained virologic response) is strongly associated with a single nucleotide polymorphism (snp) in the ifn-l promoter (rs ; cc genotype over ct or tt) and higher plasma levels of ifn-l [ ] , [ ] . furthermore, ifn-l expression is impaired in a cohort of ethnically italian individuals suffering recurrent hsv- -related herpes labialis reactivation [ ] . to determine if the clinical severity of hsv- disease is due to the observed deficiency in ifn-l expression, we screened a subset of the recurrent herpes labialis (hl) cohort and additional subjects for the ifn-l promoter polymorphism. genotypic analysis found the presence of a t (ct or tt genotype) had a dose-dependent association with clinical severity, with the homozygous tt genotype being more prevalent as disease severity increases ( figure ). in spite of the relatively small sample numbers in some clinical categories (table ) , the association of a ct or tt genotype with the most severe recurrence of herpes labialis (h+) was statistically significant (p = . ; fishers's exact t-test). as the cc genotype is directly associated with increased ifn-l levels [ ] , these data highlight a previously unknown association between the frequency/severity of recurrence of herpes labialis, the ct/tt genotype and subsequent reduction in secretion of ifn-l . it is of importance to investigate this genotype association with a larger cohort of hl patients, as well as those suffering with other hsv- -related disease, in order to determine the role of ifn-l in the full spectrum of hsv- pathogenesis. was selected for validation with deconvoluted sirnas to confirm the phenotype observed in the primary screen. the effect of the four individual sirnas ( - ) and a reconstituted smartpool (sp) were tested by reverse-transfecting into hela cells before infecting after h with hsv- -egfp (c ) and monitoring replication. replication slopes were calculated and normalized as described, and compared to the primary screen slope ( u). a heat map of replication slopes was generated where red represents inhibition (replication slope , . ) and green represents enhancement (slope . ). the phenotype was considered validated if $ sirnas produced the same or better phenotype as the primary screen. (c) virus specificity of hsv- hfs. the effect of hf sirna smartpools on the replication of vzv (a-herpesvirus), hcmv (b-herpesvirus) or semliki forest virus (sfv; rna virus) was determined and compared to hsv- . normalized replication stdev of the controls was considered inhibiting/enhancing. doi: . /journal.ppat. .g taken together, these data identify med as a novel anti-viral factor which acts as a key regulator of ifn-l expression by interacting with and enhancing the activity of irf , a major transcription factor involved in innate immunity. our observation of a link between the clinical severity of hsv- disease and, a ct/ tt genotype at a snp known to regulate ifn-l secretion demonstrates the significance of ifn-l in the control of hsv- replication in vivo. whilst this study provides no direct link between the ifn-l promoter polymorphism and med , these associations of ifn-l with hsv- disease, combined with our observations that med is required for the induction of ifn-l following hsv- infection, identifies for the first time a link between med and ifn-l, provides a clinical context for med regulation of ifn-l expression and underscores the potential biological significance of these data. the use of hsv- in a combined genome-scale screening approach has led to the identification of a regulatory axis in antiviral innate immunity, and this important finding not only highlights the power of such combined genome-scale screening approaches to identify novel host candidates for anti-herpesvirus drug discovery, but provides an invaluable dataset to the herpesvirus and scientific community at large. by nature of their scale, high-throughput screening technologies have limitations. rnai technology is limited by technical issues such as off-target effects, where an alternative gene to the intended target is degraded, and insufficient gene knockdown. similarly, y h protein interaction screens can generate both false-positive interactions, due to 'sticky' proteins and auto-activation of the reporter gene used, and false-negative interactions. whereas the number of false positives can be considerably reduced by stringent screening and selection criteria, the low sensitivity of the y h assay, which detects - % of known interactions, is inherent to the system and can only be marginally improved. this poor sensitivity is caused by factors such as structural restraints of the y h bait and prey fusion proteins, a lack of or existence of distinct protein modification in yeast cells, and cellular localization signals in bait and prey proteins preventing nuclear import [ ] . however, as all other high-throughput methods for measuring binary protein interactions possess a similarly low sensitivity, but are considerably more laborious and expensive, the y h system is still the most commonly used technology [ ] . in this study we have exploited a combined genome-wide screening approach to investigate hsv- replication and interaction with its host. this identified functional hfs modulating hsv- replication, and cellular interaction partners. in validation experiments, . % of the interactions were confirmed by co-immunoprecipitation assays in mammalian cells, and of the functional hfs identified in the sirna screen, the phenotype of . % was confirmed in deconvoluted sirna experiments. this, combined with qpcr data demonstrating a minimum gene depletion of %, suggests that the functional phenotypes on virus replication are genuine, and not due to 'off-target' effects. the confirmation of such a high proportion of selected validation candidates, in spite of the potential technical drawbacks, highlights the reliability of our primary screen datasets, and thus provides an invaluable resource for the herpesvirology research community. one interesting outcome of this study was the surprisingly low overlap between hits identified using these different technologies. of the genes in common between the sirna and cdna libraries, only ( . %) were classified as a hit by both methods. this, however, is not unexpected, as even the overlap between studies using the same technology has been reported to be low. for example, the overlap between the three previously published hiv screens was only % [ ] . furthermore, the degree of functional redundancy within the sirna library, and cellular pathways in general, the potential situation-specificity of virus-host interactions, and the possibility of indirect interactions between viral and host proteins, suggest that these methodologies detecting functional outcomes or physical interactions are linked, but complementary rather than confirmatory. the identified hfs were enriched for a range of cellular processes, such as transcription, gene expression, protein transport and cell cycle ( figure s and s ) , and involved at different stages of viral infection. we investigated hfs involved in capsid transport and ubiquitination of antiviral intrinsic host defence factors in more detail. incoming hsv- capsids are transported to nuclear pores via the microtubule-organizing centre (mtoc), mediated by capsid proteins vp (ul ) and ul binding to the dynein light chains dynlt (tctex ) and dynlt (rp ) [ , ] . our y h screen confirmed the known interaction between the capsid protein vp and the dynein light chain dynlt (text s and figure s ). combined with the sirna screen data, which found depletion of multiple light chain subunits had moderate anti-viral effects on hsv- , these data confirm propositions of redundancy in the capsid transport process which ensures successful infection in the event of viral mutations [ , ] , and provide further evidence that hsv- has evolved to be highly promiscuous in its exploitation of cellular pathways to its advantage. to overcome the intrinsic host defence, hsv- induces a proteasome-dependent degradation of anti-viral promyelocytic leukemia (pml) nuclear bodies (nd domains) by the ringfinger ubiquitin ligase icp expressed during early infection [ ] . in vitro, icp is a biochemically active e ubiquitin ligase in the presence of e ubiquitin conjugating enzymes (e s) [ube d (ubch a) and ube e (ubch )] [ ] , but which e s are used during infection has remained unclear. we identified cellular e s that are able to influence hsv- replication (text s and figure s ). depletion of ube d - , ube e - and ube n significantly increased the number of pml-positive cells postinfection, in an icp -dependent manner, indicating that icp can use multiple e s to degrade pml [ , ] . one of the multi-protein complexes affecting hsv- replication was the mediator complex, a large (. subunits) complex which links specific transcription factors to the rna polymerase ii transcription machinery [ ] . as the requirement of mediator subunits in the replication of herpes and other viruses is already well-known [ , , , ] , it was striking that depletion of the med subunit exerted the opposite phenotype and led to a strong increase in virus growth. the mediator is composed of four distinct modules termed the head, middle, tail and kinase domains, which provide the mediator with some degree of active control over transcription [ ] . as individual subunits of this large complex interact with and exert functional effects via specific transcription factors, it is not unexpected that the observed antiviral effects were specific to med [ ] . within the mediator, med forms a tight sub-complex with med and med [ , ] . the increase in virus replication observed upon depletion of med may be caused by the destabilisation of the structure of this sub-complex ( figure s b) . investigations into the mechanism of action revealed med inhibits hsv- replication by preferentially inducing a type iii interferon response (ifn-l) at the mrna and protein level. this induction was mediated via a direct interaction with the transcription factor irf , which resulted in a synergistic increase in ifn-l expression. med was unable, however, to further enhance irf -induced levels of ifn-b, suggesting an additional level of complexity to the regulation of interferon signalling. interestingly, the inhibitory effect of med was specific to hsv- , with replication of a range of other viruses including vaccinia virus and semliki forest virus being unaffected by med depletion. as vaccinia virus is resistant to ifn-l anti-viral activity [ ] , this observation further highlights the importance of ifn-l, as opposed to ifn-b, in the anti-viral effect of med . the r q mutation in med (r q in med transcript variant , used here) was unable to enhance irf -induced ifn-l expression. this mutation causes hereditary dementia [ ] , and the failure to induce ifn-l and thereby control hsv- in the brain may be a potential cofactor for the development of dementia, similar to alzheimer's disease [ ] . there is mounting evidence for a role of the ifn-l family in the regulation of virus pathogenesis [ ] , particularly in the case of hepatitis c infection where a polymorphism in the promoter region of ifn-l (il- b; polymorphism rs ), which correlates with plasma levels of ifn-l [ ] , is associated with disease and treatment outcome [ ] . individuals with recurrent hsv- reactivation have been shown to be deficient in ifn-l expression [ ] , and here we found the similar association between the ifn-l promoter polymorphism and ethnically italian patients suffering recurrent and severe reactivations of hsv- -related oral herpes outbreaks, albeit with a small sample group (n = ). furthermore, sporadic mutations and genetic polymorphisms in innate immune receptor and signalling molecules that lead to the induction of type i and iii ifns have also been shown to be associated with herpes encephalitis [ ] , as well as oral and genital herpes [ , ] . hsv infection controlled by a complex, interconnected and highly regulated network of cytokines expressed by innate immune cells. type i ifns mainly produced by hsv-infected keratinocytes [ ] and pdcs [ ] inhibit the spread from neurons to epithelial cells and between epithelial cells [ ] , similar to ifn-c. type iii ifns are also able to directly inhibit hsv- infection in primary neurons, astrocytes, macrophages and dendritic cells [ , ] . ifn-c levels produced by peripheral blood cd + t-cells correlate with the frequency of hsv- reactivation [ ] . ifn-l is able to induce expression of both itself and the type i ifns, and a similar effect has also been observed for type i ifns which induce both type i and iii ifns [ , ] . type iii ifns are mainly expressed by myeloid dendritic cells (mdc) and monocyte-derived macrophages [ ] , and signal through the heterodimeric il rb/il ra receptor complex whose expression is largely restricted to cells of epithelial origin and plasmacytoid dendritic cells (pdc), in contrast to the broadly expressed type i ifn receptor (ifn-ar / ) [ , ] . since primary hsv- infection and reactivation affects skin and mucosa in the majority of cases, ifn-l may play a much greater role in the control of hsv- pathogenesis, likely in a complex network of coregulated type i and ii ifns, than previously thought. we hypothesize that hsv-infected dcs at the site of the lesion (such as skin langerhans dcs whose role in ifn-l production is currently unknown, or intruding myeloid dcs) in individuals with the rs t/t or c/t haplotype express reduced levels of type iii ifns, and, in consequence, of type i ifns, which leads to a reduced inhibition of local hsv- replication and the occurrence of fresh skin lesions. however, the relative contribution of ifn-l and l / to the interferonmediated control of hsv- replication in vivo, and indeed the role of med in this, remains to be seen. in summary, this study provides a comprehensive and robust analysis of hfs that influence hsv- replication in vitro, which will benefit many future studies on hsv- . the identification of med as a crucial cellular component for ifn-l expression, and evidence for the significant role of type iii ifn in the innate immune control of hsv- in vitro and in vivo, demonstrates the power of combined, genome-scale studies to identify physiologically important hfs for virus pathogenesis. future studies will clarify the role of genetic variations in both med and ifn-l in hsv- -related diseases, such as meningitis, keratitis and orolabial/genital reactivations. sirna smartpools ( sirnas per gene) at . mm were dispensed in ml volumes using a rapidplate liquid handler (qiagen) into triplicate black -well plates (corning), sealed with adhesive seals (thermofisher) and plastic lids. plates were stored at uc until needed (minimum h, maximum h). on the day of transfection, assay plates were thawed at room temperature and ml transfection reagent (dharmafect , dharmacon), diluted in hank's buffered saline solution (hbss, thermofisher) to give a final concentration of . %, was added using a multidrop (thermofisher). plates were incubated for min at room temperature to allow formation of transfection complexes. during complex formation, low-passage (p - ) hela cells (ecacc) from , % confluent flasks were washed in pbs and trypsinised in trypsin-edta (lonza) before diluting in phenol red-free, antibiotic-free transfection medium (dmem/f- : / % fcs with mm hepes and l-glu; gibco). cells were counted and cells in ml were added to each well using the multidrop . plates were incubated for h at uc in a humidified incubator with % co . to infect, media was removed from plates by inversion, and ml media (as for transfection, but containing penicillin-streptomycin; lonza) or virus (hsv- -egfp strain c , diluted to moi . in infection media) [ ] was added using the multidrop . plates were incubated at uc for h before ml infection media was added and plates returned to the incubator. replication was monitored as a function of egfp fluorescence from h to h post-infection using the polarstar optima plate reader (bmg labtech). virus replication slopes over the linear phase were calculated and normalized to mock transfected wells on individual assay plates, and the mean replication slope from six replicates used for subsequent data analyses. cells were transfected as described above, and the cytotoxicity of sirnas was determined using the celltiter blue (ctb, promega) reagent, which gives a fluorescent or absorbance signal relative to the number of live cells. briefly, ml ctb was added per well using the multidrop . plates were incubated at uc in a humidified incubator with % co for h before measuring fluorescence (polarstar optima plate reader). readings were normalized to viability of mock-transfected cells, per plate, and mean cell viability over three replicates was calculated. distribution analysis of cell viability values identified median viability as %, and values , % were considered cytotoxic. the hsv- clone collection was cloned by recombinatorial (gateway tm , invitrogen) and conventional cloning into the bait vector pgbkt , and screened against a library pooled from , mgc clones [ ] in the pgadt prey vector using a semiautomated y h assay [ ] . interacting prey cdnas were identified by sequential blasting of refseq, ensembl and unigene databases. blast hits with identical parameters (score, expectation value, length of alignment) were considered indistinguishable and counted separately. a high-confidence dataset was generated from interaction pairs isolated at least twice, or where the bait interacted with two highly related, non-promiscuous preys. interactions between hsv- and human proteins were connected to a network of human protein-protein interactions (a total of , ) taken from the databases hprd [ ] (release ), biogrid [ ] , dip [ ] , mint [ ] and intact (downloaded may th ). a high-confidence interaction set ( , interactions) was compiled from interactions identified in at least two studies. betweenness centrality (g(v) of a protein v was calculated as g(v) = gs {v{t (s st (v)/s st ), where s st is the total number of shortest paths from protein s to protein t, and s st (v) is the number of those shortest paths that contain v. betweenness centrality was normalized by dividing by the total number of protein pairs in the network. enrichment for functional annotations from gene ontology (go) [ ] , kegg [ , ] , reac-tome [ , ] , and biocarta was performed using david [ ] . data on known human protein complexes was retrieved from the corum database, and complexes with subunits showing consistently stronger effects (inhibiting or enhancing) than expected by chance were detected using wilcoxon's rank-sum test. genes included in the rnai screen were ranked by their distance from the median knockdown, with the most inhibiting and enhancing genes being ranked highest. fdr was used for multiple testing correction. the hsv- replication phenotype observed in the primary screen was validated for a subset of candidates by deconvoluting the assay smartpools. the four individual sirnas targeting different regions of each gene, as well as a reconstituted smartpool, were diluted to . mm in sirna buffer and dispensed to black well plates. transfection and infection was carried out as described above. replication slopes were calculated and normalized as described, and a phenotype was considered validated if two or more of the four sirnas resulted in the same, or better, phenotype. for inter-viral comparison, sirnas were considered inhibitory or enhancing if normalized replication was stdev of the controls a) hsv- replication assays. selected sirna smartpools were diluted to . mm in sirna buffer and dispensed in black -well plates (corning). to this ml dharmafect , diluted in hbss to a final concentration of . %, was added using the multidrop . following a min incubation to enable complex formation, hela cells in ml transfection media were seeded on to the complexes bringing the final volume of transfection to ml in each well. plates were incubated for h at uc in a humidified incubator with % co before infection. to infect, media was removed from plates by inversion, and ml media (as for transfection, but containing penicillinstreptomycin; lonza) or virus (strain c , diluted to moi . in infection media) was added using the multidrop . plates were incubated at uc for h before virus was removed by plate inversion and ml infection media was added. plates were returned to the incubator before replication was monitored as a function of egfp fluorescence from h to h post-infection. virus replication slopes over the linear phase were calculated and normalized to mock transfected wells on individual assay plates, and the mean replication slope from six replicates used for subsequent data analyses. b) vzv replication assays. selected sirna smartpools were diluted to . mm in sirna buffer and dispensed in black -well plates (corning). to this, ml dharmafect diluted in hbss to a final concentration of . % was added using the multidrop . following a min incubation to enable complex formation, . mewo cells (atcc, htb- tm ) in ml media (emem/ % fcs/ % non-essential amino acids) were seeded on to the complexes bringing the final volume of transfection to ml in each well. plates were incubated for h at uc in a humidified incubator with % co before infection. to infect, media was removed from plates by inversion and , colony forming units of vzv-egfp-infected mewo cells (vaccine strain oka) [ ] diluted in mewo growth media were seeded on to the complexes using the multidrop . virus growth was measured in h intervals as a function of egfp fluorescence from to h post-infection. virus replication slopes over the linear phase were calculated and normalized to mock transfected wells on individual assay plates, and the mean replication slope from six replicates used for subsequent data analyses. c) hcmv replication assays. selected sirna smartpools were diluted to . mm in sirna buffer and dispensed in ml volumes in black -well plates (corning). to this, ml dharmafect, diluted in hbss to a final concentration of . %, was added using the multidrop . following a min incubation to enable complex formation, mrc- (atcc, ccl- tm ) in ml growth medium (phenol red-free dmem/ %fbs/l-glutamine/ % non-essential amino acids were seeded on to the complexes bringing the final volume of transfection to ml in each well. plates were incubated for h at uc in a humidified incubator with % co before infection. to infect, media was removed from plates by inversion, and ml media (as for transfection, but containing penicillin-streptomycin) or virus hcmv-gfp (strain ad ) [ ] , diluted to moi . in infection media, was added using the multidrop . plates were incubated at uc for h before ml infection media was added and plates returned to the incubator prior to monitoring virus replication. replication was monitored as a function of egfp fluorescence from h to h post-infection using the polarstar optima plate reader (bmg labtech). virus replication slopes over the linear phase were calculated and normalized to mock transfected wells on individual assay plates, and the mean replication slope from six replicates used for subsequent data analyses. d) sfv replication assays. selected sirna smartpools were diluted to . mm in sirna buffer and dispensed in ml volumes in black -well plates (corning). to this, ml dharmafect diluted in hbss to a final concentration of . % was manually added. following a min incubation to enable complex formation, hela cells in ml transfection media (dmem/ % fcs/l-glu) were seeded on to the complexes bringing the final volume of transfection to ml in each well. plates were incubated for h at uc in a humidified incubator with % co before infection. to infect, media was removed from plates by inversion, and ml media (as for transfection, but containing penicillin-streptomycin; lonza) or virus (sfv ( h)-rluc [ ] diluted in phosphate buffered saline (pbs)/ . % bovine serum albumin to an moi . ) was manually added. plates were incubated at uc for h before media (as for transfection media) was added manually to increase the volume to ml per well. plates were incubated at uc for h before cells were lysed using a passive lysis buffer and renilla luciferase levels measured with a microplate reader (promega) using a dual luciferase reporter assay kit (promega). luciferase activity, which is representative of virus genome replication, was normalized to mock-transfected cells and mean luciferase activity from six replicates used for subsequent data analyses. e) vaccinia virus replication assays. hela cells were transfected as described in primary sirna screen. plates were incubated for h at uc in a humidified incubator with % co before infection. to infect, media was removed from plates by inversion, and ml media (as for transfection, but containing penicillin-streptomycin) or ml media containing vaccinia virus strain wr with egfp-tagged a protein [ ] , diluted to moi . , was added using the multidrop . plates were incubated at uc for h before ml of media was added to each well, the plates inverted to remove the media and virus, and a final volume of ml of media added to the plates before they were returned to the incubator. replication was calculated as a function of egfp fluorescence at h post-infection using the polarstar opti-ma plate reader (bmg labtech). virus replication was normalized to mock transfected wells on individual assay plates, and the mean replication from eight replicates used for subsequent data analyses. hela cells were transfected with selected smartpool sirnas in -well plates, in triplicate, as described. after h transfection, medium was removed, cells rinsed in pbs and lysed in ml trizol (invitrogen). triplicate wells were combined, and rna extracted by standard phenol:chloroform extraction methods. mrna levels were determined by taqman qpcr, using the one-step rt-qpcr kit (thermofisher), with gene-specific primers ( table s in text s ), and probes from the universal probe library (roche). expression levels normalized to the housekeeping cellular gene hypoxanthine phosphoribosyltransferase (hprt) and calibrated to mock-transfected cells. qpcr was carried out in duplicate for each sample, and the mean of normalized expression levels calculated. proteins were transiently expressed in hek cells as hybrid proteins with the staphylococcus aureus protein a tag or renilla reniformis luciferase fused to their amino termini. ng of each expression construct were transfected into hek cells using . ml of lipofectamine (invitrogen) in -well plates. after h, medium was removed and cells were lysed on ice in ml of ice-cold lysis buffer ( mm tris ph . , mm nacl, % tritonx- , mm edta, mm dtt, protease inhibitor cocktail (roche), phosphatase inhibitor cocktail (roche), benzonase (novagen) units per ml final concentration) containing sheep-anti-rabbit iggcoated magnetic beads (invitrogen, dynabeads m , mg/ml final concentration). lysates were incubated on ice for minutes. ml of wash buffer (pbs, mm dtt) were added per well, and % of the diluted lysate was removed to determine the luciferase activity present in each sample before washing. the remaining sample was washed times in wash buffer in a tecan hydroflex plate washer. luciferase activity was measured in the lysate as well as in washed beads. negative controls were wells transfected with the plasmid expressing the luciferase fusion protein and a vector expressing two copies of protein a. for each sample, four values were measured: the luciferase present in % of the sample before washing (''input''), the luciferase activity present on the beads after washing (''bound''), and the same values for the negative controls (''input nc'', and ''bound nc''). normalized interaction signals were calculated as follows: log(bound)/log(input) -log(bound nc)/ log(input nc). normalized interaction signals were z-transformed by subtracting the mean and dividing by the standard deviation. the mean and standard deviation were calculated from large datasets of protein pairs which were not expected to interact, i.e. from negative reference sets. selected sirnas smartpools were diluted to nm in hbss and ml was incubated with ml dharmafect diluted in hbss to a final concentration of . %. after min incubation, hela cells in ml transfection medium were added, mixed with the transfection complexes and transferred to -well glass bottomed chamber slides (becton dickinson). plates were incubated for h at uc in a humidified incubator with % co before infection by removing medium and adding ml hsv- -egfp at a moi of . after incubation for h at uc, virus was removed and replaced with ml growth medium. images were acquired h post-infection. select sirnas smartpools were diluted to nm in hbss and ml was incubated with ml dharmafect diluted in hbss to a final concentration of . % in individual wells of a well plate. after min incubation, hela cells in ml transfection medium were added. plates were incubated for h at uc in a humidified incubator with % co before infection by removing medium and adding ml hsv- -egfp at a moi of . after incubation for h at uc, virus was removed and replaced with ml growth medium. after h, medium was removed, cells rinsed in pbs and dislodged by trypsinisation. cells were washed in pbs and pelleted by centrifugation for min at g. supernatant was removed and cells fixed in % paraformaldehyde before analysing for egfp expression by flow cytometry (facs diva, bd biosciences) using the cellquest software package. for transient over-expression, . hek cells were seeded in black -well plates. the following day, cells were transfected with ng pcr -med using lipofectamine tm ltx (invitrogen) and incubated for h before infection with the recombinant hsv- reporter viruses c and vp -yfp at moi . . replication growth curves were monitored, and endpoint replication (as determined by fluorescence) was normalized to untransfected cells. for stable expression, hela cells were transduced with plenti-med , generated using the virapower tm lentiviral expression system (invitrogen), as per manufacturers' instructions. stable cells were infected, and replication monitored, as above. for confirmation of overexpression, rna was extracted (qiagen rna-easy kit) and expression quantified by one-step rt-qpcr (thermofisher), with gene-specific primers (table s in text s ), and probes from the universal probe library (roche). expression levels were normalized to the housekeeping cellular gene hypoxanthine phosphoribosyltransferase (hprt) and calibrated to mock-transfected cells. qpcr was carried out in duplicate for each sample, and normalized expression levels averaged. the vp -yfp reporter virus was generated from a kos bac kindly provided by david leib using the red-mediated recombination system, where the first amino acids of vp were replaced with yfp [ , , ] . the effect of med over-expression and depletion was investigated in interferon-deficient cells. the human alveolar epithelial cell line a , and a -v, a stat -deficient derivative cell line stably expressing the v protein from simian virus [ ] , were seeded at cells per well in a -well plate, and transfected with med sirna or pcr -med and infected as described for hela cells. qrt-pcr analysis of interferon and cytokine induction a cells were transfected with ng pcr or med overexpression plasmids, or rscf or med smartpool sirna ( nm) in duplicates, in -well plates as described. rna was harvested h post-transfection, and mrna expression levels quantified by qrt-pcr, as described. induction of ifn-l by med was determined in a range of cell types by seeding cells in -well plates to be , % confluent the next day. cells were transfected in duplicates with ng pcr or pcr -med using lipofectamine ltx with plus reagent (invitrogen), in antibiotic-free medium. ifn-l levels quantified - h post-transfection. the synergistic effect of med and irfs on ifn-l induction was determined by co-transfection of pcr or pcr -med ( ng) with pcr -irf ( ng) in a cells, with lipofectamine ltx with plus reagent. the effect of med depletion on ifn-l induction was determined by transfection of a cells in -well plates with nm rscf or med sirna. ifn-l was quantified h post-transfection. ifn-l protein expression was quantified in supernatants using a commercial ifn-l duoset elisa kit (r and d systems). a point mutation (r q) was introduced into med (transcript variant ) by pcr with specific primers (see table s in text s ) and the clone verified by sequence analysis. a cells were co-transfected with ng of pcr , pcr -med or pcr -r q, ng of pcr -irf , ng of ifn-b-, ifn-l -or isre-responsive luciferase reporter constructs and ng prl-tk, a renilla luciferase transfection control, in antibiotic-free low serum ( %) medium. after h cells were lysed, and firefly (promoter reporter construct) and renilla (transfection control) luciferase activity was measured (dual-luciferase reporter kit, promega). relative luminescence activity was normalized to renilla (as a transfection control). a cells were transfected in triplicate in black -well plates, as described. after incubation at uc for h, cells were serumstarved for h by growing in low serum ( %) medium, before cells were left untreated or stimulated with ng/ml ifn-a, ifnb or ifn-c, or ng/ml ifn-l or ifn-l / in serum-reduced medium. after h cells were infected with hsv- c diluted to moi . in ifn-containing serum-reduced medium. after h incubation, virus was removed and media replaced with ml serum-reduced growth medium containing no interferon, ifn-a/b/-c at ng/ml, or ifn-l /-l / at ng/ml. replication was monitored as described and normalized to replication in mock-transfected, unstimulated cells. potential interactions between med and the irfs were determined by yeast two-hybrid analysis and confirmed by coimmunoprecipitation (co-ip) in mammalian cells. a) co-immunoprecipitation. co-immunoprecipitation was performed using the epitope-tagged plasmids pgbkt (myc epitope) and pgadt (ha epitope) containing the t promoter, and recombinant vaccinia virus vtf- expressing the t rna polymerase (nih aids repository). hek cells were seeded in cm dishes and the following day infected with vtf- (moi ) for h before transfecting mg each of empty bait (pgbkt ) or med -bait, and irf-prey (pgadt ) vectors with lipofectamine (invitrogen). after h cells were lysed on ice for min in np buffer, containing protease and phosphatase inhibitors. debris was removed by centrifugation, and protein quantified with a bca protein assay kit (thermo scientific, uk) as per manufacturers' instructions. equal protein quantities ( mg per sample) were pre-cleared by continuous mixing with ml preequilibrated protein g sepharose beads for h at uc. samples were centrifuged and supernatants halved before overnight mixing incubation with beads pre-coated with mg a-ha (roche, uk) or a-c-myc (santa cruz, uk) at uc. beads with precipitated proteins were washed three times with ice-cold np buffer, resuspended in sds protein sample buffer and boiled for min before sds-page separation on two % polyacrylamide gels. proteins were transferred onto nitrocellulose membranes overnight at uc before blocking with % milk in tbs-tween then incubating with either a-ha or a-myc antibody (diluted : in % milk/tbs-tween). membranes were washed in tbs-tween before incubation with hrp-conjugated secondary antibody (diluted : in % milk/tbs-tween). after further washes, proteins were detected by incubation with ecl western blotting detection system, exposing to x-ray film and developing films in an optimax x-ray film processor. b) yeast two-hybrid assays. haploid yeast strains ah and y were transformed with mg prey (pgadt or pgadt -irf , -irf , -irf , -irf , -irf , -irf or -irf ) or bait (pgbkt or pgbkt -med ) plasmid dna, respectively, and grown overnight in synthetic defined (sd) medium lacking either leucine (-l; prey) or tryptophan (-w; bait) before prey-and bait-expressing haploid yeast cells were mated overnight in sd-lw/ % ypda medium. haploids were selected in sd-lw for h before transferring to triple-knockout, histidine-deficient sd-lwh liquid medium containing -aminotriazol ( -at) and methylumbelliferyl-b-d-galactopyranoside ( -mux) for - days. interactions were tested in quadruplicates, detected by growth of colonies on sd-lwh agar with -at, and quantified by measurement of fluorescence released from a-galactosidase cleavage of -mux upon protein interaction. relative fluorescence (rfu) was normalized to negative control interaction (empty pgadt mated with empty pgbkt ). ethnically italian subjects with or without a history of recurrent herpes labialis (hl) gave written voluntary informed consent and were enrolled in this study at the university of rome tor vergata with the approval of the ethical committee at the university of rome tor vergata. all subjects were interviewed by medically trained investigators using an appropriate questionnaire and agreed to provide saliva and/or blood samples. data and blood sample collection was carried out as previously described [ ] . a total of healthy immunocompetent individuals ( men and women) age - years (overall median age . yr; male median age yr, range - ; female median age , range - ) participated in the study. none of the patients presented with an active lesion at the time of or in the weeks preceding saliva sample collection. for the purpose of this study, patients were characterized into no recurrence of hl (nr), low recurrence (l; - hl episodes/yr, with a maximum extension of cm, mild symptoms and healing time , days), high recurrence (h; or more hl episodes/yr, extension of lesions more than cm), very high recurrence (h+; more than hl episodes/yr, extension of lesions . cm and/or involving nose or cheek beyond the lip, more severe and long lasting associated symptoms including itch, burning, paresthesias and/or neuralgia, with healing times . days and who required antiviral therapy ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ]). saliva samples (, ml) were obtained from each subject after an overnight fast and after rinsing the mouth twice with water, split into two aliquots and frozen at uc. samples were anonymised and stored with dual code labels before shipping on dry ice to the university of edinburgh for dna extraction. saliva and pbmc samples were thawed and dna extracted using a qiaamp dna blood mini kit (qiagen) as per manufacturer's instructions, quantified using a nanodrop and il b genotype determined by melt-curve analysis pcr on a lightcycler (roche) using the lightmixh kit il b (tib molbiol) as per manufacturer's instructions. significance of genotype association was determined by fisher's exact test, comparing the frequency of the cc, ct or tt genotype in the nr group versus the l (pvalue = ), h (p-value = . ) or h+ (p-value = . ) clinical groups. below lists the geneid numbers for genes and proteins mentioned within the text of this manuscript: ifitm ( ) text s text for supporting figures. descriptive text for figures s and s validation of hsv- -host y h interactors. a subset of protein interactions identified in the hsv- -host y h screen were validated using the lumier pull-down assay in a mammalian cell system. strength of interaction was determined by z-score, where a score to represents a weak interaction and score . represents a strong interaction. (h) distribution of direct hsv- targets in the rnai screen. the proteins directly targeted by hsv- were taken from the y h data set and from the literature curation. an enrichment of literature-derived targets could be observed in the top % most inhibiting knockdowns ( . -fold enrichment; p = . ), but the same could not be observed for the y h-detected direct targets. (tif) figure s validation of hf identified by rnai. hfs identified in the hsv- perturbation screen were validated with deconvoluted sirnas and qpcr: (a) chromoboxes, (b) homeoboxes, (c) general transcription factors, (d) nuclear receptors, (e) proteasome family members, (f) topoisomerases, (g) mediator complex subunits, (h) proteins involved in vesicle transport, (i) integrins, (j) y h interactors, (k) ubiquitin e ligases, (l) vesicle transport -further candidates, (m) interferon-stimulated membrane proteins and (n) others. hsv replication is presented as normalized replication slope, and is the mean of six individual assay points. error bars represent standard deviation of the six data points. deconvoluted sirnas which had a sequence different to that in the original screen are highlighted in red. genes for which the primary screen phenotype was not confirmed in the deconvolution assay are shown in bold text. (tif) figure s hsv- hfs are involved in diverse cellular pathways and at multiple stages of the hsv life cycle. enrichment of protein functions among the hsv- hfs. the enrichment for gene ontology (go) terms and kegg, bio-carta or reactome pathway annotations among the hsv- hfs identified by (a) rnai and (b) y h assay was performed using david bioinformatics software. (c) direct interactions between human and hsv- proteins. the protein-protein interactions (ppis) depicted are from the high confidence y h data set and from literature curation. circles and diamonds correspond to human and hsv- proteins, respectively. human proteins detected in the y h screen are drawn with red borders. human genes that showed the strongest effects in the rnai screen are colored yellow (extreme %) and orange (extreme %). (d) highly interconnected regions in the human interaction network composed of hfs. highly interconnected regions in the human interaction network composed of hfs. we assembled a human interaction network using data from the major ppi databases. a subnetwork consisting of hfs was then defined by limiting the network to the hfs detected in the y h screen, the rnai (extreme %) screen, and the literature curation. highly interconnected regions in the subnetwork were sought out using the mcode algorithm. the top six scoring regions are shown. proteins displayed in red correspond to hfs that are known only from the literature, and those in green are those that were detected in either of the screens performed in this study. the three boolean values beside each gene symbol represent whether the hf is present in the y h screen, the rnai screen (extreme %), or the literature curated set, in that order. dominant functional categories could be observed in each of the regions, including transcription (e.g., mediator complex, rna polymerase ii and associated genes), translation initiation, splicing, and intracellular transport. (tif) figure s hfs involved in viral entry and capsid transport. (a) diagrammatic summary of the role of dynein chains in hsv- infection. (b) microtubule transport is required for hsv- infection. the role of dynein microtubule transport components in hsv- replication was analysed by comparing the replication slope of hsv- -egfp (c )-infected cells depleted for a range of dynein chains from the primary sirna perturbation screen. error bars represent the mean of three independent experiments done in duplicate. (c) depletion of dyneins inhibits virus particle release. the effect of dynein chain depletion on hsv- particle release was determined by quantifying virus titer in supernatants of hela cells depleted of dync h (heavy chain), dync i (intermediate chain) and dync li (light intermediate chains) in a high multiplicity (moi ; hsv- kos) growth assay. titers were compared to control transfected cells (nt, nontargeting sirna). (d) depletion of dynein chains prevents immediate-early gene expression. immediate-early (icp ) and late (vp ) viral protein expression in cells depleted of dync h , dync i or dync li was analysed and quantified by western blot. levels were compared to control transfected cells (nt, non-targeting sirna). (e) quantification of icp and vp protein expression. protein levels of icp and vp in cells depleted of dync h , dync i or dync li were quantified with an odyssey imager and normalized to protein levels in control transfected cells (non-targeting sirna). (tif) figure s e ubiquitin conjugating enzymes in hsv- immune evasion. (a) depletion of e ubiquitin ligases inhibits pml degradation following hsv- infection. hela cells mounted on coverslips were depleted for a range of e ubiquitin ligases for h before infecting with hsv- +. cells were fixed and stained for icp (green) and pml (red), analysed by confocal microscopy and pml-positive cells were counted ( fields of view per coverslip) and expressed as a mean percentage of pmlpositive cells remaining ( independent experiments). error bars represent the standard deviation over independent experiments. (b) inhibition of pml degradation by e s is icp -dependent. hela cells were seeded on coverslips, transfected as above and infected with an icp ring-finger deletion mutant (fxe). remaining pml-positive cells were quantified as above. (c) immunofluorescence staining for pml bodies in cells transfected with control sirna not incorporated into the risc complex (rscf) or cells depleted of the e ubiquitin conjugating enzymes e d or e l . the arrows highlight cells that have wt pml levels remaining in them whilst containing wt icp . (tif) figure s med is an anti-viral component of the largely pro-viral multi-protein mediator complex. (a) diagrammatic summary of the role of mediator complex subunits in virus replication. subunits are coloured according to whether hsv- replication was unchanged (grey), inhibited (top %, red; top %, orange) or enhanced upon gene knockdown (top %, light green; top %, dark green). subunits not included are white; herpesvirus proteins reported to target mediator subunits are violet; mediator subunits detected in other viral rnai screens are highlighted by coloured diamonds. (b) mediator complex subunits influence hsv- replication. individual subunits of the mediator complex and associated proteins were depleted by sirna knockdown and infected with hsv- c (moi . ). replication was monitored over multiple rounds and the slope of replication over the linear phase was calculated and normalized to controls (mock-transfected cells). grey, no significant effect; red, strongly pro-viral; green, strongly anti-viral. error bars represent the mean of six replicates. herpes simplex virus type infection: overview on relevant clinico-pathological features herpes simplex virus infects most cell types in vitro: clues to its success the family herpesviridae: a brief introduction herpesviruses and immunity: the art of evasion proteomic analysis of cells in the early stages of herpes simplex virus type- infection reveals widespread changes in the host cell proteome analysis of virion-incorporated host proteins required for herpes simplex virus type infection through a rna interference screen identification of host proteins required for hiv infection through a functional genomic screen global analysis of host-pathogen interactions that regulate early-stage hiv- replication genome-scale rnai screen for host factors required for hiv replication the ifitm proteins mediate cellular resistance to influenza a h n virus, west nile virus, and dengue virus genomewide rnai screen identifies human host factors crucial for influenza virus replication human host factors required for influenza virus replication a genome-wide genetic screen for host factors required for hepatitis c virus propagation rna interference screen for human genes associated with west nile virus infection discovery of insect and human dengue virus host factors comparative rnai screening reveals host factors involved in enterovirus infection of polarized endothelial monolayers vaccinia virus uses macropinocytosis and apoptotic mimicry to enter host cells human genome-wide rnai screen reveals a role for nuclear pore proteins in poxvirus morphogenesis host cell factors in hiv replication: meta-analysis of genome-wide studies population context determines cell-to-cell variability in endocytosis and virus infection hepatitis c virus infection protein network a physical and regulatory map of host-influenza interactions reveals pathways in h n infection epstein-barr virus and virus human protein interaction maps genome-wide analysis of vaccinia virus protein-protein interactions analysis of vaccinia virus-host protein-protein interactions: validations of yeast two-hybrid screenings the sarscoronavirus-host interactome: identification of cyclophilins as target for pancoronavirus inhibitors virus-host interactomes and global models of virusinfected cells human immunodeficiency virus type , human protein interaction database at ncbi virhostnet: a knowledge base for the management and the analysis of proteome-wide virus-host interaction networks mint: the molecular interaction database pig-the pathogen interaction gateway hpidb-a unified resource for host-pathogen interactions the human-bacterial pathogen protein interaction networks of bacillus anthracis, francisella tularensis, and yersinia pestis herpes simplex virus type promoter activity during latency establishment, maintenance, and reactivation in primary dorsal root neurons in vitro horfeome v . : a resource of human open reading frames representing over , human genes herpesviral protein networks and their interaction with the human proteome highthroughput mapping of a dynamic signaling network in mammalian cells eclipse phase of herpes simplex virus type infection: efficient dynein-mediated capsid transport without the small capsid protein vp plus-and minus-end directed microtubule motors bind simultaneously to herpes simplex virus capsids using different inner tegument structures mediator complexes and eukaryotic transcription regulation: an overview a novel docking site on mediator is critical for activation by vp in mammalian cells role of metazoan mediator proteins in interferon-responsive transcription ns proteins of avian influenza a viruses can act as antagonists of the human alpha/beta interferon response toll-like receptor expression and induction of type i and type iii interferons in primary airway epithelial cells lambda interferon (ifn-lambda), a type iii ifn, is induced by viruses and ifns and displays potent antiviral activity against select virus infections in vivo activation of toll-like receptor- induces interferon-lambda expression in human neuronal cells ifn-lambdas mediate antiviral protection through a distinct class ii cytokine receptor complex il- , il- and their class ii cytokine receptor il- r genetic variation in il b predicts hepatitis c treatment-induced viral clearance interferon-lambda serum levels in hepatitis c interferon-lambda in immunocompetent individuals with a history of recurrent herpes labialis connecting viral with cellular interactomes an experimentally derived confidence score for binary protein-protein interactions herpes simplex virus type capsid protein vp interacts with dynein light chains rp and tctex and plays a role in retrograde cellular transport walking the walk: how kinesin and dynein coordinate their steps replication of icp -null mutant herpes simplex virus type is restricted by both pml and sp herpes simplex virus type immediateearly protein icp and is isolated ring finger domain act as ubiquitin e ligases in vitro the degradation of promyelocytic leukemia and sp proteins by herpes simplex virus is mediated by the ubiquitinconjugating enzyme ubch a the metazoan mediator co-activator complex as an integrative hub for transcriptional regulation transcription control by e a and map kinase pathway via sur mediator subunit the trap component of the trap/mediator complex is essential in broad transcriptional events and development inhibition of type iii interferon activity by orthopoxvirus immunomodulatory proteins med mutation links intellectual disability to dysregulation of immediate early gene expression alzheimer's disease gene signature says: beware of brain viral infections herpes simplex encephalitis in children with autosomal recessive and dominant trif deficiency association of tlr -hyporesponsiveness and functional tlr l f polymorphism with recurrent herpes labialis polymorphisms in tlr are associated with increased viral shedding and lesional rate in patients with genital herpes simplex virus type infection beta interferon produced by keratinocytes in human cutaneous infection with herpes simplex virus toll-like receptor -mediated recognition of herpes simplex virus- by plasmacytoid dendritic cells alpha and gamma interferons inhibit herpes simplex virus type infection and spread in epidermal cells after axonal transmission interferon lambda inhibits herpes simplex virus type i infection of human astrocytes and neurons induction of cytokine expression by herpes simplex virus in human monocyte-derived macrophages and dendritic cells is dependent on virus replication and is counteracted by icp targeting nf-kappab and irf- the cycle of human herpes simplex virus infection: virus transport and immune control expression profiles of human interferon-alpha and interferon-lambda subtypes are ligand-and cell-dependent interferon-lambda: a new addition to an old family ifn-lambda (ifn-lambda) is expressed in a tissue-dependent fashion and primarily acts on epithelial cells in vivo automated yeast two-hybrid screening for nuclear receptor-interacting proteins human protein reference database- update the biogrid interaction database: update the database of interacting proteins: update gene ontology: tool for the unification of biology. the gene ontology consortium kegg: kyoto encyclopedia of genes and genomes the kegg resource for deciphering the genome reactome: a knowledgebase of biological pathways reactome: a knowledge base of biologic pathways and processes systematic and integrative analysis of large gene lists using david bioinformatics resources varicella-zoster virus infection of a human cd -positive t-cell line regulation of the transcription and replication cycle of human cytomegalovirus is insensitive to genetic elimination of the cognate nf-kappab binding sites in the enhancer properties of non-structural protein of semliki forest virus and its interference with virus replication vaccinia virus cores are transported on microtubules live visualization of herpes simplex virus type compartment dynamics construction and characterization of bacterial artificial chromosomes containing hsv- strains and kos en passant mutagenesis: a two step markerless red recombination system we are grateful to tomozumi imamichi (national cancer institute, bethesda), rick randall (university of st andrews) and geoffrey l. smith (university of cambridge) for providing reagents, as well as george sorensen, tobias bergmann, gabriella siszler, frank schwarz, melanie ott, verena raschbichler, hannah striebinger, philippa beard and susanne bailer for assistance. key: cord- -italbsed authors: desai, tanay m.; marin, mariana; chin, christopher r.; savidis, george; brass, abraham l.; melikyan, gregory b. title: ifitm restricts influenza a virus entry by blocking the formation of fusion pores following virus-endosome hemifusion date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: italbsed interferon-induced transmembrane proteins (ifitms) inhibit infection of diverse enveloped viruses, including the influenza a virus (iav) which is thought to enter from late endosomes. recent evidence suggests that ifitms block virus hemifusion (lipid mixing in the absence of viral content release) by altering the properties of cell membranes. consistent with this mechanism, excess cholesterol in late endosomes of ifitm-expressing cells has been reported to inhibit iav entry. here, we examined iav restriction by ifitm protein using direct virus-cell fusion assay and single virus imaging in live cells. ifitm over-expression did not inhibit lipid mixing, but abrogated the release of viral content into the cytoplasm. although late endosomes of ifitm -expressing cells accumulated cholesterol, other interventions leading to aberrantly high levels of this lipid did not inhibit virus fusion. these results imply that excess cholesterol in late endosomes is not the mechanism by which ifitm inhibits the transition from hemifusion to full fusion. the ifitm 's ability to block fusion pore formation at a post-hemifusion stage shows that this protein stabilizes the cytoplasmic leaflet of endosomal membranes without adversely affecting the lumenal leaflet. we propose that ifitm interferes with pore formation either directly, through partitioning into the cytoplasmic leaflet of a hemifusion intermediate, or indirectly, by modulating the lipid/protein composition of this leaflet. alternatively, ifitm may redirect iav fusion to a non-productive pathway, perhaps by promoting fusion with intralumenal vesicles within multivesicular bodies/late endosomes. the recently identified interferon-induced transmembrane proteins (ifitms) inhibit infection of diverse enveloped viruses [ ] [ ] [ ] . ectopic expression of ifitm , - and - restricts a growing number of unrelated viruses, including iav [ , , [ ] [ ] [ ] [ ] . ifitm has been shown to potently restrict infection by iav and the respiratory syncytial virus in vivo [ ] [ ] [ ] . in contrast, arenaviruses and some retroviruses, such as murine leukemia virus (mlv), are resistant to ifitm restriction [ , ] . the ifitms have been reported to inhibit hiv- entry, albeit less potently than iav and apparently in a cell type-dependent manner [ ] [ ] [ ] . the mechanism by which ifitms inhibit infection of diverse viruses is not fully understood. ifitm and - are predominantly found in late endosomes (le) and lysosomes [ , ] , whereas ifitm is also found at the cell periphery [ , ] . different membrane topologies of ifitms have been proposed [ ] , but recent data suggests that ifitm is a type ii transmembrane protein [ ] . accumulating evidence implies that ifitms may interfere with virus-endosome fusion [ , , , , ] . the fact that ifitms seem to expand acidic intracellular compartments [ ] indicates that the fusion block is downstream of the low ph trigger. effective restriction of viruses that enter from the le, such as iav, ebola virus (ebov) and sars coronavirus seems consistent with the cellular localization of ifitm and - proteins. however, these proteins also restrict vesicular stomatitis virus (vsv) that appears to fuse with early endosomes [ ] . ifitms have been reported to curtail viral infection by modifying properties of cellular membranes, such as fluidity and spontaneous curvature [ , , ] . these effects could be related, in part, to the accumulation of cholesterol in le as a result of ifitm-mediated disruption of the interaction between the vesiclemembrane-protein-associated protein a (vapa) and oxysterolbinding protein (osbp) [ ] . since lipids play an important role in membrane fusion, these findings offer an attractive paradigm for a broad antiviral defense mechanism that involves altering the lipid composition of cellular membranes. the recent finding that amphotericin b, which forms complexes with sterols [ ] , rescues iav infection in ifitm -and ifitm -expressing cells [ ] is in line with the notion that cholesterol may be directly or indirectly involved in iav restriction. however, lipid composition-based models do not readily explain the lack of restriction of amphotropic mlv and arenaviruses, which enter cells via distinct endocytic routes [ , ] . these findings indicate that ifitms may restrict virus entry from a subset of intracellular compartments. in order to define the mechanism of ifitm restriction, it is important to identify the viral entry step(s) targeted by these proteins, define compartments in which restriction occurs, and elucidate potential changes in intracellular membranes that may be responsible for this phenotype. here, we examined the mechanism of ifitm restriction of iav using single particle imaging and a direct virus-cell fusion assay. our results show that ifitm does not inhibit the lipid mixing stage of iav fusion but blocks the release of viral contents into the cytosol, and that this phenotype does not correlate with cholesterol accumulation in intracellular compartments. specifically, ifitm inhibits the conversion of hemifusion to fusion through a mechanism that does not rely on cholesterol accumulation. together these findings reveal a previously unappreciated view of ifitm-mediated restriction and suggest new avenues of investigation to delineate the mechanism by which these proteins block infection. we chose to focus on ifitm to study the mechanism of iav restriction because this protein potently inhibits infection in vitro and in vivo [ ] [ ] [ ] . since published data suggest that ifitm likely inhibits the viral fusion step, a direct virus-cell fusion assay was employed to evaluate the extent of restriction in different cell lines [ ] . hiv- particles carrying the b-lactamase-vpr (blam-vpr) chimera and pseudotyped with the influenza ha and na proteins from the h n a/wsn/ strain (referred to as iavpp) were allowed to fuse with cells transduced with an empty vector or with an ifitm -expressing vector. the resulting cytosolic blam activity was measured as previously described [ ] . out of several cell lines tested, a and mdck cells over-expressing ifitm were least permissive to iavpp fusion (fig. a) . in agreement with the previous reports [ , ] , we found that ifitm overexpression partially inhibited vsv g glycoprotein-mediated fusion of pseudoviruses (vsvpp) carrying the blam-vpr chimera (fig. a) . similar to inhibition of iavpp fusion, the ifitm mediated restriction of vsvpp was most potent in a and mdck cells. as expected, fusion of particles pseudotyped with the lassa fever virus glycoprotein (lasvpp), which directs virus entry through an ifitm -resistant pathway [ , ] , was not considerably affected by ifitm over-expression. we next checked if the strong suppression of virus fusion in a and mdck cells was related to the level of ifitm expression. immunostaining for ifitm in these and cho cells which exhibited modest restriction of viral fusion (fig. a ) did not reveal a clear correlation between ifitm expression and inhibition of iavpp or vsvpp fusion (fig. b) . of note, potent iav restriction in a and mdck cells was not related to the usage of hiv- corebased pseudoviruses. influenza virus-like particles containing the iav blam-m chimera [ ] also failed to efficiently fuse with a -ifitm and mdck-ifitm cells while fusing well with vectortransduced cells (fig. c) . we also found that both vectortransduced a and mdck cells were highly susceptible to iav infection, as determined by virus titration (see materials and methods). these two cell lines were therefore chosen for studies of ifitm -mediated restriction described below. ifitm-based restriction has been studied using a cell-cell fusion model, as well as by forcing viral fusion with the plasma membrane by lowering the ph [ , ] . since fusion with the plasma membrane is more amenable to mechanistic studies than endocytic entry, we asked whether ifitm can restrict forced iav fusion. exposure to acidic buffer induced iavpp fusion with a -vector cells pretreated with bafilomycin a (bafa ), which blocked low ph-dependent entry from endosomes (fig. d ). the extent of forced fusion was lower compared to the conventional entry route. by contrast, forced iavpp fusion with a -ifitm cells was , -fold more efficient than endocytic fusion with cells not treated with low ph or bafa , showing that ifitm does not restrict iavpp fusion at the cell surface. interestingly, ifitm suppressed iavpp-plasma membrane fusion at low ph (fig. d ), in agreement with the jaagsiekte sheep retrovirus (jsrv) and iav fusion data [ , ] . the inability of ifitm to block iav fusion with the plasma membrane is consistent with its lower abundance at the cell surface [ , , ] and shows that the mechanism of restriction must be studied in intracellular compartments. preponderance of evidence implies that hemifusion is a universal intermediate (reviewed in [ , ] ) that precedes the formation of a fusion pore. having shown that ifitm overexpression inhibits viral fusion (fig. a, c) , we asked whether this protein also blocks the upstream hemifusion step. this was accomplished by labeling the a/pr/ / virus membrane with a self-quenching concentration of vybrant did (vdid), using a modification of the previously published protocol [ ] . incorporation of self-quenching quantities of a lipophilic dye enables the visualization of single lipid mixing events based on the marked increase in fluorescence upon dye redistribution to an endosomal membrane (see for example [ , ] ). significantly, to control for fluctuations in the vdid fluorescence caused by deviation from a focal plane, the viral surface proteins were labeled with the amine-reactive alexafluor- (af ) dye. the relatively steady af signal before and after hemifusion is allowed correcting for the vdid intensity fluctuations due to moving in and out of focus. the vdid/af co-labeling interferon-induced transmembrane proteins (ifitms) block infection of many enveloped viruses, including the influenza a virus (iav) that enters from late endosomes. ifitms are thought to prevent virus hemifusion (merger of contacting leaflets without formation of a fusion pore) by altering the properties of cell membranes. here we performed single iav imaging and found that ifitm did not interfere with hemifusion, but prevented complete fusion. also, contrary to a current view that excess cholesterol in late endosomes of ifitm -expressing cells inhibits iav entry, we show that cholesterol-laden endosomes are permissive for virus fusion. the ability of ifitm to block the formation of fusion pores implies that this protein stabilizes the cytoplasmic leaflet of endosomal membranes, either directly or indirectly, through altering its physical properties. ifitm may also redirect iav to a non-productive pathway by promoting fusion with intralumenal vesicles of late endosomes instead of their limiting membrane. protocol only modestly (, -fold) reduced iav infectivity compared to the mock-labeled viruses (fig. s a ). immunofluorescence staining of af -labeled virions with anti-ha antibodies revealed an excellent co-localization of the two signals (fig. s b , c), thus supporting the notion that af /vdid-labeled particles are bona fide virions. . cells were fixed, permeabilized and immunostained for ifitm (red), as described in materials and methods. the nuclear stain, hoechst- , is shown in blue. (c) ifitm restricts fusion of influenza virus-like particles containing b-lactamase reporter protein fused to the influenza matrix protein- (blam ). experiments were carried out as described above. data are means and sem from independent triplicate experiments. (d) exposure to low ph overcomes the ifitm -mediated block of iavpp fusion. to force pseudovirus fusion at the plasma membrane, a cells transduced with ifitm , ifitm or an empty vector were pretreated with nm bafa for min at uc or left untreated. iavpp/blam-vpr pseudoviruses (moi = ) were bound to cells of in the cold and exposed to either a prewarmed ph . mes-citrate buffer or neutral buffer for min at uc and further incubated in growth medium (with or without bafa ) for min at uc. data are means and sem from independent triplicate experiments. ***, p, . by two-tailed t-test. doi: . /journal.ppat. .g labeled viruses were allowed to enter a -vector cells, and the resulting lipid mixing activity was examined by single particle tracking. a fraction of virions exhibited a marked increase in the vdid signal ( fig. a, b) . redistribution of vdid was mediated by low ph-dependent conformational changes in the iav ha glycoprotein, as evidenced by potent inhibition of lipid mixing by anti-ha antibodies (fig. c ) and by nh cl (fig. a) . without simultaneous monitoring of the viral content release into the cytoplasm, vdid dequenching does not discriminate between hemifusion (operationally defined as lipid mixing without content transfer [ ] ) and full fusion. to avoid over-interpreting dequenching events, we will refer to these events as lipid mixing or hemifusion. a similar vdid dequenching pattern was observed in mdck cells transduced with an empty vector (data not shown). analysis of lipid mixing showed that . . % and . . % of cell-bound particles released vdid in a and mdck cells, respectively (fig. a) . by comparison, a much greater fraction of virions ( . . %) hemifused with cho cells (data not shown), in agreement with the previously reported data [ ] . importantly, iav lipid mixing was readily detected in ifitm + a and mdck cells (figs. d-g and a). not only was lipid mixing not inhibited in a -ifitm cells, but a . -fold greater fraction of particles released vdid in these cells compared to control cells ( fig. a , p, . ). by comparison, ifitm overexpression in mdck cells did not significantly promote vdid dequenching (fig. a) . thus, contrary to the cell-cell fusion results [ ] , ifitm does not inhibit and can even promote iav lipid mixing, consistent with the block of virus entry at a posthemifusion stage. accordingly, the addition of oleic acid, which augments hemifusion by altering spontaneous membrane curvature, did not rescue iavpp or vsvpp fusion with a -ifitm cells (fig. s ). this is in agreement with the recent infectivity results [ ] , but in contrast with the rescue of fusion between jsrv env-and ifitm-expressing cells by this fatty acid [ ] . the higher frequency of vdid dequenching in a -ifitm cells could be caused by the increased endosome acidity compared to control cells [ ] . however, the distribution of waiting times to the onset of lipid mixing was independent of ifitm expression or the type of target cells (a vs. mdck, fig. b , p = . ). the fact that the kinetic curves do not reach plateau indicates that iav entry into a and mdck cells is not completed within the first hour. our results thus demonstrate that ifitm restricts the iav fusion at a post-hemifusion step, most likely at the point of fusion pore opening, as evidenced by the dramatic decrease of the blam signal in a and mdck cells expressing this protein (fig. a ). under our conditions, vdid dequenching was typically completed within a few minutes for both control and ifitm + cells (fig. ) . this dequenching rate is much slower than sudden increases in fluorescence of the iav membrane markers described previously [ , ] . while a portion of vdid dequenching could be completed within seconds (fig. s ), these fast events were not common. slow dequenching was also typical with the vdid/ af -labeled x virus, as well as with the x virus labeled with a -fold excess of did, using the published protocol for single virus imaging [ ] (data not shown). slow vdid dequenching during the first hour of virus-cell coincubation did not appear to result from iav degradation in le/ lysosomes, since the surface-exposed af label persisted long after vdid dequenching was completed and because anti-ha antibodies blocked vdid dequenching (fig. ). in addition, we did not detect any correlation between the lag before the onset of lipid mixing and the vdid dequenching slope (fig. s a ). this result reinforces the notion that late lipid mixing events are mediated by ha and not by virus degradation. control experiments, in which samples were not exposed to laser light during the first min at uc, did not reveal fast dequenching events reaching completion in less than min (data not shown). this control argues against phototoxicity-related attenuation of virus fusogenicity as the cause for sluggish lipid redistribution. since free vdid diffusion between a virus and a small endosome should be completed in less than a second [ , ] , an initial membrane connection between iav and an endosome must severely impair lipid movement. to assess whether early fusion intermediates in control and ifitm + cells restrict vdid diffusion to the same extent, we examined the rate of vdid dequenching. single particle analysis revealed that, in a cells, the average vdid dequenching profile (fig. c ) was independent of ifitm expression, as were the initial slopes of vdid dequenching ( fig. s b , p. . ). these results indicate that ifitm over-expression does not affect the properties of fusion intermediates responsible for vdid redistribution, such as the size and/or architecture of a hemifusion site (e.g., [ , ] ). we then asked whether the rate of vdid dequenching varied depending on the cell type. the average rate of vdid fluorescence increase in mdck cells was , -fold greater than in a cells (figs. c and s b, p, . ). this demonstrates our ability to detect changes in the rate of vdid transfer and shows that lipid transfer lasts several minutes irrespective of the cell type. we also examined the final extent of vdid dequenching, which is proportional to the surface area of a target membrane over which it redistributes. this parameter was not significantly affected by ifitm expression in a cells or by the cell type (mdck vs. a cells, fig. d ). together, similar kinetics and extents of viral lipid dilution in control and ifitm + cells suggest that neither the size/architecture of early fusion intermediates nor the surface area of target endosomes is considerably affected by ifitm expression. to investigate the relationship between lipid mixing and productive iav infection, we compared the fraction of cells ''receiving'' at least one vdid dequenching event in live cell imaging experiments to the fraction of cells that got infected under the same conditions. the only difference was that virus imaging was not continued beyond h after initiation of fusion, whereas infection proceeded overnight. we found that one or more vdid dequenching events occurred in % of a cells while % of cells got infected (fig. s ). under the same conditions, % of mdck cells ''hosted'' one or more dequenching events and % were infected. the greater fraction of infected cells compared to those permissive to hemifusion is likely due to the shorter time widow for single virus imaging, which is likely to miss late vdid dequencing events (fig. b ). the lower apparent fraction of cells supporting vdid dequenching could also be caused by the presence of viruses that did not incorporate self-quenching amounts of vdid. importantly, the comparable efficiencies of lipid mixing and infection, indicate that the former events likely culminate in productive infection. to determine whether ifitm impairs the iav's ability to form small fusion pores, we attempted to load the virus with a content marker by soaking in a concentrated solution of sulforhodamine b, as described in [ ] . however, only a small fraction of af -labeled particles stained with sulforhodamine, and the retained dye was lost in live cell experiments under conditions that blocked iav fusion (data not shown). we therefore ifitm blocks influenza a virus fusion pore plos pathogens | www.plospathogens.org resorted to using hiv pseudoviruses bearing a/wsn/ ha and na glycoproteins and co-labeled with the capsid marker, yfp-vpr, and the content marker, gag-icherry [ , ] . upon virus maturation, the ''internal'' mcherry is proteolytically cleaved off the hiv- gag-icherry and released through a fusion pore, as manifested by the loss of the red signal ( fig. and [ ] ). the yfp-vpr signal, which remained associated with the viral core after fusion, provided a reference signal for single particle tracking. under our conditions , % of double-labeled pseudoviruses entering a -vector cells lost their content marker, while approximately % fused with mdck-vector cells. in sharp contrast, the mcherry release in ifitm + a and mdck cells or in vector-transduced cells in the presence of nh cl could not be detected (fig. e , p, . ). thus, ifitm does not adversely affect iav hemifusion but severely inhibits viral content release into the cytoplasm. together these findings suggest that the mechanism of ifitm -mediated restriction arises from the entrapment of viruses at a hemifusion intermediate prior to fusion pore formation. a recent study has shown that, through disrupting the interaction between vapa and osbp, ifitm causes cholesterol accumulation in le [ ] . based on this finding, the authors proposed that high levels of endosomal cholesterol may inhibit iav fusion and/or the release of nucleocapsid. staining with filipin revealed that ifitm + a cells exhibited increased levels images of vdid dequenching (extended projections) and particle fluorescence intensities obtained by tracking virions in a cells. a schematic illustration of iav hemifusion with an endosome (gray), which leads to vdid dequenching, is overlaid on the graph. i and i are fluorescence intensities immediately before dequenching and at the peak of dequenching, respectively. (c) iav lipid mixing activity in a -vector cells is blocked in the presence of anti-ha antibody. af and vdid-labeled iav were pre-incubated with mg/ml of polyclonal anti-iav antibody (millipore, billerica, ma) for h at room temperature. viruses were then bound to a -vector cells in the cold by spinoculation, and entry was initiated with warm imaging buffer supplemented with mg/ml of the antibody. images were collected from fields and the average fraction of af particles with the vdid signal above the threshold level was determined and normalized to control conditions without the antibody. ***, p, . . (d, e) representative images and analysis of lipid mixing in a -ifitm cells. (f, g) representative images and analysis of lipid mixing in mdck-ifitm cells. the ratio of vdid and af signals (blue line) shows robust increase in the red signal in spite of variations in the green channel caused by axial displacement of the virus. thick lines were obtained by smoothing raw fluorescence intensity data (thin lines). cell contours are shown by dashed lines in a and d. see also corresponding movies s , s and s . doi: . /journal.ppat. .g of intracellular cholesterol (fig. a) . however, the filipin signal was still primarily associated with the plasma membrane and the total cellular cholesterol was not elevated in ifitm + cells (fig. s ). in addition, the overall intensity of intracellular cholesterol poorly correlated with the level of ifitm expression (fig. c ). by comparison, pretreatment of a -vector cells with u a, which inhibits transport of ldl-derived cholesterol from le/lysosomes (reviewed in [ ] ), resulted in a dramatic shift in the filipin staining pattern from the plasma membrane to endosomes (fig. b ). aberrant accumulation of cholesterol in le is also known to occur in cells lacking the functional npc cholesterol transporter [ ] . we therefore knocked down npc expression in a cells using shrna (shnpc , fig. d ) and examined the resulting cholesterol distribution (fig. b) . reduced npc expression correlated with excess cholesterol in intracellular compartments, which was also much more pronounced than endosomal filipin staining in a -ifitm cells. we next asked whether the cholesterol accumulation induced by u a pretreatment or by down regulation of npc can phenocopy the ifitm -mediated restriction of viral fusion. neither iav lipid mixing (vdid dequenching) nor fusion (blam signal) was inhibited by silencing npc in a cells (fig. e, f) . vsvpp also fused with shnpc -transduced cells as efficiently as with control cells (fig. e) . these results show that excess cholesterol does not inhibit viral fusion or hemifusion. in control experiments, silencing the npc expression potently suppressed fusion of ebola gp-pseudotyped particles (ebovpp, fig. e ), which use npc as a receptor [ , ] . similar to the npc knockdown phenotype, pretreatment of a cells with mm u a, which caused cholesterol buildup in endosomes (fig. b) , did not inhibit fusion of iavpp or vsvpp (fig. g) . as will be shown below for mdck cells, higher doses of u a can inhibit viral fusion (fig. g) , but this effect is due to elevation of endosomal ph as opposed to cholesterol accumulation in endosomes. to generalize the effects of excess cholesterol in a cells, we tested whether endosomal cholesterol can inhibit viral fusion in mdck cells. as in a cells, ifitm over-expression in mdck cells caused moderate accumulation of cholesterol in endosomes (fig. a) , while pre-treatment with u a caused a much more dramatic buildup of intracellular cholesterol (fig. b) . however, unlike a cells, iavpp and vsvpp fusion was significantly inhibited in u a-treated mdck cells (fig. c ). since prolonged exposure to u a has been reported to raise endosomal ph [ ] , we sought to determine if insufficiently acidic ph could prevent iav hemifusion/fusion with pretreated mdck cells. the ph in iav-carrying endosomes was measured using virions co-labeled with the ph-insensitive af (green) and cypher e (red), which fluoresces brighter at acidic ph [ ] (fig. s a) . cells were incubated with viruses for min, and the red/green signal ratio from individual particles was measured (fig. s b) . the average ph in virus-containing endosomes of mdck-ifitm cells was slightly less acidic than in control cells: . . (n = ) vs. . . (n = ), respectively ( fig. d and f, p, . ). interestingly, as shown in figure e , endosomal ph in u a-treated mdck cells was markedly shifted to neutral values ( . . , n = , p, . ). since the ph threshold for triggering a/pr/ / fusion is reported to be around . [ ] , elevation of endosomal ph in u a-treated mdck cells is the likely cause of inhibition of viral fusion. together our results we also took advantage of the available cho cell line that does not express npc [ ] to further ascertain the role of endosomal cholesterol in iav fusion. these cells (designated cho-npc ) exhibited exaggerated endosomal cholesterol staining, in sharp contrast to a peripheral staining pattern in parental cho cells (fig. a) . in spite of the high endosomal cholesterol content in cho-npc cells and of the elevated level of total cholesterol (fig. s ), iavpp fused with these cells as efficiently as with parental cells (fig. c) . the npc -null cells also supported iav lipid mixing, albeit at somewhat reduced level compared to control (figs. d and s ) . pretreatment of cho cells with u a also trapped cholesterol in endosomes and raised the total cholesterol content (figs. b and s ), but only modestly diminished the extent of iavpp or vsvpp fusion (fig. e) . interestingly, in contrast to the decreased endosome acidity in mdck cells, endosomes in u a-treated cho cells were more acidic than in control cells (fig. s ) . in control experiments, both the lack of npc expression and u a pretreatment blocked ebovpp fusion (fig. c, e) , consistent with its reliance on npc receptor and high sensitivity to disruptions of cholesterol transport [ ] . together, our results show that the cholesterol accumulation achieved through two different interventions -u a pretreatment and npc silencing -does not phenocopy ifitm mediated restriction of viral fusion. this implies that (i) elevated levels of endosomal cholesterol do not generally confer resistance to viral fusion, and (ii) the mechanism by which ifitm blocks transition from hemifusion to full fusion is not through the mislocalization of cholesterol. the ifitms restrict the cellular entry of multiple pathogenic enveloped viruses. recent studies lead to a model that ifitms inhibit virus-host hemifusion [ ] and that the membranerigidifying properties of cholesterol may contribute to antiviral actions [ ] . in contrast to these studies, our results now demonstrate that ifitm prevents the release of viral genomes into the cytosol by inhibiting viral entry after hemifusion but prior to fusion pore formation (fig. ) . moreover, we found that ifitm can promote hemifusion in some cells, perhaps secondary to its acidifying the endosomal pathway. ifitm therefore does not negatively regulate the properties of contacting leaflets involved in hemifusion, but stabilizes the cytoplasmic leaflet of the endosomal membrane, thereby disfavoring the formation of fusion pores [ ] . in one potential scenario ifitm is located directly at the site of arrested hemifusion, perhaps ''toughening'' the endosomal membrane to create a barrier to viral entry (pathway ). a considerable colocalization of ifitm with internalized iav ( [ ] and fig. s ) is consistent with pathway 's direct mechanism of inhibition. alternatively, ifitm might arrest hemifusion through an indirect mechanism, perhaps involving modulation of lipid and/or protein composition of the cytoplasmic leaflet (pathway ). recent findings that changes in global membrane properties interfere with productive entry would appear to support an indirect mechanism [ , ] . lipids, such as unsaturated fatty acids and cholesterol that confer negative spontaneous curvature to membranes can promote hemifusion (a net negative curvature structure) and disfavor a fusion pore (a net positive curvature intermediate), as has been previously shown for oleic acid [ ] . although this prediction is consistent with efficient lipid mixing in endosomes of ifitm + cells observed in our imaging experiments, several studies [ , [ ] [ ] [ ] and our own results do not support cholesterol accumulation as playing a role in fusion inhibition. we found that cholesterolladen endosomes in cells pretreated with u a or expressing undetectable/low levels of npc supported efficient viral fusion. it is thus possible that ifitm interferes with cellular functions of vapa other than the interaction with osbp, such as regulation of snares and modulation of lateral mobility of membrane proteins (reviewed in [ ] ). ifitm appears to induce the formation multivesicular bodies and increase the number of ilvs [ , ] . one can therefore envision that ifitm may inhibit infection by redirecting viruses to a non-productive pathway, perhaps involving fusion with ilvs instead of the limiting membrane of le (fig. , pathway ) . if, as suggested in [ ] , ifitm disallows back fusion of ilvs with the limiting membrane, then virus-ilv fusion products will likely be degraded. indeed, back fusion has been implicated in the vsv core release into the cytosol following the virus-ilv fusion [ ] . it should be stressed that this ''fusion decoy'' model does not explain the ability of ifitm to interfere with fusion at the cell surface ( [ ] and fig. d ). it is also not clear why the old world arenaviruses, which have been reported to enter from mvbs [ ] , are not restricted by ifitms. the indistinguishable extents of vdid dequenching in control and ifitm + cells (fig. d) indicate that target endosomes have similar sizes. while this appears to argue against redirection of iav fusion to small ilvs, the lack of a post-hemifusion decay of vdid fluorescence in a and mdck cells (figs. and s ) is consistent with iav fusion with abundant ilvs in endosomes of ifitm + cells. this is because a lipophilic dye in the limiting membrane of an endosome should be quickly removed through membrane trafficking [ , , ] . because post-dequenching decay was not observed irrespective of the level of ifitm expression, it is possible that iav may infect several cell lines by fusing with small intralumenal vesicles followed by the nucleocapsid release through back fusion (fig. , dashed black arrows) . this pathway could explain the similar extents and rates of vdid dequenching in control and ifitm -expressing cells, which are indicative of similar lipid intermediates and of the size of a target membrane, respectively. as discussed above, slow vdid dequenching observed by single iav imaging can be rationalized in the context of fusion with the limiting membrane of endosomes (pathways and ), as well as in the context of fusion with ilvs (pathway ). slow dilution of this dye in pathway could occur through multiple rounds of iav fusion with small ilvs, whereas pathways and would predict restricted lipid diffusion through early fusion intermediates formed at the limiting membrane. although the latter notion is in agreement with the reported restriction of lipid movement through hemifusion sites and small fusion pores [ , , , ] , these intermediates are usually short-lived under physiological conditions and tend to resolve into larger structures that do not impair lipid movement [ , , ] . clearly, more detailed studies of virusendosome hemifusion and fusion are needed to understand the nature of slow lipid redistribution between iav and endosomes. the ifitms may now arguably be one of the most broadly acting and clinically relevant restriction factor families [ , ] . while both ifitm 's membrane-associated topology and its localization to the site of viral attenuation suggest it acts to restrict viral entry via a direct mechanism, additional work remains to be done to fully elucidate its actions. nonetheless, as the primary effector of ifn's anti-iav actions, ifitm represents a previously unappreciated class of restriction factor that prevents viral entry by stabilizing a hemifusion intermediate, likely comprised of an invading virus fatally tethered to the interior of the endosome's limiting membrane. future single virus experiments combining the detection of both viral lipid and content release events (see for example [ ] ) should provide further insights into iav entry pathways and the mechanism of ifitm -mediated restriction. indeed, such efforts may also bring to light unknown viral countermeasures, which are perhaps employed by the ifitmresistant new and old world arenaviruses. cell lines, plasmids and reagents hek t/ cells and human lung epithelial a cells were obtained from atcc (manassas, va) and grown as previously described [ ] . wild-type cho cells and cho-npc cells, a gift from dr. l. liscum (tufts university) [ ] , were grown in alpha-mem (quality biological inc, gaithersburg, md) supplemented with % fbs and penicillin-streptomycin. the a , mdck, helah and cho cells stably expressing ifitm or ifitm were obtained by transducing with vsv-g-pseudotyped viruses encoding wild-type ifitm and ifitm or with the vector pqcxip (clontech) and selecting with puromycin, as described previously [ ] . the pr denv, blam-vpr, pcrev, hiv- gag-icherrydenv and pmdg vsv g expression vectors were described previously [ , ] . the yfp-vpr was a gift from dr. t. hope (northwestern university). the pcaggs vectors encoding influenza h n wsn ha and na were provided by donna tscerne and peter palese, and the pcaggs blam (wsn) plasmid was a gift from dr. a. garcia-sastre (mount sinai). vectors expressing phcmv-gpc lassa and pcdna . -ebola gp (zaire) were gifts from dr. f.-l. cosset (université de lyon, france) [ ] and dr. l. rong (university of illinois) [ ] , respectively. u a was from tocris bioscience (bristol, uk). poly-llysine, filipin, sulphorhodamine b bafilomycin a and the cholesterol kit were from sigma-aldrich. alexafluor- amine-reactive carboxylic acid, vybrant-did (vdid, , -dioctadecyl- , , , -tetramethylindodicarbocyanine, -chlorobenzenesulfonate salt), hoechst- and live cell imaging buffer were purchased from life technologies (grand island, ny). cypher e mono nhs ester was from ge healthcare (pittsburgh, pa). antibodies used were rabbit anti-ifitm (to n-terminus) from abgent (san diego, ca), mouse anti-iav-np and goat anti-iavpolyclonal antibodies from millipore (billerica, ma), rat antimouse-igg-fitc from ebioscience (san diego, ca), and goat anti-rabbit-cy from jackson immunoresearch (west grove, pa). pseudovirus production and titration were described previously [ ] . pseudoviruses were produced by transfecting hek t/ cells using jetprime transfection reagent (polyplus-transfection sa, ny). for lasv and ebov pseudoviruses, mg of the phcmv-gpc lassa or mg of the pcdna . -ebola gp was included in the transfection mixture. fluorescently labeled influenza pseudoviruses were produced using mg of pr denv, mg of hiv- gag-icherrydenv [ ] , mg of yfp-vpr, mg of pcrev, and mg of each wsn ha-and na-expressing vectors. ebola gp pseudoviruses were concentrated , using lenti-x tm concentrator (clontech, mountain view, ca). to generate influenza blam vlps, hek t cells were transfected with pcaggs-blam ( mg) and . mg of each pcaggs-wsn ha and pcaggs-wsn na. after h, the transfection reagent was removed, and cells were further cultivated in phenol red-free growth medium. the influenza virus surface proteins and the lipid membrane were labeled with af and vdid, respectively. a hundred mg of influenza virus from the purified h n a/pr/ / stock ( mg/ ml, charles river, ct) was diluted in ml of sodium bicarbonate buffer (ph . ) supplemented with mm af . the mixture was incubated for min at room temperature, after which time, ml of vdid (from mm stock in dmso) was added followed by an additional incubation for min in the dark at room temperature with mild agitation. the labeled viruses were purified through a nap- gel filtration column (ge healthcare) in mm nacl solution buffered with mm hepes, ph . . approximately % of af -labeled particles incorporated detectable amounts of vdid with minimal contamination by free dye aggregates. the infectious iav titer was determined in mdck or a cells after incubation with serially diluted inoculum for h at uc. cells were fixed, permeabilized, blocked and incubated with rabbit r anti-wsn ha antibody (a gift from dr. d. steinhauer, emory university) for h at room temperature. cells were then washed and incubated with secondary cy -conjugated goat anti-rabbit antibodies (jackson immunoresearch, pa) in % fbs-containing buffer supplemented with mg/ml hoechst- for h. the number of infected cells per image field was determined by fluorescence microscopy and normalized to the total number of cells (stained nuclei). the infectious titer (iu/ml) was calculated by taking into account the ratio of the area of well and the image area and correcting for dilution and volume of viral inoculum. the b-lactamase (blam) assay for virus-cell fusion was carried out as described previously ( [ ] and methods s ). briefly, pseudoviruses bearing b-lactamase-vpr chimera (blam-vpr) were bound to target cells by centrifugation at uc for min at g. unbound viruses were removed by washing, and fusion was initiated by shifting to uc for min, after which time cells were placed on ice and loaded with the ccf -am substrate (life technologies). the cytoplasmic blam activity (ratio of blue to green fluorescence) was measured after an overnight incubation at uc, using the synergy ht fluorescence microplate reader (bio-tek, germany). iav was pre-bound to a -ifitm cells in the cold, followed by incubation at uc for min and immunostaining with mouse anti-iav-np (millipore, billerica, ma) (when applicable) and rabbit anti-ifitm antibody (n-terminus, abgent, san diego, ca), as described in [ ] . rat anti-mouse-igg-fitc (ebioscience, san diego, ca) and goat anti-rabbit-cy antibodies were used for secondary staining. cellular distribution of cholesterol was examined by incubation with . mg/ml filipin added during the incubation with secondary antibodies. images were collected on a lsm laser scanning microscope (carl zeiss, germany) using a oil immersion objective. all staining methods involved fixation with % paraformaldehyde, permeabilization with . % triton-x , blocking in with % fbs and dilution in phosphate buffered saline (with calcium and magnesium), and sequential incubation with primary and secondary antibodies for h and h, respectively. to silence the npc gene, a cells were transduced with five shrnas encoded by plk . lentiviral vector (sigma) and selected with puromycin. the samples for western blotting were processed as described in [ ] . the npc protein band was detected with rabbit anti-npc (abcam, cambridge, ma) and horseradish peroxidase-conjugated protein g (bio-rad, hercules, ca), using a chemiluminescence reagent from ge healthcare. stage of an lsm confocal microscope. virus entry was initiated by adding . ml of pre-warmed imaging buffer and imaged at uc using a c-apo / . na water-immersion objective. three z-stacks separated by , mm were acquired every - s through the multitime macro (carl zeiss). to block iav hemifusion and fusion, experiments where performed in hbss supplemented with mm hepes/ mm nh cl (ph . ) or containing nm of bafa . the time lapse images were first visually inspected to identify vdid dequenching or loss of mcherry events. the number of relevant events in each experiment was independently determined by two trained individuals. particle trajectories and their mean/total fluorescence intensities were obtained using volocity (perkinelmer, ma). the onset of lipid mixing and the initial slope of vdid dequenching were determined by fitting to a pair of straight lines (fig. s ). iav particles were co-labeled with the af dye (phinsensitive) and cypher e, which fluoresces brighter at acidic ph. the ratios of the cypher e and af signals were converted to ph values using a calibration curve obtained by exposing coverslip-immobilized viruses to citrate-phosphate buffers of different acidity (fig. s ). images were collected from different fields, and sum of single-particle fluorescence was calculated. the mean ratios of cypher e to af signals as a function of ph were used for the calibration curve. cells were inoculated with labeled viruses for min at uc, as described above. images were collected from at least different fields, and single particle-based ratio of fluorescence signals was calculated. outliers with a near-background cypher e signal were rejected to reduce the uncertainty in ph measurements. statistical significance was assessed using the pairwise t-test or rank sum test. single-particle fusion events in control and ifitm expressing cells were compared by the z-test. figure s relationship between iav lipid mixing activity and infection. the fraction of a cells where at least one lipid mixing event was observed within h at uc, and the fraction of cells that became infected within h at uc were estimated as described in methods s . infectivity data were collected from image fields each, with . cells per field. particle-to infectivity ratio was calculated from the fraction of infected cells and the average number of virions bound to cells. figure s calibration of labeled iav as a ph-sensor. af -and cypher e-labeled iav particles were attached to poly-l-lysine coated coverslips, and the ratio of two fluorescence signals was measured in citrate-phosphate buffers of different acidity. (a) top and bottom panels are images of labeled iav at neutral ph and low ph, respectively. (b) the total signal for each dye was determined after thresholding and the cypher e/af ratio at different ph are plotted. error bars are standard deviations for different imaged fields for each ph value. the line indicates a first order polynomial fit to the data, which served as a ph calibration curve. (pdf) figure s an example of single iav lipid mixing event in cho cells. (a) image panels show entry of an af (green) and vdid (red) labeled virus into a cho cell that culminates in vdid dequenching (arrow). (b) fluorescence intensity profiles of af and vdid obtained by tracking the virion shown in panel a. (pdf) figure s ph distribution in iav carrying endosomes of cho cells. shown are the distributions of endosomal ph in cho cells pretreated with mm of u a for h or left untreated. cells were incubated with af /cypher e-labeled iav, and endosomal ph was measured as described in materials and methods. u a increased endosomal acidity (p, . ). (pdf) figure s incoming iav tends to colocalize with ifitm -positive endosomes. a -ifitm cells were allowed to internalize iav for min at uc and immunostained for the iav-np using mouse antibody (millipore, billerica, ma) and for ifitm . the enlarged boxed area is shown on the right. iav and ifitm puncta were identified by thresholding and object identification. the extent of colocalization was estimated by counting iav puncta, which exhibited a volumetric overlap of at least % with ifitm puncta, and normalizing over all iav puncta. the number in the right corner is the mean % colocalization and standard deviation for image fields. (pdf) figure s a line-fitting approach to determining the onset and the initial rate of vdid dequenching in single iav fusion experiments. fitting the vdid dequenching traces with two straight lines yields the time of hemifusion (t h ) and the initial slope of dequenching. (pdf) methods s description of additional methods employed in this study. movie s lipid mixing between single vdid-labeled iav and an endosome in a cells. iav co-labeled with af (green) and vdid (red) was incubated with a cells at uc. the lipid mixing event (hemifusion) is manifested in marked increase of vdid fluorescence. the numbers in the upper right corner show time after raising the temperature (min:sec:msec). scale bar is mm. for details, see fig. a movie s lipid mixing upon entry of single vdidlabeled iav into an mdck-ifitm cell. iav co-labeled with af (green) and vdid (red) was incubated with cells at uc. lipid mixing (hemifusion) is seen as marked increase in the vdid signal. the numbers in the upper right corner show time after raising the temperature (min:sec:msec). for details, see the broad-spectrum antiviral functions of ifit and ifitm proteins the ifitm proteins mediate cellular resistance to influenza a h n virus, west nile virus, and dengue virus ifitms restrict the replication of multiple pathogenic viruses ifitm- and ifitm- but not ifitm- restrict rift valley fever virus ifitm proteins restrict viral membrane hemifusion distinct patterns of ifitm-mediated restriction of filoviruses, sars coronavirus, and influenza a virus identification of five interferon-induced cellular proteins that inhibit west nile virus and dengue virus infections ifitm limits the severity of acute influenza in mice ifitm restricts the morbidity and mortality associated with influenza defining the range of pathogens susceptible to ifitm restriction using a knockout mouse model characteristics of ifitm, the newly identified ifninducible anti-hiv- family proteins the ifitm proteins inhibit hiv- infection ifitm inhibits influenza a virus infection by preventing cytosolic entry the antiviral effector ifitm disrupts intracellular cholesterol homeostasis to block viral entry the cd domain of ifitm is required for both ifitm protein association and inhibition of influenza a virus and dengue virus replication palmitoylome profiling reveals s-palmitoylation-dependent antiviral activity of ifitm interferon-induced transmembrane protein is a type ii transmembrane protein differential requirements of rab and rab for endocytosis of influenza and other enveloped viruses interaction of polyene antibiotics with membrane lipids: physicochemical studies of the molecular basis of selectivity amphotericin b increases influenza a virus infection by preventing ifitm -mediated restriction different mechanisms of cell entry by human-pathogenic old world and new world arenaviruses caveola-dependent endocytic entry of amphotropic murine leukemia virus a sensitive and specific enzymebased assay detecting hiv- virion fusion in primary t lymphocytes hiv enters cells via endocytosis and dynamin-dependent fusion with endosomes an enzymatic virus-like particle assay for sensitive detection of virus entry membrane hemifusion: crossing a chasm in two leaps the energetics of membrane fusion from binding, through hemifusion, pore formation, and pore enlargement visualizing infection of individual influenza viruses characterization of the early events in dengue virus cell entry by biochemical assays and single-virus tracking gpi-anchored influenza hemagglutinin induces hemifusion to both red blood cell and planar bilayer membranes viral membrane fusion and nucleocapsid delivery into the cytoplasm are distinct events in some flaviviruses observation of single influenza virus-cell fusion and measurement by fluorescence video microscopy diffusion and redistribution of lipid-like molecules between membranes in virus-cell and cell-cell fusion systems membrane flux through the pore formed by a fusogenic viral envelope protein during cell fusion the pathway of membrane fusion catalyzed by influenza hemagglutinin: restriction of lipids, hemifusion, and lipidic fusion pore formation singleparticle kinetics of influenza virus membrane fusion fusion of mature hiv- particles leads to complete release of a gag-gfp-based content marker and raises the intraviral ph cellular mechanism of u a-mediated apoptosis in cultured murine cortical neurons: bridging niemann-pick disease type c and alzheimer's disease lipid and cholesterol trafficking in npc ebola virus entry requires the cholesterol transporter niemann-pick c small molecule inhibitors reveal niemann-pick c is essential for ebola virus infection regulation of the v-atpase along the endocytic pathway occurs through reversible subunit association and membrane localization ph-dependent hemolysis by influenza, semliki, forest virus, and sendai virus the transport of low density lipoprotein-derived cholesterol to the plasma membrane is defective in npc cells multiple cationic amphiphiles induce a niemann-pick c phenotype and inhibit ebola virus entry and infection cholesterol promotes hemifusion and pore widening in membrane fusion induced by influenza hemagglutinin sterols and sphingolipids strongly affect the growth of fusion pores induced by the hemagglutinin of influenza virus multiphasic effects of cholesterol on influenza fusion kinetics reflect multiple mechanistic roles non-vesicular lipid transport by lipid-transfer proteins and beyond endosome-tocytosol transport of viral nucleocapsids old world arenaviruses enter the host cell via the multivesicular body and depend on the endosomal sorting complex required for transport imaging single retrovirus entry through alternative receptor isoforms and intermediates of virus-endosome fusion the lipid-anchored ectodomain of influenza virus hemagglutinin (gpi-ha) is capable of inducing nonenlarging fusion pores restricted movement of lipid and aqueous dyes through pores formed by influenza hemagglutinin during cell fusion inhibition of hiv- endocytosis allows lipid mixing at the plasma membrane, but not complete fusion characterization of lassa virus cell entry and neutralization with lassa virus pseudoparticles comprehensive analysis of ebola virus gp in viral entry multifaceted mechanisms of hiv- entry inhibition by human alphadefensin we wish to thank dr. david steinhauer (emory university) for the gift of r antibody, dr. laura liscum (tufts university) for cho-npc cells, dr. f.-l. cosset (université de lyon, france) and dr. l. rong (university of illinois) for lassa virus and ebola virus gp-expressing vectors, respectively. we are also grateful to dr. n. gahlaut for help with image analysis and lauren byrd-leotis for the initial efforts to label and image iav, as well as to dr. leonid chernomordik (nichd) and the members of melikyan laboratory for stimulating discussions. key: cord- -cpryxa p authors: lello, laura sandra; utt, age; bartholomeeusen, koen; wang, sainan; rausalu, kai; kendall, catherine; coppens, sandra; fragkoudis, rennos; tuplin, andrew; alphey, luke; ariën, kevin k.; merits, andres title: cross-utilisation of template rnas by alphavirus replicases date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: cpryxa p most alphaviruses (family togaviridae) including sindbis virus (sinv) and other human pathogens, are transmitted by arthropods. the first open reading frame in their positive strand rna genome encodes for the non-structural polyprotein, a precursor to four separate subunits of the replicase. the replicase interacts with cis-acting elements located near the intergenic region and at the ends of the viral rna genome. a trans-replication assay was developed and used to analyse the template requirements for nine alphavirus replicases. replicases of alphaviruses of the semliki forest virus complex were able to cross-utilize each other’s templates as well as those of outgroup alphaviruses. templates of outgroup alphaviruses, including sinv and the mosquito-specific eilat virus, were promiscuous; in contrast, their replicases displayed a limited capacity to use heterologous templates, especially in mosquito cells. the determinants important for efficient replication of template rna were mapped to the ' region of the genome. for sinv these include the extreme '- end of the genome and sequences corresponding to the first stem-loop structure in the ' untranslated region. mutations introduced in these elements drastically reduced infectivity of recombinant sinv genomes. the trans-replicase tools and approaches developed here can be instrumental in studying alphavirus recombination and evolution, but can also be applied to study other viruses such as picornaviruses, flaviviruses and coronaviruses. a a a a a the genus alphavirus (family togaviridae) comprises approximately known virus species. many of these are "arboviruses", infecting vertebrate hosts and are transmitted through the bite of an arthropod vector, commonly mosquitoes. many alphaviruses present a threat to human health. these include chikungunya virus (chikv), which recently (re-)emerged in asia, africa and america [ ] , o'nyong-nyong virus (onnv) that is widespread in africa [ ] , ross river virus (rrv) which is epidemic in australia/oceania [ ] and venezuelan equine encephalitis virus (veev) which is found in the americas [ , ] . in addition to arboviruses there are also horizontally transmitted alphaviruses such as salmon pancreas disease virus (salmonid alphavirus, sav) infecting aquatic species. some alphaviruses, such as eilat virus (eilv), lack a vertebrate host and infect arthropods exclusively [ , ] . the members of the genus alphavirus are divided into several groups (complexes) that form three major clades [ ] . alphaviruses having vertebrate hosts are also often divided according to their geographical distribution and pathogenesis associated with their infection. new world alphaviruses, exemplified by veev and western equine encephalitis virus (weev), cause encephalitis while old world alphaviruses, including sindbis virus (sinv, type member of the genus), chikv, onnv, rrv and barmah forest virus (bfv) cause fever, rash and arthritic symptoms. this division is supported by molecular biological evidence indicating that old and new world alphaviruses have different mechanisms to suppress host cell transcription and to counteract host antiviral mechanisms [ ] . there are also notable differences in the host factors these viruses require for their genome replication [ ] . however, the correlation between current geographical distributions and categorization as new or old world alphaviruses is not absolute. for example, mayaro virus (mayv) found in south america belongs to the semliki forest virus (sfv) complex of old world alphaviruses [ ] . similarly, phylogenetic analysis suggests that sinv has its origin in the new world regions [ ] . thus, it is likely that alphaviruses have spread from one geographical area to another, possibly in migratory birds [ , ] . weev has its ancestry in a recombination event between eastern equine encephalitis virus and sinv-like viruses [ ] . taken together, the evolutionary history of alphaviruses is complex and only partially understood. alphaviruses have a positive strand rna genome of approximately kb in length, with a ' type- n -methylguanosine cap structure, a ' poly(a) tail and contain two open reading frames (orf). the first orf encodes four non-structural proteins (nsp - ) as polyprotein precursors p or p , with p being synthesized by read-through of a stop codon between nsp and nsp [ ] . these polyproteins are processed by the protease activity of the compensated for by compensatory changes in sequences at the ends of the virus genome [ ] . conversely, mutations in the ' utr result in adaptive changes in virus-encoded proteins [ ] . the alphavirus replicase possesses a high activity in trans and is capable of replicating rnas containing suitable structures at their ' and ' ends including defective interfering (di) rnas [ ] [ ] [ ] [ ] . if the di rna contains sg promoter sequences, sg rnas are also synthesized. these properties have been utilized to develop a packaging systems for alphavirus replicon vectors [ , ] and, more recently, trans-replicase systems for different alphaviruses [ ] [ ] [ ] . it has also allowed uncoupling of the replicase protein synthesis from its mrna replication. this property was used to analyse template requirements of sfv and sinv replicases [ ] . however, an overall picture of the cross-utilisation of template rnas by replicases of different alphaviruses and their host-cell type dependence has been lacking, hampering analysis of the organization of alphavirus replicase complex and replicase protein/rna interactions. information on the potential for cross-utilisation of template rnas by related viruses is important for understanding the basic properties of (alpha)virus infection such as superinfection exclusion or rescue. this study used extremely sensitive trans-replicase systems, constructed previously for different alphaviruses [ ] . the cross-utilisation of the template rnas by the different replicases was analysed in both human and mosquito cells. this analysis revealed the existence of templates with different promiscuity. in general, the cross-utilisation of rna templates was found to be similar in human and mosquito cells. replicases of alphaviruses not belonging to the sfv complex showed high preference for their own rna templates, with their capacity for using heterologous templates being more limited in mosquito cells compared to human cells. the sequence determinants responsible for the capacity of sinv, rrv and chikv replicases to use each other's templates were shown to locate in the ' region of the template rna. for sinv replicase they mapped to the extreme ' end of the genome and the first sl structure. mutations introduced into these elements had a severe impact on template rna replication and on the rescue of recombinant sinv from infectious transcripts. in addition to revealing the broader picture of rna template cross-utilisation by alphavirus replicases, our study also provides new and highly efficient tools to generate replicating rnas that can be used for analysis of their structure in cells. this also opens new possibilities for genetic attenuation of alphaviruses and for development and analysis of compounds targeting critical regions of alphavirus rna genomes. we have previously shown that replicases of eight arbovirus members of the alphavirus genus are capable of replicating and transcribing their cognate template rnas in human cells [ ] . here, we additionally created replicase and template rna expression constructs for the mosquito-specific eilv. in order to allow the analysis of rna replication in mosquito cells, template rna and replicase expression plasmids for ae albopictus cells were constructed using designs previously described for chikv [ ] . altogether, this resulted in nine sets of template rna/replicase expression plasmids for each virus in human ( fig a) and ae albopictus cells (fig b) . in order to compare replication in two different host cell types, replicase expression constructs used in this study were based on native coding sequences rather than host-cell adapted coding sequences. sfv, mayv, rrv, chikv and onnv belong to the sfv complex while sinv, veev, bfv and eilv represent outgroup alphaviruses. the relative degrees of similarity between the replicases of sfv complex and outgroup viruses are indicated in fig c. the differences between outgroup viruses and those belonging to the sfv complex had an impact on the design of the template rnas. the ' region of the chikv genome contains seven sl structures that affect genome replication in mammalian and/or mosquito cells [ ] . as similar cmv, immediate early promoter of human cytomegalovirus; li, leader sequence of the herpes simplex virus thymidine kinase gene with artificial intron; sv ter, simian virus late polyadenylation region; hspoli, a truncated promoter (residues − to − ) for human rna polymerase i; mmter, a terminator for rna polymerase i in mice. (b). schematic representation of constructs for aedes albopictus cells. ubi, polyubiquitin promoter of aedes aegypti; ul, leader sequence of aedes aegypti polyubiquitin gene with a natural intron; albpoli-truncated promoter (residues − to − ) for aedes albopictus rna polymerase i; albter-putative terminator for aedes albopictus rna polymerase i. (a, b) utr, full length ' utr of an alphavirus; ' utr, truncated (last residues) utr of an alphavirus; sg-sg promoter spanning (with respect to termination codon of nsp ) from position - to the end of intergenic region, nsp n � -region encoding the n-terminal to amino acid residues of nsp , depending on the virus; hdv rz-antisense strand ribozyme of hepatitis delta virus. red arrow indicates the location of the gdd motif in nsp ; in polymerase negative constructs this was replaced by gaa. the vector backbones are not shown and drawings are not to scale. (c) phylogenetic tree of replicases of analysed alphaviruses. phylogenetic tree was constructed using evolutionary analysis by maximum likelihood method and jtt matrix based model. the tree is drawn to scale, with branch lengths measured in the number of substitution per site. this analysis involved sequences of p of indicated viruses. evolutionary analysis was conducted using mega-x software. https://doi.org/ . /journal.ppat. .g cross-utilisation of template rnas by alphavirus replicases structures can be predicted for rnas of other members of the sfv complex (s fig), the ' ends of the template rnas of sfv, mayv, rrv and onnv had an identical design to that of the previously constructed chikv template, i.e. the ' utr was followed by nt encoding the n-terminus of nsp [ ] . in contrast, the predicted final downstream sl in the ' genomic region of the outgroup viruses (equivalent to sl in chikv [ ] ) align less well or, for bfv and sinv, is predicted to have different secondary structure (s fig) . therefore, based on our previously published predictions for the structure and position of secondary structure elements homologous to those in chikv, longer coding regions of nsp were incorporated into the template rnas of these viruses. these had variable lengths, i.e. nt for eilv, nt for veev, nt for bfv and nt for sinv. for simplicity, hereafter the full-length rna serving as template for fluc expression is termed "genomic rna" (and its synthesis as "replication"), the rna synthesized from the sg promoter serving as template for gluc expression is termed "sg rna" (and its synthesis as "transcription") and all rnas synthesized by trans-replicases are referred to as "viral rnas". the levels of fluc and gluc expression in human cells, transfected with plasmids expressing template rna and corresponding polymerase negative control replicase (p gaa ), were similar for trans-replicases derived from different viruses and the same was observed for ae albopictus cells. therefore, the efficiency of replication and transcription were estimated by fold changes ("boost") of corresponding reporter expression i.e. reporter activity in cells expressing native p of alphavirus relative to those expressing its polymerase-negative p gaa variant. in human cells, the expression kinetics of the gluc marker were highly similar for all eight trans-replicases of arbovirus members of the genus (fig a) . based on these data, a single time point, set at h post transfection (h p.t.), was used in subsequent experiments. as expected, the mosquito-restricted eilv replicase was inactive at ˚c, however, when the experiment was performed at ˚c, significant activity was observed ( fig b) . thus, the eilv replicase has a temperature-sensitive phenotype in human cells, but has no absolute requirement for mosquito-specific factors. with two exceptions, the replicases of all analyzed viruses boosted the expression of fluc and gluc markers to high levels. the activity of onnv replicase was clearly lower than that of other replicases, changing the time point for measurement (fig a) or the ratio of replicase and template rna expression plasmids failed to increase the boost in marker expression. it cannot be excluded that the low efficiency reflects a low level of onnv replicase expression, for example due to rare codons and/or cryptic splicing sequences present in the coding sequence. however, it is more likely that the relatively low activity of the onnv transreplicase is a feature caused by the specific properties of onnv p . veev trans-replicase, albeit efficiently boosting expression of gluc, was relatively less efficient in boosting expression of fluc, indicating only modest template rna replication (fig b) . in order to confirm that the quantification of fluc and gluc activities indeed correlate with the synthesis of viral rnas, rna synthesis was analyzed using northern blot. the observed levels of viral rnas clearly correlated with those deduced from analysis of reporter activities. all highly active trans-replicases synthesized high levels of negative and positive strand rnas. in contrast, synthesis of viral rnas by eilv replicase was below the limit of detection at both ˚c and ˚c. synthesis of negative strand rnas by onnv and veev replicases was also close to the detection level. in contrast, positive strands made by these replicases were clearly detected, although onnv replicase was found to synthesize relatively little sg rna (fig c) , which is consistent with the modest boost of gluc expression (fig b) as well as with its absolute activity (fig a) . the amount of sg rna made by the veev replicase was much lower t. growth media aliquots were collected the activity of secreted gluc measured. means of relative luminescence units (rlu) per μl of sample + standard deviation (sd) of three independent experiments are shown. (b) hek t cells in -well plates were co-transfected with matching pairs of cmv-p and hspoli-fg plasmids and, as negative control, cmv-p gaa , which lacks polymerase activity, instead of cmv-p . cells were incubated at ˚c and lysed h post transfection (p.t.); cells transfected with plasmids containing sequences from eilv were also incubated at ˚c and lysed h p.t. fluc (marker of replication, left panel) and gluc (marker of transcription, right panel) activities produced by active replicases were normalized to the p gaa controls. value obtained for p gaa controls was taken as ; activities lower than that observed for p gaa are also shown as . means + sd of three independent experiments are shown; p< . �� ; p< . ��� (student's unpaired t-test). (c) hek t cells in -well plates were co-transfected and incubated as described for panel a; control cells were mock-transfected. total rna was extracted and analysed by northern blotting. full-length "genomic" template rna of positive (+) and negative (-) polarity and subgenomic rna are indicated. note that transcripts made by human rna polymerase i using hspoli-fg plasmids as templates co-migrate with replicase-generated positivestrand genomic rna and are detected by the same probe. the experiment was repeated twice with similar results; data from one experiment is shown. https://doi.org/ . /journal.ppat. .g cross-utilisation of template rnas by alphavirus replicases compared to the amounts of sg rnas made by replicases of sinv or sfv. compared with sinv or sfv there was approximately -fold difference in gluc expression (fig a) , this is however less prominent than the differences observed between corresponding sg rna levels ( fig c) . furthermore, the boosts of gluc expression observed for the replicases of veev, sinv and sfv were similar (compare fig b and c ). most likely the somewhat elevated expression levels of gluc in the veev trans-replication system originated from basic properties of the veev replicase. nsp and nsp proteins of old world alphaviruses inhibit transcription and translation in vertebrate cells [ , ] . in new world alphaviruses the most prominent inducer of cytotoxic effects is the capsid protein [ ] which is absent in the veev trans-replicase. it has been observed that in the absence of host cell shutdown the sg rnas of alphaviruses lose their competitive advantage over the host cell mrnas but, nonetheless, the absolute translation efficiency of the sg rna increases [ ] [ ] [ ] . therefore, it is reasonable to assume that sg rnas made by the veev replicase are more efficiently translated than those made by trans-replicases of old world alphaviruses. except for onnv all trans-replicases were found to display similar gluc expression profiles in ae albopictus cells (fig a) , enabling us to use a single time point, set at h p.t., for subsequent experiments performed in c / cells. analysis performed in c / cells also revealed that the activities of alphavirus trans-replicases were approximately - -fold lower than in human cells. the boost of fluc expression was typically as low as -fold while the boost of gluc expression was around -fold. not surprisingly, the exception to this rule was the eilv trans-replicase, for which both replication and transcription in c / cells were higher than these obtained in human cells under similar conditions (compare figs b and b). it was also observed that the trans-replicase activities differed across different alphaviruses. notably, the activation of gluc and fluc expression by onnv replicase occurred only at very low level ( fig b) . combined with low absolute levels of gluc expression ( fig a) this indicates that onnv trans-replicase was virtually inactive in ae albopictus cells. this cell-type specific defect most likely reflects the fact that, in its natural cycle, onnv is transmitted by anopheles and not by aedes mosquitoes [ ] . no boost in fluc expression and only a very modest boost of gluc expression were observed with the bfv trans-replicase ( fig b) . again, low activity may reflect the fact that bfv is mostly transmitted by culex annulirostris or aedes vigilax mosquitoes [ , ] . interestingly, a very low boost of fluc expression was also observed for replicases of rrv and mayv (fig b) , which are associated with culex annulirostris and aedes vigilax [ ] or haemagogus mosquitos [ ] , respectively. thus, there appears to be at least some correlation between vector preference of an alphavirus and the activity of the corresponding transreplicase in c / cells. however, this correlation should not be over-emphasized as for rrv and mayv the boost of gluc expression (transcription) in c / cells were relatively high ( fig b) . furthermore, alphaviruses can use different vectors for transmission [ ] and these viruses are often capable of replicating in cultivated cells of aedes mosquitoes even when the cells are not from a field-relevant vector species. consistent with the modest boost of gluc expression, only very low levels of sg rna were detected in cells transfected with the bfv trans-replicase ( fig c) . trans-replicases of sfv, chikv, rrv, mayv, veev, sinv and eilv synthesized sg rnas at high levels ( fig c) . the highest sg rna levels were observed for sinv, mayv and sfv trans-replicases ( fig c) , correspondingly the absolute levels of gluc expression were also highest for these three trans-replicases ( fig a) . thus, expression of gluc serves as a reliable marker for replicasemediated transcription also in ae albopictus cells. interestingly, however, production of viral fig b and c ). hence, for some of the alphaviruses the boost of fluc expression in ae albopictus cells cannot be used as a reliable marker for levels of genomic rnas generated by their trans-replicases (compare left panel of fig b and fig c) . consistent with our previous observations [ ] , levels of negative strand rnas from transfected c / cells were below the detection limit of northern blot analysis. this analysis also failed to detect any positive-strand viral rna in cells transfected with the onnv trans-replicase ( fig c) confirming that it is, at best, only weakly active in c / cells. the reduced boosts of reporter expression, the low levels of negative strand rnas and the poor correlation of the boost in fluc expression and the genomic rna synthesis revealed by northern blot all indicate that trans-replicases of arboviruses are much less efficient in c / cells than in human cells. when hek t and c / cells were transfected with cmv-egfp and ubi-egfp plasmids, respectively, a higher percentage of egfp positive cells was obtained for hek t cells (approximately %) than for c / cells (approximately %). next, the gluc marker in the hspoli-fg-chikv and albpoli-fg-chikv plasmids was replaced with a zsgreen marker. experiments performed with the chikv trans-replicase revealed that approximately % of human but only . % of mosquito cells become zsgreen positive. at the same time the expression levels of zsgreen in replication-positive human and mosquito cells were similar (s fig, s fig) . taken together, these data indicate that lower activities of trans-replicases in ae albopictus cells were due to a reduced number of cells in which rna replication was initiated, rather than low levels of replication per cell. normalizing for transfection efficiency, it was estimated that the template rna replication was initiated in~ % of transfected hek t cells but only in~ . % of transfected c / cells (fig d) . the trans-replicase systems allow i) analysis of rna replication activities for the various alphaviruses, independent of entry-related host cell restrictions and factors associated with translation, as well as ii) analyses in the absence of virus adaptation. these properties permitted the comparative analysis of templates originating from the same alphavirus (hereafter "homologous template" or "homologous combination") to those of other alphaviruses (hereafter "heterologous templates" or "heterologous combinations"). this allowed, for the first time, an extensive analysis of cross-utilization of rna templates by alphavirus replicases. in order to include the most distant known member of the genus alphavirus, the fishinfecting salmonid alphavirus (sav), a hspoli-fg-sav template was additionally constructed and used together with the nine templates described in the previous sections. however, it was found that none of the heterologous replicases was able to use the sav template rna. to characterize the reasons behind this effect, a working functional homologous combination of sav replicase and template would be needed but as we were lacking the facilities to cultivate strand rnas were detected using northern blotting. "genomic" indicates the full-length template rna. note that transcripts made by mosquito rna polymerase i using albpoli-fg plasmids as template co-migrate with replicase-generated positivestrand genomic rnas. "subgenomic" indicates the sg rnas synthesized by replicases using the sg promoter. the experiment was repeated twice with similar results; data from one experiment is shown. (d) hek t cells (left panel) were co-transfected with hspoli-fzsg-chikv and cmv-p -chikv; c / cells (right panel) were co-transfected with albpoli-fzsg-chikv and ubi-p -chikv. control cells were transfected with plasmid expressing egfp from a cmv promoter (hek t) or from a polyubiquitin promoter (c / ). at h (hek t) or h (c / ) p.t. cells were collected and analyzed with an attune nxt acoustic focusing cytometer. the number of zsgreen-expressing cells is shown as a proportion of the number of egfp expressing cells, to control for the different transfection efficiency of the two cell types. all transfections were performed in triplicate, means + sd are shown. https://doi.org/ . /journal.ppat. .g cross-utilisation of template rnas by alphavirus replicases fish cells this could not be obtained. therefore, the exact reasons why replicases of other alphaviruses lack the capacity to use sav template rna remain unknown. it can be speculated that these reasons may include use of unsuitable host cells (human instead of fish cells), differences in critical cis-sequences required for sav replication, temperature (sav is normally propagated at temperatures below ˚c) affecting formation of essential rna secondary structures, and/or very high specificity of the sav template to its own replicase. in contrast to the sav template, templates from other alphaviruses could be cross-utilized by heterologous replicases. in general, replicases of viruses belonging to the sfv complex had the capacity to use each other's templates (fig a- e; s fig) . a correlation with the phylogenetic relationship of these viruses was also observed within the group. thus, replicases of chikv and onnv used each other's templates efficiently; furthermore, the use of chikv and onnv templates by replicases of other alphaviruses was very similar (compare fig a and b ). these templates were also efficiently used by mayv replicase and, to a lesser extent, by replicases of rrv and sfv. on hspoli-fg-chikv the transcription efficiencies of rrv and sfv replicases were significantly lower than chikv replicase (p< . for both of sfv and rrv). the same was also observed for hspoli-fg-onnv template which was transcribed significantly more efficient by onnv replicase then by sfv or rrv replicases (p< . at both cases). in addition, compared to chikv replicase, the rrv replicase was significantly less efficient for replication of chikv and onnv template rnas (fig a and b ) (p< . and p< . respectively). template rnas of rrv and sfv also behaved similar to each other (compare fig c and d ) and, with the exception of onnv, were efficiently transcribed by replicases of viruses from the sfv complex. replication of these template rnas was also similar, except that chikv and mayv replicases used the sfv template significantly more efficient (p< . for chikv and p< . for mayv replicase) than the rrv template (compare fig c and d ). again, with the exception of that of onnv, the mayv template was efficiently replicated by replicases from viruses belonging to the sfv complex. for transcription it was also noted that rrv and sfv replicases used this template significantly less efficient than the chikv replicase (p< . at both cases), indicating that the mayv sg promoter is not optimal for sfv and rrv replicases ( fig e) . taken together, it can be concluded that viruses belonging to the sfv complex can replicate and transcribe each other's templates. in contrast, no replicase of the outgroup viruses was capable of comparable utilization of any of the sfvcomplex templates. the highest replication efficiencies were observed for the sinv replicase on mayv and sfv templates (s fig) . utilization of the chikv and especially onnv template by replicases of outgroup viruses was very inefficient (fig a- d ). cross-utilization of sfv and sinv templates has been previously studied using corresponding replicon vectors and di rna type reporters. in contrast to our findings ( fig d) this analysis revealed that sinv replicase was virtually unable to use a sfv template [ ] . the differences probably originate from the use of different tools, including the use of self-replicating rna as source of replicase, in the earlier experiments. the utilization of rna templates of outgroup viruses was clearly different. surprisingly, the templates of these evolutionary distant alphaviruses ( fig c) behaved rather similar to each other. the sinv and bfv templates were efficiently used by replicases of all viruses except eilv, veev and onnv (fig a and b ). the veev template was replicated well by all replicases except that of onnv and at least three of the heterologous replicases (bfv, chikv, and sfv) significantly outperformed the homologous one (p< . for bfv, p< . for chikv and sfv replicases). the boost of gluc expression was, however, greatest for the homologous replicase (p< . compared to any heterologous replicase), an effect that may be the result of the lower cytotoxicity of veev non-structural proteins for human cells. only the sinv and eilv replicases failed to transcribe the veev template (fig c) , likely because of the inability cross-utilisation of template rnas by alphavirus replicases normalized to the paired cmv-p gaa control cells. value obtained for p gaa controls was taken as ; activities lower than that observed for p gaa are also shown as . x-axis represents different replicases, means + sd are from three independent experiments. https://doi.org/ . /journal.ppat. .g cross-utilisation of template rnas by alphavirus replicases to use the veev sg promoter. taken together, it could be concluded that in contrast to templates from viruses belonging to the sfv complex the templates of sinv, bfv and veev can be efficiently used, albeit with some exceptions, by replicases of different alphaviruses. surprisingly, it was found that the same also applies to the template rna of insect specific eilv. this template was efficiently used by replicases from all alphaviruses except veev (fig d) . replication of the template by eilv and veev replicases was inefficient and not significantly different between the two (p> . ). in contrast, transcription of the template was clearly and significantly (p< . ) more efficient for homologous replicase (fig d) . overall, the use of eilv template was very similar to that of sinv (compare fig a and d) , supporting the observation that these viruses are phylogenetically related ( fig c) and harbor similar secondary structures at the ' end region of genome (s fig) . in order to reveal the molecular basis of incompatibility of some replicase/template pairs an analysis of viral rnas was performed. it was concluded from the reporter expression that the veev replicase cannot use the sinv template while the sinv replicase can only replicate, but not transcribe, the veev template ( fig a; s fig) . these effects were confirmed by analysis of the viral rnas. the veev replicase was found to be unable to synthesize any detectable rna using the sinv template while the sinv replicase efficiently synthesized veev negative and positive strand genomic rnas but failed to synthesize sg rna (fig b) . similarly, analysis of reporter expression lead to the conclusion that the chikv template can be replicated by the sinv or bfv but not by the veev replicases and that none of them was able to use the chikv sg promoter (fig a) . again, the data obtained using northern blot fully supported these conclusions (compare fig a and b) . in contrast to veev, the replicases of sinv, and to lesser extent of bfv, were indeed able to synthesize negative and positive genomic rnas from the chikv template. at the same time, none of these heterologous replicases produced chikv sg rna at detectable levels. thus, the sg promoter of many alphaviruses is rather selective: the veev replicase cannot use the sg promoter of sinv, and the sinv and bfv replicases cannot use the sg promoter of chikv. the effect was not reciprocal as the chikv replicase used the sg promoters of sinv and bfv as efficiently as the homologous replicases ( fig a and b , in both cases p> . ). previous studies have shown that cis-active sequences and/or rna secondary structures in alphavirus rnas may have different functionalities in vertebrate and mosquito cells [ , , ] . therefore, the cross-utilization experiments described in the section above were also performed in ae albopictus cells. these experiments confirmed that the onnv replicase was essentially inactive in c / cells (figs and ) because the boost of fluc expression was typically undetectable and the boost of gluc expression did not exceed - -fold. this finding contrasts sharply with the observation in human cells where the onnv replicase, albeit typically less efficient than replicases of other viruses, was able to boost fluc expression approximately -fold and gluc expression > -fold (fig a and b) . the difference in reporter gene expression observed between human and mosquito cells appears too large to be solely attributed to the less efficient initiation of replication in c / cells (fig d) and therefore presumably originate from some intrinsic property of onnv. at the same time, replicases of chikv and mayv used the onnv template efficiently (fig b) . thus, the replication defect of onnv in c / cells was due to the virus replicase and not the template rna. cross-utilization of template rnas of viruses belonging to the sfv complex in c / cells was similar to that in human cells except that differences between alternative replicases were cross-utilisation of template rnas by alphavirus replicases more pronounced, possibly due to a lower efficiency of trans-replicases in c / cells. templates of chikv and onnv were efficiently replicated and transcribed by chikv and mayv replicases. chikv template was used, albeit less efficient, also by the replicase of sfv. the rrv replicase was virtually unable to use either of these templates (fig a and b ). in contrast, the templates of sfv, rrv and mayv were similarly and efficiently replicated and transcribed by all viral replicases (except the one from onnv) of the sfv complex (fig c- e ). for the replicases of rrv and mayv it was observed that the boost of fluc expression from the homologous templates was relatively modest and replication of heterologous templates was often much more efficient (s fig). however, the significance of this, if any, is not clear as the effect probably originates from the low sensitivity of the fluc reporter-based replication assay in c / cells (fig b) . no replicase from outgroup alphaviruses was capable of using template rnas from the sfv complex (fig a- e) . in c / cells, the sinv template was used in the same way as in human cells. it was efficiently replicated by several heterologous replicases, among which the replicases of chikv, mayv and sfv significantly outperformed the homologous replicase (p< . , p< . and p< . , respectively). these three replicases were also efficient in sinv template transcription though for this their activities were significantly lower than that of homologous replicase (p< . in all three cases). in contrast to human cells, where the sinv template was also efficiently replicated and transcribed by the rrv replicase, this replicase performed poorly in c / cross-utilisation of template rnas by alphavirus replicases cross-utilisation of template rnas by alphavirus replicases p gaa controls was taken as ; activities lower than that observed for p gaa are also shown as . x-axis represents different replicases, means + sd from three independent experiments are shown. https://doi.org/ . /journal.ppat. .g cross-utilisation of template rnas by alphavirus replicases cells (fig a) . the template of bfv was in general less efficiently used than other templates. it was replicated relatively efficiently only by replicases of chikv and sfv. however, replicases of viruses from the sfv complex (except onnv), the homologous replicase and that of veev transcribed the bfv template (fig b) . the veev template was efficiently used only by its own replicase and, to a smaller extent, by replicases of viruses from the sfv complex ( fig c) . although a similar trend was observed in human cells (fig c) , it was evident that in c / cells the veev template is preferentially transcribed by the homologous replicase. the eilv template rna was efficiently used by sinv, chikv, mayv and sfv replicase and poorly by the veev replicase. unlike the situation in human cells, it was also poorly used by the replicase of bfv, rrv and onnv (fig d) . it was also observed that while the eilv replicase was very active using the homologous template, it failed to use the templates of bfv, veev and viruses from sfv complex (s fig). the only heterologous template used by eilv replicase to any extent was that of sinv ( fig a, s fig) . thus, in contrast to the promiscuous utilization of eilv template rna, the eilv replicase has a limited capacity to use heterologous templates. in summary, our findings revealed similarities and differences of template rna use in human and ae albopictus cells. in general, in c / cells alphavirus replicases had a reduced capacity to use templates other than their own. this was less pronounced for viruses belonging to the sfv complex but very clear for replicases of outgroup viruses, none of which could use templates of viruses from the sfv complex. it was clear that in c / cells, the replicases of sinv, bfv, veev and eilv could typically replicate and transcribe only homologous template rnas. the only exception was that the sinv replicase could also use the template rna of eilv (s fig). in the trans-replicase system, replication of the chikv template by the sinv replicase was inefficient and no synthesis of sg rna was observed (fig ) . at the same time the replicase of chikv was capable of using the sinv template efficiently (fig a; s fig) . thus, the crossutilization of templates by the chikv and sinv replicases is, as already described for sfv and sinv [ ] , not reciprocal. therefore, the chikv and sinv templates were chosen for mapping the determinants important for their use by their corresponding replicases. to increase the sensitivity of the assay subsequent experiments were performed in u os cells that have been found to be the most efficient for chikv [ ] and sinv trans-replicase assays. the ' regions in the chikv and sinv templates used in previous experiments had different lengths, i.e. nt for chikv and nt for sinv. the differences mostly originate from the extra nt derived from the nsp orf that were included into the sinv template. even though this sequence has no known function for alphavirus rna replication/transcription, we wanted to exclude any possible bias caused by the presence or absence of this region. therefore, an hspoli-fg-c � cc plasmid, extending the chikv template ' region to an extent similar to the sinv template, was constructed. it was found that the template rnas encoded by hspoli-fg-c � cc and hspoli-fg-chikv were both replicated and transcribed with high efficiency by the chikv replicase and with low efficiency by the sinv replicase, and no statistically significant difference between two templates was observed for either replicase. hspoli-fg-c � cc and hspoli-fg-sinv were subsequently used for swapping the ' regions, sg promoters and ' regions, resulting in six swapped chikv/sinv template constructs ( fig a) . as expected, the chikv replicase was able to replicate and transcribe not cross-utilisation of template rnas by alphavirus replicases only the sinv and chikv template rnas but also all six swapped variants (fig b) , confirming that it does not discriminate between cis-elements in the homologous template and these in the sinv template. at the same time, the sinv replicase replicated and transcribed its own template at high efficiency but was much less efficient on the chikv template (fig c) . replacement of the ' region of sinv with that of the chikv template (resulting in s-s-c template) did not reduce the ability of the sinv replicase to use the template (fig c, compare s-s-s and s-s-c). similarly, swapping of ' region of the chikv template with its counterpart from sinv (resulting in c � -c-s template) did not increase the utilization by the sinv replicase ( fig c, compare c � -c-c and c � -c-s). these findings demonstrate that the ' regions of the chikv and sinv templates do not contain any major determinants for sinv replicase specificity. in contrast, swapping of the sinv sg promoter with that of chikv drastically diminished the ability of the sinv replicase to transcribe the resulting s-c-s template while replication was slightly but significantly increased (fig c compare s-s-s and s-c-s). the reciprocal swap increased transcription and also significantly reduced the replication of the resulting c � -s-c template by the sinv replicase ( fig c, compare c � -c-c and c � -s-c). taken together these findings confirm that the sinv replicase has a strong preference for its own sg promoter. furthermore, our data suggests that there may be competition between genomic and sg promoters that leads to reduced replication of the c � -s-c template and somewhat increases replication of the s-c-s template. consistent with findings using the sfv and sinv templates [ ] , swapping of their ' regions had a major effect on replication of corresponding rnas by the sinv replicase. the c � -s-s template was used very inefficiently while the s-c-c template was replicated by the sinv replicase as efficiently as sinv's own template rna (fig c) . thus, the ' region of the rna template contains determinants for replication by the sinv replicase. next, we wanted to analyze whether these findings also apply for pair of alphaviruses belonging to the sfv complex. advantage was taken from the finding that both replication and transcription of the chikv template by the rrv replicase occurred only at a modest level (fig a) . furthermore, use of the rrv template by the chikv replicase was also clearly less efficient than its utilization by the rrv replicase, the difference being especially evident for replication ( fig c) . it was found that these phenotypes were also preserved in u os cells ( fig d and e) . the use of swapped templates again revealed that replacement of the ' region of the template with its counterpart from a related alphavirus significantly reduced replication of such a template. replacement of the sg promoter of chikv in c-c-c template with that of rrv did not have a significant effect on transcription of such templates. reciprocal substitutions in the rrv templates somewhat reduced their transcription by the rrv replicase. the observed differences reached statistical significance only in case of the r-c-c and c-c-r templates. the c-c-r template was also characterized by reduced replication, thus its reduced transcription may, at least in part, be due to the swap of the ' region. the r-c-c template on other hand, replicates similar to the r-r-r template and its reduced transcription is likely due to the presence of a heterologous sg promoter. finally, swapping of the ' region of the cross-utilisation of template rnas by alphavirus replicases templates had no detectable effect on replication or transcription of such templates by the chikv or rrv replicases (fig d and e) . taken together, these experiments confirmed that the determinants responsible for preferential use of templates by replicases of viruses belonging to the sfv complex are also located at the ' region of the template rna. analyses of sinv/sfv chimeric templates have previously convincingly demonstrated that replacement of the sinv ' utr with that of sfv drastically reduces the use of the resulting template by the sinv replicase [ ] . to analyze whether the same applies for sinv/chikv chimeras we took advantage of the shape derived and reverse genetically-verified structure of the ' end of the chikv genome [ ] and designed hybrid templates shown in fig a. it was found that templates where the sl and sl regions (ccs-s-s), the sl and the nsp orf regions (csc � -s-s) or only the sl region (css-s-s) originated from chikv were inefficiently replicated by the sinv replicase, similar to the c � -c-c and c � -s-s templates. in contrast, all chimeric templates that contained the sinv ' sequences to the end of the sl element (ssc � -s-s, scc � -s-s and scs-s-s) were replicated as efficiently as the sinv template by the sinv replicase. thus, in order to replicate the sinv/chikv chimeric templates efficiently, the sinv replicase needs homologous sequences located at the ' end of the template and/or in sl . consistent with earlier findings [ ] the sequences of the sinv ' utr downstream of nucleotide had little, if any, impact on the replication efficiency. the comparison of the ' ends of the efficiently replicated s-s-s template and the very inefficiently replicated css-s-s template highlighted that the former is longer due to three insertions of (t, position ), (tatt, positions - ) and (acagccgaccaatt, positions - ) nucleotides ( fig a) . therefore, we hypothesized that the shorter length of the ' utr may be the reason for the low replication efficiency of the css-s-s template. indeed, adding the nucleotide fragment to this template resulted in significant increase of its replication efficiency. however, the reciprocal mutation, resulting in the s Δ -s-s template, had no effect on the replication of the truncated sinv template. this contrasts with the previous findings indicating that deletions of nucleotides or results in an impaired, temperature-sensitive, phenotype [ ] . the addition of nucleotides to the css-s-s template or their removal from the s-s-s template had minimal, if any, effect on their replication efficiencies. a similar lack of effect has been previously observed for analogous mutations introduced in the sinv genome [ ] . in sharp contrast, deletion of a single nucleotide from position of the sinv template drastically reduced the replication efficiency of the resulting rna (fig c) . deletion of a single nucleotide from position of the sinv template rna results in a very similar effect [ ] and deletion of residues - of the sinv genome is lethal for the virus [ ] . thus, it should be concluded that the length and/or sequence of the ' extreme of sinv genome is crucial for the use of the template rna by the sinv replicase. interestingly however, experiments using the css-s-s template only partly confirmed this hypothesis. addition of a t residue after nucleotide in the construct for expression of this template rna resulted in a minor but significant increase of its replication efficiency. the same was observed when this change was combined with the addition of a nt sequence. only the addition of a third missing element, the tatt sequence, resulted in a prominent increase of the replication efficiency of the corresponding template rna (fig c) . this data indicates that for conversion of the chikv-type template to the sinv-type the synergistic effect of several insertions was required. all the effects described above were specific for the sinv replicase as the chikv replicase used all templates shown in fig a with high and the trans-replicase assays, though very efficient and sensitive, are based on an artificial system. to exclude the possibility that the observed effects may represent artifacts of the system we introduced the selected mutations into an infectious cdna plasmid of sinv resulting in plasmids designated ptoto Δt , ptoto css and ptoto ct/tatt/ ss . when the effects of the introduced mutations were analysed using an infectious centre assay (ica) it was found that transcripts from ptoto ct/tatt/ ss had an infectivity of . x pfu/μg rna which was only slightly lower than the infectivity of transcripts from wild type ptoto ( . x cross-utilisation of template rnas by alphavirus replicases pfu/μg rna). the infectivity of transcripts from ptoto Δt was strongly reduced ( . x pfu/μg rna) and that of transcripts from ptoto css was close to the limit of detection of the assay (< pfu/μg rna). thus, the infectivity of transcripts correlated, at least in terms of rank order, with the observed replication efficiencies of the corresponding templates in the trans-replication assay (fig b and c) . furthermore, the difference observed between transcripts of ptoto Δt and these of ptoto css may reflect the complexity of reversions/ adaptations. for ptoto Δt transcripts, an addition of a single u residue to the ' end of the rna will restore its length and generate wild type like sequence. addition of a and u residues to the ends of imperfect genomes is an adaptation commonly found in alphaviruses [ ] . in contrast, in order to increase replication of the css-s-s template at least three insertions, none of which had major effect on its own, were required ( fig c) . apparently, re-creating the recovery pathway-or, more likely, creating one of its own-was more time consuming and this was reflected in low infectivity of corresponding transcripts. whatever solution(s) were developed by mutant viruses they were clearly effective as all recombinant viruses grew to high final titres (> . x pfu/ml) that were similar to these of wild type sinv. no correlation between the final titres and the efficiency of the corresponding template replication was observed. trans-replicases were developed for nine alphaviruses across the alphavirus genus. compared with actual viruses these represent safe and easy-to-use systems. trans-replicases of viruses of different origin can be used in the same cell types and the relative ease of quantifying reporter enzyme activities, compared with viral rna levels, allows use of the system for studies that generate large panels of data, such as screening for antiviral compounds or for host factors essential for virus replication. the sensitivity of the trans-replication system depends on the amplitude of "boost" of marker expression. the "boost" was always larger for transcription marker (gluc) expression and, in general, showed an excellent correlation with the actual sg rna production (figs , and ). nevertheless, it should be noted that the rna/marker protein ratio is susceptible to the virus-induced shutoff of cellular transcription and translation that may be captured by trans-replicases or be host cell type specific. compared to gluc, the boost of the replication marker (fluc) was almost always smaller. in human cells, however, it was prominent enough to serve as proxy of viral genomic rna synthesis (fig ) . in mosquito cells the boost of fluc is low or modest and its correlation with genomic rna synthesis is less pronounced (fig ) generally hampering comparison of different viruses. it is, however, still useful for comparison of the effects of mutations introduced into replicase of a specific alphavirus [ ] . furthermore, the low boost of fluc expression makes it useful for analysis of mutations or other effects that increase replication efficiency [ ] . in this study several heterologous replicase/template combinations that resulted in increased rna replication in mosquito cells were revealed: rrv replicase replicated mayv template~ -fold more efficiently and mayv replicase replicated eilv template~ -fold more efficiently than their own templates (s fig). accordingly, this trans-replication assay could be applied for analysis of molecular mechanisms behind these effects. taken together, certain limitations of trans-replication assay may, depending on circumstances, also be regarded as benefits. the new tools were used to assess the compatibility of template rnas and replicases of different alphaviruses in human and ae albopictus cells. our analysis of determinants allowing efficient use of template by sinv replicase (figs and ) correlated well with data previously obtained using infectious virus [ ] or alphavirus replicons and di rna templates [ ] . the high degree of correlation indicates that the alphavirus trans-replication system is not only useful for analysis of alphavirus proteins [ , , ] and to study the importance of host factors cross-utilisation of template rnas by alphavirus replicases [ , ] but is also fully suitable to study the interactions between alphavirus replicases and template rnas. several previously unknown properties, revealed in this study, considerably expand our understanding regarding the alphavirus replication process and may provide novel insight in the phylogeny of the genus. one of the key findings in this study is that replicases of alphaviruses from the sfv complex can use rna templates from each other as well as those from outgroup viruses. we recently demonstrated that sl structures located in the ' region of the chikv template rna are essential for genome replication, indicating that they are recognized by components of chikv replicase complex [ ] . the determinants of sequence specificity for viruses from the sfv complex are also located at the ' region of the template rna. the determinants of rrv are preferentially recognized by the rrv replicase and reduce the utilization of the corresponding template by the chikv replicase, while the determinants located at the ' region of the chikv template have an opposite effect. interestingly, sl structures located in ' regions both chikv and rrv are predicted to be extremely homologous (s fig), suggesting that determinants of template selectivity for these replicases are most likely associated with primary rna sequence rather than secondary structure. the situation with template rnas of outgroup viruses is somewhat different: the sinv template accommodates both sinv and chikv replicases. the secondary structures at the ' ends of genomes of outgroup viruses were predicted (s fig) to be thermodynamically less stable (from - . to - . kj/mol) than those at the ' ends of genomes of viruses from sfv complex (from - . to - . kj/mol). however, no obvious correlation was observed between the capacity of replicase to use a heterologous template and stability of the secondary structure at its ' end (s fig, s fig, s fig) . interestingly, small changes in ' end of sinv template rna could dramatically reduce its use by the homologous replicase even in the absence of an impact on the stability of secondary structures (s fig). furthermore, the predicted secondary structure of the ' extreme of s-s-s and c t/tatt/ ss-s-s template rnas (that are efficiently used by sinv replicase) are similar to those of their inefficiently used counterparts, s Δt -s-s and c t/ ss-s-s (s fig). this raises the possibility that the determinants required for use as template rnas in general are different from those required for recognition by specific replicases. evidence suggests that the former acts via a mechanism involving multiple rna secondary structure elements, while the latter is likely based on rna length and/or primary sequence. another interesting, yet still unanswered, question is whether the ability of alphaviruses to use different templates represents a biological advantage or a restriction. we have previously demonstrated that the replicase of sfv can use cellular rnas in order to generate doublestranded rnas that are recognized by rig-i and induce expression of type-i interferons [ ] . the efficiency of synthesis of such rnas is different for different alphaviruses, being much higher for the sfv and sinv replicases than for the replicase of rrv [ ] . thus, the ability of the replicase to synthesize such interferon-inducing rnas does not correlate with the ability of the replicases of these viruses to use heterologous templates (figs , and ) . furthermore, experiments with the sav-template clearly demonstrate that the ability of alphavirus replicases to use heterologous rnas as templates has its limits. chikv and mayv have been shown to be able to co-infect mosquitoes, at least in the laboratory [ ] . co-infection of mosquitoes by different arboviruses creates the possibility of co-transmission from mosquito to human [ ] . co-infection can also occur if humans are bitten by mosquitoes carrying different alphaviruses. a study from kenya revealed that % of people who had been exposed to onnv and/or chikv have, in fact, been exposed to both of them [ ] . given the similarity between these viruses and findings that a chikv vaccine (and, presumably, chikv infection) confers protection against onnv [ , ] it seems plausible that many of these people have been coinfected by these viruses. the ability of alphaviruses to cross-utilize each other's templates, both in human and mosquito cells, can facilitate recombination between virus genomes, ensuring that resulting recombinant rna genomes can be used by replicase(s) of parental viruses or by chimeric replicase created as a result of the recombination. thus, while the benefits from the ability to cross-utilize templates of other alphaviruses are not obvious for an individual infection event, the ability may represent a potential factor for alphavirus evolution and may be the key for successful recombination between distantly related members of the genus. cross-utilization of templates in mosquito cells, coupled with the promiscuous nature of template rna of mosquito-specific alphaviruses, creates the possibility of recombination between divergent alphaviruses. this may include recombination between mosquito-specific and arbovirus members of the genus, potentially leading to novel viruses of either type. the increase of our knowledge about mosquito-specific alphavirus genomes may reveal if such recombination events have occurred and what type of viruses originated from such events. the ability of the alphavirus replicase to specifically recognize its own ' utr and sg promoter elements in the template rna indicates that some component(s) of the virus replicase are capable of recognizing primary sequence motifs and/or secondary structures. the helicase region of chikv nsp has been co-crystallized with an rna oligonucleotide corresponding to the ' end of the chikv genome [ ] . however the interaction of this protein with rna is not sequence-specific and, based on our data (fig ) , alphavirus replicases do not differentiate between each other's ' utr sequences. specific interactions with sequences corresponding to both utr regions and the sg promoter have been reported for the central domain of nsp [ ] . nevertheless, the strongest candidate as a specificity factor for template recognition is nsp , the rna polymerase subunit of the replicase. its ability to bind viral rnas is dependent on other ns-proteins but clearly occurs in a very specific manner, i.e. it is possible to block the interaction of nsp with the sg promoter without affecting its interaction with the genomic promoter [ , ] . unfortunately, recombinant nsp of chikv has very low solubility and activity [ ] , which complicates in vitro analysis of nsp -rna interactions. it is also of great interest to reveal which rna elements-sequences, secondary structures and/or long-range interactions-are crucial for specific template recognition. as alphavirus rna can adopt alternative secondary structures [ ] , which most likely change in response to the interaction with different host and viral components, advantage could be taken of novel methods, allowing analysis of rna structures and rna-rna interactions inside the cells. for such analysis, the trans-replicase system allows manipulation of sequence and secondary structures of template rnas in a more efficient way than using infectious virus genomes. it was observed that the trans-replicase of onnv has low activity in human cells. it is possible that the poor activity of its trans-replicase may reflect a natural property of onnv, or at least of the onnv isolate used in this study. interestingly, onnv replicates reasonably well in c / cells, a property that contrasts sharply with a near complete lack of activity of its transreplicase in these cells. we have previously observed such a combination for trans-replicase/ infectious virus of chikv harbouring mutations accelerating processing of p polyprotein precursor [ ] . most likely these findings are linked and represent a consequence of an additional hurdle for the formation of functional replicase complexes by trans-replicase-compared to those of infectious virus. here we have revealed that such a hurdle, likely involving finding and binding template rna provided in trans, has a much more prominent effect in ae albopictus than in human cells (fig d) . interestingly, alterations in the speed of p processing also has a much stronger impact on the trans-replicase activity in mosquito cells than in human cells [ ] . the cell type specificity of this effect strongly argues for involvement of cellspecific conditions and/or cell-specific factors. thus far the only known host factor present in both human and mosquito cells and absolutely required for the replication of chikv and onnv is g bp and its mosquito ortholog called rasputin [ ] . these proteins interact with the c-terminal region of nsp of old world alphaviruses [ , ] . interestingly, a chikv trans-replicase harbouring mutant nsp which is unable to interact with rasputin, is virtually inactive in c / cells [ ] and therefore similar to the trans-replicase of onnv. however, onnv nsp does have sites for interaction with rasputin and onnv is competent for replication in mammalian cells that express g bp. it is plausible that onnv replicase can bind ae albopictus rasputin but uses it efficiently only in the context of infectious virus and not in the context of the trans-replicase. thus, the replication defect, albeit detected in an artificial system, may actually be connected to the most important in vivo property of onnv i.e. its transmission by anopheles mosquitoes. interestingly, the determinant of infection rates in anopheles gambiae has also been mapped to nsp of onnv [ ] . thus, this study adds another important piece of information to the puzzle regarding alphavirus nsp , host g bps/ rasputin proteins and the mechanism(s) surrounding the formation and functioning of virus replicase complexes and ultimately its impact on alphavirus vector transmission. trans-replicase systems, that reproduce, and possibly even emphasise, natural properties of alphavirus replicases, represent a valuable tool for studying such interactions. interesting findings were also obtained for eilv. firstly, in sharp contrast to the template rna of fish-infecting sav, the template of mosquito-specific eilv was efficiently used by replicases of arbovirus members of alphaviruses in both mosquito and human cells. at the same time, eilv replicase was highly specific to its own template rna. both of these properties are similar to sinv, furthermore replicases of both viruses were able to use each other's templates better than any other heterologous template. it is plausible that these functional similarities originate from the phylogenetic relationship between these viruses ( fig c) and similar folding of ' regions of these template rnas (s fig). this raises an intriguing question: did a sinv-like ancestor lose its ability to replicate in vertebrates or did an eilv-like ancestor acquire the ability to replicate in a vertebrate host? the number of recognized insect-specific alphaviruses is rapidly increasing and the same is the case for other virus groups traditionally considered as arboviruses. given that insect-specific viruses from these groups are widespread and abundant, they may represent ancestors of arbovirus members of corresponding groups. here we show that, in order to become an arbovirus, they do not need to acquire the ability of replicating in mammalian cells: this activity already exists-at least for eilv (fig ) . increased replication efficiency and adaptation to elevated temperatures are effects frequently observed for alphavirus temperature sensitive mutants propagated in cell culture; consequently, it seems plausible that insect-specific alphaviruses have, perhaps repeatedly, adapted to infection of vertebrate hosts. thus, trans-replicase assays are versatile tools to examine the poorly understood relationship between insect-specific viruses and arboviruses that cause infections in humans. as template switching by the viral replicase represents the most efficient mechanism for recombination between viruses, the potential for cross-utilization of templates could shed light on the mechanisms of (alpha)virus recombination and evolution. the trans-replication approach developed here is applicable to other virus families containing major human pathogens and/or emerging viruses such as picornaviridae, flaviviridae and coronaviridae. transreplicases are flexible, easy to use systems that offer an alternative to study viral recombination and evolution when infectious clones are not available. their use also eliminates the need for high containment facilities and allows convenient assessment of template cross-utilization, and thus potential for recombination, without the need for creating recombinant viruses in the lab. better understanding of the template rna requirements of (alpha)virus replicases creates numerous possibilities. promiscuous templates can be used for analysis of interactions between replicase proteins of (alpha)viruses, rna-replicase interactions and for reconstruction of (alpha)virus replication complexes. sub-optimal combinations of ' utr sequences and replicases can be used to generate recombinant attenuated viruses with potential use as vaccine candidates. the trans-replicases of alphaviruses can be used as tools for inducible expression of proteins of interest. the use of replicases from old world alphaviruses may be hampered by the cytotoxic effects of their nsps. however, here we demonstrate that the less cytotoxic replicase of veev produces a comparable boost in protein expression via activity of the sg promoter (fig ) . this property, likely shared by replicases of other new world alphaviruses, may be utilized for development of efficient expression systems with extremely high induced/uninduced expression ratios (i.e. ratio of expression levels with and without replicase). furthermore, constructs expressing promiscuous templates can be used to obtain transgenic cell lines that can serve as universal biosensors of alphaviruses. this approach may be applied to the detection and identification of unknown alphaviruses, especially those belonging to the sfv complex and capable of using different rna templates. other virus families with trans-active replicases may take advantage of this technology. it may even be possible to extend this approach to transgenic mosquitoes, potentially providing rapid, robust biosensors not dependent on access to tissue culture facilities, and perhaps providing a new route to develop methods to modify wild vector populations with specific, engineered response elements to arboviruses allowing detection and/or reduced vector competence. conversely, alphavirus group-specific templates and sg promoters can be utilized in biosensors that allow discrimination between alphaviruses belonging to different serocomplexes and potentially even between different species of alphaviruses. finally, the critical sequences and rna secondary structures required for replication are interesting new and potentially specific targets for antiviral interference with molecules such as locked nucleic acids and morpholino oligonucleotides. u os human bone osteosarcoma cells (atcc htb- ) were maintained in iscove's modified dulbecco's medium (gibco) containing % fetal bovine serum (fbs) and mm l-glutamine at ˚c in a % co atmosphere. hek t cells (atcc crl- ) were maintained in dulbecco's modified eagle medium (dmem) with mm l-glutamine and % fbs at ˚c in a % co atmosphere. bhk- cells (atcc ccl- ) were grown in glasgow's minimal essential medium (gibco) containing % fbs, % tryptose phosphate broth (tpb) and mm hepes ph . at ˚c in a % co atmosphere. aedes albopictus derived c / cells were maintained in eagle's minimum essential medium containing % fbs at ˚c with no extra co . all media were supplemented with u/ml penicillin and . mg/ml streptomycin. plasmids containing native (not codon optimized) sequences encoding for p of chikv (ecsa genotype, lr -opy isolate) or its polymerase negative variant p gaa , harboring gdd to gaa substitution in the active site of nsp , under the control of immediately early promoter of human cytomegalovirus (cmv) have been previously described [ ] . similar plasmids designed for the expression of p of sfv (sfv strain), sinv (toto strain), rrv (rrv-t strain), mayv (trvl strain), onnv (chad isolate), bfv and veev (v strain) as well as their polymerase negative variants in mammalian cells have also been described [ ] . the plasmid for expression of p of eilv in mammalian cells has similar design. it was assembled from synthetic dnas (genscript, usa) and restriction fragments of corresponding infectious cdna clone which was a gift from scott weaver (utmb) [ ] ; its polymerase negative variant was obtained using site-directed mutagenesis and sub-cloning procedures. for simplicity, and in order to avoid confusion with each other, these plasmids cross-utilisation of template rnas by alphavirus replicases were designated as cmv-p -chikv, cmv-p gaa -chikv and so on. to obtain constructs for expression of replicases in ae albopictus cells, the p encoding regions were cloned under the control of polyubiquitin promoter from aedes aegypti as previously described for chikv trans-replicase [ ] ; obtained clones were designated as ubi-p -chikv, ubi-p gaa -chikv and so on. human rna polymerase i promoter-based plasmids for the production of replication competent rna templates of chikv (originally named hspoli-fluc-gluc), sfv, onnv, sinv, rrv, bfv and veev have been previously described [ , ] . the plasmids for expression of rna templates of eilv and sav had the same design and were assembled from synthetic dna fragments (genscript, usa). to obtain constructs for expression of corresponding template rnas in ae albopictus cells the design developed for chikv (originally named albpoli-fluc-gluc [ ] ), was adapted to all above listed alphaviruses except sav. for simplicity these plasmids were designated as hspoli-fg-chikv, albpoli-fg-chikv and so on. for detection of individual cells positive for rna replication the gaussia luciferase (gluc) marker in hspoli-fg-chikv and albpoli-fg-chikv was replaced with zsgreen; resulting in vectors designated hspoli-fzsg-chikv and albpoli-fzsg-chikv. for making sinv/chikv swapped templates the region corresponding to the ' end of chikv template rna was extended from nucleotide to nucleotide ; obtained plasmid was designated as hspoli-fg-c � cc. the swapping of ' region ( ' utr+ region encoding for n-terminus of nsp ), sg promoter region and ' region (truncated ' utr) between hspo-li-fg-c � cc and hspoli-fg-sinv or hspoli-fg-chikv and hspoli-fg-rrv was performed using standard cloning procedures. obtained constructs were designated as explained on fig a, hspoli-fg-sss, hspoli-fg-c � ss and so on. corresponding template rnas were designated as s-s-s, c � -s-s and so one. note that plasmid designated as hspoli-fg-ccc is the same as hspoli-fg-chikv, hspoli-fg-sss is the same as hspoli-fg-sinv and hspo-li-fg-rrr is the same as hspoli-fg-rrv. plasmids for the expression of templates with changes in the ' region were constructed using synthetic dnas (genscript, usa) and subcloning procedures. the first set of constructs contained swaps of sl , sl regions and nsp orf parts in template rna encoded by hspo-li-fg-sss (fig a, left panel) . the obtained plasmids were designated as hspoli-fg-ssc � ss, hspoli-fg-ccsss and so on; the first small letter in the name of plasmid reflects the origin of the ' extreme and sl , second small letter reflects the origin of the region corresponding to chikv sl ; the third small letter reflects the origin of the region encoding the n-terminus of nsp ; c � indicates use of longer fragment of chikv nsp orf. in the second set of plasmids the ' ends and sl regions of templates encoded by hspoli-fg-sss and hspoli-fg-cssss were modified as shown on fig a (right panel) . constructs based on hspoli-fg-sss were designated as hspoli-fg-s Δt ss, hspoli-fg-s Δtatt ss and hspoli-fg-s Δ ss; constructs based on hspoli-fg-cssss were designated as hspoli-fg-c t ssss, hspoli-fg-c tatt ssss, hspoli-fg-c ssss, hspoli-fg-c t/ ssss and hspoli-fg-c t/tatt/ ssss. plasmid ptoto was used to construct infectious cdna clones of sinv harbouring modifications in the ' utr. the deletion of a residue corresponding to the position of sinv genome was performed using site-directed mutagenesis and subcloning procedures, corresponding plasmid was designated ptoto Δt . replacements identical to these used in hspoli-fg-cssss and hspoli-fg-c t/tatt/ ssss template-rna encoding plasmids were made using synthetic dnas (genscript) and subcloning procedures; obtained plasmids were designated ptoto css and ptoto ct/tatt/ ss , respectively. sequences of all plasmids were verified using sanger sequencing and are available from the authors upon request. virus rescue in bhk- cells and ica were performed as previously described [ ] . briefly, bhk- ( × ) were transfected with in vitro transcripts by electroporation with a bio-rad gene pulser ii unit (two pulses at v and μf) in . cm cuvettes (thermo fisher scientific). after h of incubation at ˚c, the cells were overlaid with ml of gmem supplemented with % fbs and containing % bacto tm agar (bd biosciences). for virus rescue experiments virus stocks were collected at h (wild type and ptoto c+t/tatt/ ss ) or at h (ptoto -t and ptoto css ) post transfection (p.t.). obtained stocks were clarified by centrifugation at xg for minutes and virus titers were determined using standard plaque assay on bhk- cells. the trans-replication assay in u os, hek t and c / cells was performed as previously described [ ] . in order to determine the optimal time point for analysis of reporter expression, the hek t and c / cells were transfected with homologous pairs of plasmids ( u os cells grown on -well plates were co-transfected with μg of hspoli-fg-chikv or other plasmid encoding for template rna and μg of cmv-p -chikv or other plasmid encoding for replicase; in control cells the later was replaced with plasmid encoding a polymerase negative version of replicase protein (cmv-p gaa -chikv and so on). transfected cells were incubated at ˚c for h. in experiments involving hspoli-fg-eilv and cmv-p -eilv plasmids the transfected cells were also incubated at ˚c for h. all experiments were repeated at least three times. for hek t and c / cells the trans-replication assay carried out using -well plate format. briefly, approximately , cells per well were co-transfected with . μg of the template and . μg of appropriate replicase expression plasmids; in control cells the later was replaced with a plasmid expressing the polymerase negative version of replicase. transfections were performed using lipofectamine ltx reagent. hek t cells were generally incubated at ˚c for h. hek t cells transfected using eilv cdna derived plasmid as well as all transfected c / cells were incubated at ˚c for h. all transfections were performed in triplicate and experiments were repeated at least twice. after incubation, cells were lysed and firefly luciferase (fluc) and gluc activities were measured using the dual-luciferase-reporter assay (promega). fluc and gluc activities measured for cells transfected using plasmids expressing active replicases were normalized to these obtained for corresponding control cells. hek t cells grown in -well plates were co-transfected with μg of hspoli-fzsg-chikv and μg of cmv-p -chikv; c / cells grown in -well plates were co-transfected with μg of albpoli-fzsg-chikv and μg of ubi-p -chikv. control hek t cells were transfected with μg of plasmid expressing egfp from cmv promoter (cmv-egfp) and control c / cells were transfected with μg of plasmid expressing egfp from polyubiquitin promoter (ubi-egfp). transfections were performed in triplicate using lipofectamine ltx reagent. at h (hek t) or h (c / ) p.t. cells were collected in μl pbs and analyzed with an attune nxt acoustic focusing cytometer. for each sample , events were recorded. the acquired data was analyzed using attune nxt software. hek t and c / cells grown in -well plates were co-transfected with μg of template rna expression plasmid and μg of replicase expression plasmid using lipofectamine ltx reagent; control cells were mock-transfected. at h (hek t) or h (c / ) p.t. total rna was extracted using trizol reagent (life technologies). μg of total rna was used for detection of positive strands and μg of total rna was used for detection of negative strands. rnas were denatured for min at ˚c in x rna loading dye (thermo scientific), cooled on ice and separated on a denaturing gel ( % agarose/ % formaldehyde) using x mops buffer. rna was transferred to a hybond-n+ filter (ge healthcare) and fixed using a uv stratalinker (stratagene). digoxigenin (dig)-labelled rna probe complementary to residues - of the sequence encoding for gluc marker was used to detect positive-strand rnas; probe corresponding to residues - of the sequence encoding for fluc marker was used to detect negative-strand rnas. filters were hybridized overnight; blots were washed and developed according to the manufacturer's (roche) protocols. in silico thermodynamic rna structure and free energy predictions were carried out using unafold at ˚c on default settings (version . ) [ ] . rna structures were visualised using the varna software package [ ] . statistical analysis was performed using graphpad prism software. data were analyzed using student's unpaired one tailed t-test. p values of � . ( � ), � . ( �� ), � . ( ��� ) and � . (' ��� ) were used to represent degrees of significance for each mutant compared to wild-type. each experiment was repeated to gain a minimum of independent biological repeats. raw data used to generate graphs is presented in s data. chikungunya virus: an update on the biology and pathogenesis of this emerging pathogen o'nyong-nyong fever: a neglected mosquito-borne viral disease. pathog glob health ross river virus: ecology and distribution evolution and spread of venezuelan equine encephalitis complex alphavirus in the americas re-emergence of epidemic venezuelan equine encephalomyelitis in south america. vee study group ictv virus taxonomy profile: togaviridae eilat virus, a unique alphavirus with host range restricted to insects by rna replication the old world and new world alphaviruses use different virus-specific proteins for induction of transcriptional shutoff new world and old world alphaviruses have evolved to exploit different components of stress granules, fxr and g bp proteins, for assembly of viral replication complexes phylogeographic structure and evolutionary history of sindbis virus a comparison of the nucleotide sequences of eastern and western equine encephalomyelitis viruses with those of other alphaviruses and related rna viruses evolutionary genetics and vector adaptation of recombinant viruses of the western equine encephalitis antigenic complex provides new insights into alphavirus diversity and host switching alphavirus rna synthesis and non-structural protein functions polypeptide requirements for assembly of functional sindbis virus replication complexes: a model for the temporal regulation of minus-and plusstrand rna synthesis phosphatidylinositol -kinase-, actin-, and microtubuledependent transport of semliki forest virus replication complexes from the plasma membrane to modified lysosomes functional sindbis virus replicative complexes are formed at the plasma membrane timeliness of proteolytic events is prerequisite for efficient functioning of the alphaviral replicase macromolecular assembly-driven processing of the / cleavage site in the alphavirus replicase polyprotein fhl is a major host factor for chikungunya virus infection the ' and ' ends of alphavirus rnas-non-coding is not non-functional chikungunya virus ' untranslated region: adaptation to mosquitoes and a population bottleneck as major evolutionary forces whole-genome sequencing analysis from the chikungunya virus caribbean outbreak reveals novel evolutionary genomic elements chikungunya virus evolution following a large utr deletion results in host-specific molecular changes in protein-coding regions sindbis virus usurps the cellular hur protein to stabilize its transcripts and promote productive infections in mammalian and mosquito cells the alphaviruses: gene expression, replication, and evolution cis-acting rna elements at the ' end of sindbis virus genome rna regulate minus-and plus-strand rna synthesis sequence studies of several alphavirus genomic rnas in the region containing the start of the subgenomic rna utilization of heterologous alphavirus junction sequences as promoters by sindbis virus identification of the amino acid sequence in sindbis virus nsp that binds to the promoter for the synthesis of the subgenomic rna distinct sites on the sindbis virus rna-dependent rna polymerase for binding to the promoters for the synthesis of genomic and subgenomic rna the '-terminal sequences of the genomic rnas of several alphaviruses defined mutations in the ' nontranslated sequence of sindbis virus rna structural and phenotypic analysis of chikungunya virus rna replication elements a viral rna structural element alters host recognition of nonself rna modification of the ' terminus of sindbis virus genomic rna allows nsp rna polymerases with nonaromatic amino acids at the n terminus to function in rna replication studies of defective interfering rnas of sindbis virus with and without trnaasp sequences at their ' termini deletion mapping of sindbis virus di rnas derived from cdnas defines the sequences essential for replication and packaging common sequence elements in structurally unrelated genomes of defective interfering semliki forest virus selection of functional ' cis-acting elements promoting efficient sindbis virus genome replication a new generation of animal cell expression vectors based on the semliki forest virus replicon sindbis virus expression vectors: packaging of rna replicons by using defective helper rnas assembly of alphavirus replication complexes from rna and protein components in a novel trans-replication system in mammalian cells versatile trans-replication systems for chikungunya virus allow functional analysis and tagging of every replicase protein sensitivity of alphaviruses to g bp deletion correlates with efficiency of replicase polyprotein processing design and use of chikungunya virus replication templates utilizing mammalian and mosquito rna polymerase i-mediated transcription sindbis virus nonstructural protein nsp is cytotoxic and inhibits cellular transcription sindbis virus infection causes cell death by nsp -induced transcriptional shutoff or by nsp -dependent translational shutoff dual mechanism for the translation of subgenomic mrna from sindbis virus in infected and uninfected cells viral translation is coupled to transcription in sindbis virus-infected cells mutations in the nuclear localization signal of nsp influencing rna synthesis, protein expression and cytotoxicity of semliki forest virus o'nyong nyong virus molecular determinants of unique vector specificity reside in non-structural protein deployable molecular detection of arboviruses in the australian outback wetlands, climate zones and barmah forest virus disease in queensland identification of natural molecular determinants of ross river virus type i ifn modulation infection with mayaro virus in a french traveller returning from the amazon region, brazil anopheles mosquitoes may drive invasion and transmission of mayaro virus across geographically diverse regions plos pathogens cross-utilisation of template rnas by alphavirus replicases plos pathogens changes of the secondary structure of the ' end of the sindbis virus genome inhibit virus growth in mosquito cells and lead to accumulation of adaptive mutations requirements at the ' end of the sindbis virus genome for efficient synthesis of minus-strand rna a chikungunya virus trans-replicase system reveals the importance of delayed nonstructural polyprotein processing for efficient replication complex formation in mosquito cells adp-ribosyl-binding and hydrolase activities of the alphavirus nsp macrodomain are critical for initiation of virus replication structural insights into rna recognition by the chikungunya virus nsp helicase vcp/p is a proviral host factor for replication of chikungunya virus and other alphaviruses rig-i and mda- detection of viral rna-dependent rna polymerase activity restricts positive-strand rna virus replication decreased virulence of ross river virus harboring a mutation in the first cleavage site of nonstructural polyprotein is caused by a novel mechanism leading to increased production of interferon-inducing rnas. mbio infection pattern of mayaro virus in aedes aegypti (diptera: culicidae) and transmission potential of the virus in mixed infections with chikungunya virus arbovirus coinfection and co-transmission: a neglected public health concern? high rates of o'nyong nyong and chikungunya virus transmission in coastal kenya cross-protective immunity against o'nyong-nyong virus afforded by a novel recombinant chikungunya vaccine multiple roles of the non-structural protein (nsp ) alphavirus unique domain (aud) during chikungunya virus genome replication and transcription chikungunya virus nsp rna-dependent rna polymerase core domain displays detergent-sensitive primer extension and terminal adenylyltransferase activities the c-terminal repeat domains of nsp from the old world alphaviruses bind directly to g bp mosquito rasputin interacts with chikungunya virus nsp and determines the infection rate in aedes albopictus. parasit vectors mutations conferring a noncytotoxic phenotype on chikungunya virus replicons compromise enzymatic properties of nonstructural protein mfold web server for nucleic acid folding and hybridization prediction varna: interactive drawing and editing of the rna secondary structure the authors have declared that no competing interests exist. key: cord- -l kocy f authors: liang, jingjing; sagum, cari a.; bedford, mark t.; sidhu, sachdev s.; sudol, marius; han, ziying; harty, ronald n. title: chaperone-mediated autophagy protein bag negatively regulates ebola and marburg vp -mediated egress date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: l kocy f ebola (ebov) and marburg (marv) viruses are members of the filoviridae family which cause outbreaks of hemorrhagic fever. the filovirus vp matrix protein is essential for virus assembly and budding, and its ppxy l-domain motif interacts with ww-domains of specific host proteins, such as nedd and itch, to facilitate the late stage of virus-cell separation. to identify additional ww-domain-bearing host proteins that interact with vp , we used an ebov ppxy-containing peptide to screen an array of mammalian ww-domain-bearing proteins. using this unbiased approach, we identified bcl associated athanogene (bag ), a member of the bag family of molecular chaperone proteins, as a specific vp ppxy interactor. here, we demonstrate that the ww-domain of bag interacts with the ppxy motif of both ebov and marv vp and, unexpectedly, inhibits budding of both evp and mvp virus-like particles (vlps), as well as infectious vsv-ebov recombinants. bag is a stress induced protein that regulates cellular protein homeostasis and cell survival through chaperone-mediated autophagy (cma). interestingly, our results show that bag alters the intracellular localization of vp by sequestering vp away from the plasma membrane. as bag is the first ww-domain interactor identified that negatively regulates budding of vp vlps and infectious virus, we propose that the chaperone-mediated autophagy function of bag represents a specific host defense strategy to counteract the function of vp in promoting efficient egress and spread of virus particles. introduction ebola (ebov) and marburg (marv) viruses are virulent pathogens that cause severe hemorrhagic disease in humans and non-human primates. there are currently no fda approved vaccines or antiviral drugs to prevent or treat infections by these category a niaid priority pathogens [ ] . the recent catastrophic outbreak of ebov in west africa underscores the urgent need to better understand the biology and pathogenesis of this global public health threat, and to decipher the molecular mechanisms by which ebov interacts with the host to cause disease. the filovirus matrix protein vp is the most abundant protein in the virion and is essential for virus assembly and egress. indeed, expression of vp alone is sufficient to form viruslike particles (vlps), which are morphologically indistinguishable from infectious virions and are released from mammalian cells in a manner that recapitulates the release of authentic virus [ ] [ ] [ ] [ ] [ ] . although not required for ebov replication [ ] , late (l) domains (which contain ptap and/or ppxy amino acid sequence motifs) are conserved within ebov and marv vp and promote efficient egress of vlps and virus by recruiting host proteins that facilitate virus-cell separation [ , , , [ ] [ ] [ ] [ ] . for example, ebov and marv vp l-domains hijack specific host proteins associated with the escrt pathway, including tsg , alix, and nedd [ , , [ ] [ ] [ ] [ ] [ ] [ ] . viral proteins bearing ppxy motif each interact with a unique repertoire of ww-domain bearing host proteins with diverse functions [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . for example, the ppxy l-domain within evp , mvp , and other viral matrix proteins interacts specifically with ww-domains of: ) host nedd ; a hect family e ubiquitin ligase that is linked with the cellular escrt machinery, ) host itch; a hect family e ubiquitin ligase involved in immune regulation and inflammatory signaling, and ) host iqgap ; a multifunctional scaffolding protein involved in regulating cell motility, actin polymerization, and filopodia formation [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . in general, these previously characterized viral ppxy/ww-domain interactions promote efficient virus production. here, we sought to identify additional ww-domain bearing proteins that interact with the evp ppxy motif by screening a gst array of host proteins containing one or more ww-domains [ ] with an ebov ppxy-containing peptide. using this unbiased approach, we identified ww-domain containing protein bag as a novel evp interactor. bag is a stress-induced molecular co-chaperone involved in regulating cellular protein homeostasis by cma. since in general, viral ppxy-containing proteins tend to bind ww-domains with good specificity and selectivity [ ] , our identification of bag suggests that this protein may play a biologically relevant role in the lifecycle of ebov. indeed, we confirmed that hypothesis by first using co-ip to validate the specificity of the ppxy/ww-domain physical interaction between vp (both evp and mvp ) and bag , and functionally demonstrated that expression of bag inhibited vp vlp production, as well as budding of a vsv recombinant virus containing the ebov vp ppxy l-domain motif. to our knowledge, this is the first identification of a vp -interacting mammalian ww-domain bearing protein that negatively regulates budding. mechanistically, our data suggest that bag binds vp and not only sequesters it away from the site of budding at the plasma membrane, but also directs a fraction of vp into aggresomes, thus reducing vlp egress. screening of proline-rich motif reading array identifies bag as an ebov vp ppxy interactor the "proline-rich" reading array represents a powerful tool that we have used previously to identify ww-domain proteins that physically and functionally bind to viral ppxy motifs ( fig ) [ , ] . here, we used biotinylated peptides containing either the evp wt ppxy motif (mrrvilptappeymeai) or an evp ppxy mutant motif (mrrvilptaaaeameai) to screen gst-ww domain and gst-sh domain containing proteins (fig ) . while the ppxy mutant evp peptide did not interact with any of the arrayed ww or sh domains, the wt evp peptide bound robustly to a number of host ww domains, including previously identified proteins nedd , rsp (yeast ortholog of nedd ), and itch ( fig b) . intriguingly, the wt evp peptide also bound to a select number of ww-domains not identified previously, including the ww-domain of host protein bag (fig b, panel g) . we used purified gst-ww domain fusion proteins and a gst pull down assay to confirm the specificity of the bag -vp interaction (fig ) . briefly, protein extracts from hek t cells expressing either evp -wt, evp -Δpt/py (ppxy deletion mutant), or mvp -wt were incubated with beads containing gst alone or gst-bag ww (fig ) . input and pulldown proteins were detected by western blot. indeed, we found that gst-bag ww, but not gst alone, pulled down evp in a ppxy-dependent manner (fig a, top right panel) , and also pulled down mvp that contains a single ppxy l-domain motif (fig b, top right panel) . in sum, these data indicate that, compared to other ppxy-ww domain interactions interrogated previously [ ] , the evp ppxy motif possesses a relatively high and significant degree of specificity, suggesting that bag is likely a novel, biologically relevant interactor with vp of ebov and marv. next, we used a co-ip approach to determine whether evp and mvp interact with fulllength bag expressed in mammalian cells. to test this, hek t cells were co-transfected with bag plus wt or ppxy mutants of evp (evp -Δpt/py) and mvp (mvp -p>a) (fig a and b ). cell extracts were immunoprecipitated with non-specific igg, anti-evp antiserum, or anti-mvp antiserum as indicated (fig a and b) , and his-cmyc-tagged bag was detected in precipitated samples by western blot using either anti-cmyc or anti-his antisera (fig a and b ). exogenously expressed bag was detected in evp -wt precipitates ( fig a, lane ) , but was not detected in preimmune igg, nor evp -Δpt/py precipitates ( fig a, lanes and , respectively) . similarly, exogenously expressed bag was detected in mvp -wt precipitates (fig b, lane ) , but was not detected in preimmune igg, nor mvp -p>a precipitates (fig b, lanes and , respectively) . these results indicated that both evp and mvp interact with full length bag in a ppxy-dependent manner in transiently transfected hek t cells. next, we asked whether evp and mvp could interact with full length endogenous bag in hek t cells. hek t cells were transfected with either evp -wt (fig c) or mvp -wt (fig d) , and once again cell extracts were immunoprecipitated with either nonspecific igg, or anti-vp antiserum as indicated. endogenous bag was detected in precipitated samples by western blot using anti-bag antiserum (fig c and d ). endogenously expressed bag was detected in both evp -wt ( fig c, lane ) and mvp -wt ( fig d, lane ) precipitates, but not in preimmune igg precipitates (fig c and d , lanes ). together, these results correlate well with those from the gst pulldown assays and indicate that evp and mvp interact with full length bag in a ppxy-dependent manner. finally, we sought to identify the functional domains of bag that mediate its interaction with evp and mvp . bag contains a number of functional domains including: a single n-terminal ww-domain, two ipv regions that bind to hspb and function in protein quality control, multiple pxxp motifs that binds to sh domains, and a c-terminal bag domain that interacts with the atpase domain of hsp ( fig a) [ ] [ ] [ ] [ ] [ ] . briefly, hek t cells were co- extracts from hek t cells transfected with evp or evp -Δpt/py plus bag -wt were first immunoprecipitated (ip) with either normal rabbit igg or polyclonal anti-evp antisera as indicated. bag was detected in the precipitates by western blot (wb) using mouse anti-myc antiserum. expression controls for evp -wt, evp -Δpt/py, his-myc-tagged bag and gapdh are shown. b) extracts from hek t cells transfected with flag-tagged mvp or mvp (p>a) plus bag -wt were first immunoprecipitated (ip) with either normal mouse igg or anti-flag antisera as indicated. bag was detected in the precipitates by western blot (wb) using rabbit anti-his antiserum. expression controls for mvp , mvp (p>a), his-myc-tagged bag and β-actin are shown. c) extracts from hek t cells transfected with evp -wt alone were first immunoprecipitated with either normal rabbit igg or polyclonal anti-evp antisera as indicated. endogenous bag was detected in the precipitates by western blot using polyclonal anti-bag antiserum. expression controls for evp -wt, endogenous bag , and gapdh are shown. d) extracts from hek t cells transfected with mvp -wt alone were first immunoprecipitated with either normal rabbit igg or anti-mvp antisera as indicated. endogenous bag was detected in the precipitates by western blot using polyclonal anti-bag antiserum. expression controls for mvp -wt, endogenous bag , and β-actin are shown. transfected with evp plus his/myc-tagged bag wt, bag -Δn (ww-domain deletion mutant), or bag -Δc (bag domain deletion mutant) (fig a and b ). cell extracts were immunoprecipitated with either non-specific igg, or anti-evp antiserum as indicated ( fig b) , and cmyc-tagged bag was detected in precipitated samples by western blot using anticmyc antiserum (fig b) . bag -wt (lane ) and bag -Δc (lane ) were detected in evp precipitates; however, bag -Δn (lane ) was not. no appreciable levels of bag wt, bag -Δn, and bag -Δc were detected in preimmune igg precipitates ( fig b, lanes - ) . similar results were obtained in hek t cells transfected with mvp plus the indicated bag plasmids ( fig c) . indeed, bag -wt ( fig c, lane ) and bag -Δc ( fig c, lane ) were detected in mvp precipitates, whereas bag -Δn lacking the ww-domain was not detected (fig c, lane ) . western blots of the three bag proteins, evp , mvp , and actin are shown as expression controls (fig b and c ). these data demonstrate that the bag wwdomain specifically mediates interactions with both evp and mvp in mammalian cells. given the highly specific and select ppxy/ww-domain interaction between vp and bag , we hypothesized that this physical interaction would have a biological consequence. to test this, we used our well-established vp vlp budding assay to determine whether expression of bag -wt, bag -Δn or bag -Δc would affect vp vlp egress. briefly, hek t cells were transfected with evp or mvp alone, or in combination with either bag -wt, bag -Δn or bag -Δc, and both cell extracts and supernatants containing vlps were harvested at hours post transfection (fig ) . all proteins were detected at equivalent levels in cell extracts (fig a and c cells) . interestingly, we found that expression of bag wt or bag -Δc consistently resulted in a significant decrease in egress of both evp (fig a and b ) and mvp (fig c and d ) vlps. importantly, we did not observe a significant decrease in egress of either evp or mvp vlps in the presence of ww-domain deletion mutant bag -Δn (fig a, compare lanes and ; fig c, compare lanes and ) . intriguingly, the inhibitory effect of bag appeared to be more pronounced on budding of mvp vlps (fig d) , compared to that on evp vlps. this may reflect the presence of a single ppxy l-domain within mvp in contrast to the presence of overlapping ptap and ppxy motifs within evp . to determine whether the novel inhibitory effects of bag on vp vlp egress were dosedependent, we transfected hek t cells with a constant amount of evp or mvp plus increasing amounts of bag -wt, bag -Δn or bag -Δc. cell extracts and supernatants were harvested as described above (fig ) . appropriate expression levels for all proteins were confirmed by western blotting of cell extracts (fig , cells) . we observed a clear dose-dependent inhibition of both evp (fig a and b ) and mvp (fig d and e ) vlps in the presence of increasing amounts of either bag -wt or bag -Δc. in contrast, increasing expression of ww-domain deletion mutant bag -Δn had no effect on budding of either evp or mvp vlps (fig c and f ). once again, the level of inhibition mediated by bag -wt and bag -Δc appeared to be more pronounced on mvp vlps compared to that on evp vlps, with mvp vlps being virtually undetectable in samples expressing the highest amounts of bag . taken together, these results demonstrate a functional role for bag as a negative regulator of evp and mvp vlp budding via a ww-domain dependent mechanism. to our knowledge, this is the first host ww-domain containing protein shown to inhibit filovirus vp egress. we next asked whether knockdown of endogenous bag would result in an increase in vp vlp egress. for this, we used an sirna approach to knockdown levels of endogenous bag in hek t cells expressing evp (fig ) . hek t cells were transfected with evp plus either random or bag -specific sirnas, and both cell extracts and vlps were harvested and analyzed by western blotting (fig ) . we were able to achieve suppression of endogenous bag by approximately % (fig a, cells) , which led to a reproducible and significant increase in evp vlp egress compared to control sirna samples (fig a and b ). it should be noted that a similar~ -fold increase was also observed for mvp vlps from cells treated with bag -specific sirnas. these results correlate well with those described above (figs and ) and further confirm the inhibitory effect of bag on vp -mediated budding. as efficient egress of vlps requires localization and self-assembly of vp at the plasma membrane (pm), we next asked whether bag -mediated alterations in vp localization correlated with egress inhibition. for this, we utilized confocal microscopy of live hek t cells transfected with a gfp-evp fusion construct in the absence (vector alone) or presence of a bag -mcherry fusion construct (fig a) . representative images of live cells transfected with gfp-evp alone revealed the expected pattern of expression in the cytoplasm with pronounced localization around the cell periphery and in pm projections (fig a, top row) . in contrast, the pattern of gfp-evp changed in cells co-expressing bag -mcherry, resulting in a more diffuse pattern of cytoplasmic localization with little to no pm projections (fig a, middle and bottom rows) . indeed, this contrast in vp localization can be visualized in neighboring cells expressing gfp-evp plus either low or high levels of bag -mcherry ( fig a, middle row) . thus, these representative live cell images suggest that localization and accumulation of evp at the site of budding at the pm and in pm projections is reduced in the presence of bag . as bag can sequester target proteins into aggresomes during cma, we next sought to determine whether this altered localization pattern of evp was due in part to sequestration of evp by bag into aggresomes. to examine this possibility, we utilized confocal microscopy of live hela cells transfected with gfp-evp + bag -wt + mcherry-tagged human microtubule-associated light chain- (lc ) protein, a well-characterized marker for aggresomes ( fig b) [ , ] . representative images of live hela cells once again revealed the typical pattern of gfp-evp predominantly at the pm in cells expressing mcherry-lc , but lacking expression of bag -wt (fig b, top row) . once again, the altered and more diffuse cytoplasmic pattern of gfp-evp was observed in cells co-expressing bag -wt and mcherry-lc ; however, in addition, a fraction of gfp-evp was observed to co-localize with mcherry-lc in puncta most likely representing cellular aggresomes (fig b, bottom two rows) . together, these representative live cell confocal images suggest that the cma function of bag sequesters a fraction of evp away from the pm and into aggresomes, leading to a reduction in vlp egress. to further support the imaging data described above, we used a biochemical approach to determine whether the levels of evp and mvp in pm fractions of transfected cells would be reduced in the presence of bag -wt, but not in the presence of bag -Δn. for this, hek t cells were transfected with vector (pcaggs) alone, vp + vector, vp + bag -wt, or vp + ww-domain deletion mutant bag -Δn, and both cytosol and pm fractions were harvested and subjected to western blot analysis (fig ) . na/k atpase was used as a positive expression control for the pm fraction, and β-actin was used as a cytosol control (fig a and c ). as expected, both evp and mvp were detected at equivalent levels in all cytosol fractions (fig a and c, lanes - ) ; however, the levels of evp and mvp in pm fractions from cells expressing bag -wt were consistently reduced by - fold (fig a and c, lane ; and fig b and d ) compared to that detected in the pm fractions of cells expressing vp alone (lanes ) or vp + bag -Δn (lanes ). in addition to cell fractionation studies, we examined evp on the cell surface using indirect immunofluorescence and xzy scanning of confocal microscopy ( fig e) . as expected, evp alone localized robustly to the pm (fig e, top row) . in contrast, evp localization at the pm was less pronounced in the presence of bag -wt compared to control cells (fig e, middle row) . importantly, the pm localization pattern of evp in the presence of bag -Δn (fig e, bottom row) was virtually identical to that in cells expressing evp alone (top row). the levels of evp , bag -wt and bag -Δn detected at the cell surface by indirect immunofluorescence and confocal microscopy correlate well with results from the cell fractionation studies. taken together, our results suggest that the general mechanism by which bag inhibits vlp egress is unique and involves sequestration of a fraction of evp away from the pm and into aggresomes in a ppxy/ww domain-dependent manner. although we focused on the pm, it is important to note that we cannot completely rule out the possibility that sequestration of evp away from internal membranes may also contribute to the mechanism by which bag inhibits vlp egress. finally, we sought to determine whether expression of bag would inhibit egress of infectious virus. toward this end, we utilized our live infectious vsv recombinants; vsv-m and vsv-m -p a [ ] . recombinant vsv-m expresses the wt l-domain motifs (ptap-pey) and flanking residues from evp in place of the l-domain of vsv m protein and buds efficiently, whereas recombinant vsv-m -p a expresses mutated evp l-domain motifs (ptaaaey) and is budding defective [ ] . briefly, hek t cells were first transfected with vector alone, bag -wt, or bag -Δn for hours, and then infected with either vsv-m or vsv-m -p a at an moi of . for hours (peak time of budding). infected cell extracts were analyzed by western blot for expression controls, and virus production was quantified by standard plaque assay (fig ) . we found that the levels of infectious vsv-m released from mock-and bag -Δn-transfected cells were virtually identical; however, titers of vsv-m released from cells expressing bag -wt were consistently and significantly reduced by > % compared to controls (fig a) . in contrast, expression of bag -wt did not have any significant effect on budding of mutant vsv-m -p a (fig c) . importantly, these data demonstrate that the inhibitory effect of bag on budding extends to infectious virus, and confirms the involvement of the viral ppxy/bag ww-domain interaction in this negative regulatory mechanism. as the major filovirus matrix protein, vp plays a central role in directing virion assembly and egress from infected cells. three minimal functional domains of vp are required for efficient vlp egress, including a membrane (m) binding region, a self-interaction (i) domain, and one or more late (l) domain motifs. the l-domain motifs hijack or recruit specific host cell proteins that facilitate or promote efficient virus-cell separation [ , , , [ ] [ ] [ ] [ ] [ ] [ ] . here, we hek t cells were transfected with gfp-evp (green) plus either vector, or bag -mcherry (red), and cells were imaged at hours post transfection using a leica sp flim inverted confocal microscope. representative images are shown with arrows highlighting the typical localization pattern of gfp-evp at the plasma membrane and in pm projections, while arrowheads highlight the altered diffuse cytoplasmic localization pattern of evp observed in bag expressing cells. cell nuclei were stained with nucblue. scale bars = μm. b) hela cells were transfected with gfp-evp (green) plus mcherry-lc (red) and vector alone (top row), or bag (bottom two rows), and cells were imaged at hours post transfection using a leica sp flim inverted confocal microscope. representative images are shown with white arrows highlighting the colocalization of gfp-evp and mcherry-lc in aggresomes. cell nuclei were stained with nucblue. scale bars = μm. doi: . /journal.ppat. .g have identified host ww-domain containing protein bag as a novel interactor with the ppxy l-domain motif of both evp and mvp . moreover, we confirmed the physical and functional interaction between the ww-domain of bag and the viral ppxy l-domain motif by using l-domain and ww-domain mutants in gst-pulldowns, co-immunoprecipitation, sirna analysis, and/or vlp/virus budding assays. intriguingly, unlike previously identified host ww-domain proteins that interact with vp , including nedd [ , , , ] and itch [ ] , bag is the first host ww-domain interactor to negatively regulate egress of evp and mvp vlps, as well as infectious virus containing the evp ppxy l-domain motif. bag is a member of the bag family of proteins (bag -bag ) which are characterized by a bag domain that interacts with the atpase domain of heat shock protein (hsp) [ ] . were fixed at hrs post-transfection, and then incubated with rabbit anti-evp antiserum and mouse anti-myc antiserum (to detecting bag -wt and bag -Δn). cells were then stained with alexa fluor goat anti-rabbit and goat anti-mouse secondary antibodies. microscopy was performed using a leica sp flim inverted confocal microscope and xzy scanning. representative images displaying evp (green) and bag -wt (red) or bag -Δn (red) localized at the pm are shown. cell nuclei were stained with nucblue. scale bars = μm. doi: . /journal.ppat. .g bag is the only member of this family that contains a single n-terminal ww-domain. as a co-chaperone and cell survival protein, bag regulates multiple cell pathways, including, apoptosis, autophagy, cell development and cytoskeleton organization [ , , ] . indeed, bag is induced under conditions of cell stress and plays a major role in sequestering misfolded and/ or foreign proteins to the proteasome for degradation by cma [ , , ] . our data imply that the cma function of bag acts as a novel host defense/response mechanism to sequester a fraction of vp from the site of budding at the pm and into aggresomes, thus reducing vlp/ virus egress and spread. whether sequestration of vp away from the pm results in degradation of vp remains to be determined. interestingly, selective autophagy of the endoplasmic reticulum (er-phagy) was recently shown to regulate ebov replication in murine cells [ ] . bag has been associated with the lifecycles of other rna and dna viruses including, hiv- , varicella zoster virus, herpes simplex virus, african swine fever virus, papillomavirus, polyomaviruses, coronavirus, adenovirus, and epstein barr virus [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . however, in contrast to our findings with vp , bag primarily exerts positive effects on the lifecycles of these other viruses. for example, gout et. al [ ] reported that the adenovirus penton base protein interacted with the ww-domain of bag via its ppxy motif to promote adenovirus entry and virus progeny production. results from cell fractionation and confocal microscopy suggest that the mechanism by which bag inhibits vp -mediated egress involves, at least in part, relocalization of vp away from the site of budding at the pm and accumulation of a portion of vp into lc -containing aggresomes, which are visualized more definitively in hela cells rather than hek t cells. indeed, bag has been shown to regulate an aggresome-targeting pathway by interacting with the microtubule-motor dynein to selectively direct target proteins to the aggresome [ , , ] . a more precise determination of whether sequestration of vp correlates with its degradation, and/or whether bag may disrupt trafficking of vp along the cytoskeletal architecture of the cell to the pm, remains to be determined. since bag interacts with the viral ppxy motif, we cannot completely rule out the possibility that bag may competitively inhibit vp from interacting with other host ww-domain containing proteins such as e ubiquitin ligases, nedd and itch, which may impair ubiquitination and subsequent egress of vp vlps. this is likely not the primary mechanism of budding inhibition since the intracellular localization of vp was altered significantly in the presence of bag as judged by pm fractionation and confocal microscopy. in sum, we identified bag as a novel, ww-domain interactor with the ppxy motif of vp leading to inhibition of vlp and infectious virus egress in a ppxy/ww-domain dependent manner. as a stress-induced, cell survival protein, bag may represent a key component of a novel host defense mechanism to dampen virus egress via cma and protein sequestration. these findings provide new insights into the roles that host proteins play in regulating filovirus vp -mediated egress, and a more comprehensive understanding of these virus-host interactions may be helpful in the design of future antiviral therapies. for example, it may be possible to identify small molecules that could bind to vp in a manner that mimics the inhibitory effect of bag . alternatively, the ww domain of bag alone (per se) could be used to inhibit vlp and virus egress as documented previously for cis-expressed yap ww domain that inhibited ppxy-mediated budding of rous sarcoma virus [ ] . hek t (american type culture collection; atcc), hela (american type culture collection; atcc), and bhk- (american type culture collection; atcc) cells were maintained in dulbecco's modified eagle's medium (dmem) (corning) supplemented with % fetal bovine serum (fbs) (gibco), penicillin ( u/ml)/streptomycin ( μg/ml) (invitrogen) and the cells were grown at ˚c in a humidified % co incubator. the plasmids encoding evp -wt, evp -Δpt/py were described previously [ , , ] . flag-tagged mvp -wt and ppxy mutant mvp (p>a) were kindly provided by s. becker (institut für virologie, marburg, germany). the pcdna myc-his-bag -wt ( - ), pcdna myc-his-bag -Δn ( - ) and pcdna myc-his-bag -Δc ( - ) plasmids were kindly provided by k. khalili (temple university). plasmid pdest-mcherry-bag was kindly provided by e. sjøttem (university of tromsø). mcherry-hlc b-pcdna . was a gift from david rubinsztein (addgene plasmid # ) [ ]. the proline rich motif "reading" array consisted of almost all known ww domains ( domains) from mammalian proteins (and yeast). we prepared biotinylated peptides harboring either the ebov vp wt ppxy motif (mrrvilptappeymeai) or mutated ppxy motif (mrrvilp-taaaeameai). both of the biotinylated peptides were fluorescently labeled and used to screen the specially prepared "proline-rich" reading array. gst alone and gst-bag ww domain fusion protein were expressed in bl- cells and subsequently conjugated to glutathione (gsh) beads (ge healthcare). hek t cells were transfected with evp -wt, evp -Δpt/py or flag-tagged-mvp , respectively. at hours after transfection, the cell extracts were incubated with the gsh beads described above at ˚c for hours with continuous rotating. the proteins complexes were pulled down with beads via centrifugation. the rabbit evp antiserum (prosci), mouse anti-flag monoclonal antibody (sigma), mouse anti-gst monoclonal antibody (sigma) were used to detect evp -wt, evp -Δpt/py, mvp , gst, or gst-bag ww proteins in input and pulldown samples by western blotting. hek t cells were transfected with the indicated plasmids combinations using lipofectamine reagent (invitrogen). at hours post transfection, cells were harvested and lysed, and cell extracts were incubated with either rabbit or mouse igg, evp , or mvp specific antisera as indicated. protein a or g agarose beads were then added to the mixtures and incubated overnight at ˚c. after incubation, beads were collected via centrifugation and washed x. proteins were then detected by western blotting with polyclonal anti-bag (proteintech), polyclonal anti-his (cell signaling), or monoclonal anti-cmyc (millipore) antisera as indicated. filovirus vp vlp budding assays in hek t cells were described previously [ , , , , ] . evp and mvp proteins in vlps and cell extracts were detected by sds-page and western blotting, and quantified using nih image-j software. the anti-evp antiserum was used to detect evp -wt and evp -Δpt/py mutant, and anti-flag monoclonal antibody was used to detect flag-tagged mvp . for bag titration experiments, hek t cells were transfected with . μg of evp or mvp and increasing amounts of bag -wt or bag -Δc ( . , . , . μg), or bag -Δn ( . and . μg). the total amounts of transfected dna were equivalent in all samples. supernatants and cell extracts were harvested at hours post transfection. hek t cells seeded in well plates were transfected with human bag -specific or random sirna (dharmacon) at a final concentration of nm per well using lipofectamine reagent (invitrogen). at hours post transfection, cells were transfected again with . μg of evp or mvp plasmid. vlps and cell extracts were harvested at hours post transfection, and proteins were detected by western blotting. hek t cells were transfected with gfp-evp plus mcherry-bag or vector (pcaggs), and cells were monitored by leica sp flim inverted confocal microscope at hrs posttransfection. hela cells were transfected with gfp-evp plus mcherry-lc and either bag -wt or vector alone, and cells were monitored by leica sp flim inverted confocal microscope at hrs post-transfection. cell nuclei were stained by nucblue live cell ready probes (life thchonologies). the intracellular localization of gfp-evp , mcherry-bag , and mcherry-lc in the live cells were imaged using xyz scanning. to generate series images through the whole cell, serial optical planes of focus (at approximately μm intervals) were taken through the z stacks from the top to bottom of the cell, and the collected images were merged into one using the leica microsystems (las af) software. hek t cells were transfected with the indicated plasmid combinations. at hours post transfection, cells were washed with cold pbs and fixed with % formaldehyde for min at room temperature, then permeabilized with . % triton x- . after washing x with cold pbs, cells were incubated with polyclonal anti-evp antiserum and mouse anti-cmyc antiserum to detect his-myc tagged bag and its mutants. next, cells were stained with alexa fluor goat anti-rabbit and goat anti-mouse secondary antibodies (life techonolo-gies). cell nuclei were stained with nucblue fixed cell ready probes (life techonolo-gies). microscopy was performed using a leica sp flim inverted confocal microscope and an xzy scanning model. serial optical planes of focus were taken on the y-axis and the collected images were merged into one by using the leica microsystems (las af) software. hek t cells were transfected with indicated plasmid combinations, and cells were scraped and washed with cold pbs at hours post-transfection. cells were then collected via low speed centrifugation. the cytosol, organelle membrane and plasma membrane protein fractions were isolated sequentially using the "minute plasma membrane protein isolation kit" (invent) following the manufacturer's instructions. proteins within the cytosol and plasma membrane fractions were detected via sds-page and western blotting. the β-actin and sodium potassium atpase were used as cytosol and plasma membrane controls and were detected using mouse anti β-actin (sigma) and rabbit anti na/k atpase (abcam) monoclonal antibodies. transfection/infection assays hek t cells were first transfected with bag , bag -Δn or vector for hours, and then subsequently infected with either vsv-m or vsv-m -p a at a moi of . . supernatants and infected cell extracts were harvested at hours post-infection. released vsv-m and vsv-m -p a virions were titrated in duplicate via standard plaque assay on bhk- cells. cellular proteins were detected by western blotting using specific antibodies. filovirus research: knowledge expands to meet a growing threat a ppxy motif within the vp protein of ebola virus interacts physically and functionally with a ubiquitin ligase: implications for filovirus budding filovirus budding filovirus assembly and budding filoviruses: interactions with the host cell. cellular and molecular life sciences no exit: targeting the budding process to inhibit filovirus replication ebola virus vp late domains are not essential for viral replication in cell culture role of multivesicular bodies and their components in the egress of enveloped rna viruses mechanisms for enveloped virus budding: can some viruses do without an escrt? functional characterization of ebola virus l-domains using vsv recombinants viral and host proteins that modulate filovirus budding late budding domains and host proteins in enveloped virus release alix rescues budding of a double ptap/ ppey l-domain deletion mutant of ebola vp : a role for alix in ebola virus egress the ww domain: a signalling site in dystrophin the ww domain of yes-associated protein binds a proline-rich ligand that differs from the consensus established for src homology -binding modules characterization of a novel protein-binding module-the ww domain the ww module competes with the sh domain structure and function of the ww domain functions of ww domains in the nucleus the ww domain: linking cell signalling to the membrane cytoskeleton a map of ww domain family interactions new wrinkles for an old domain rhabdoviruses and the cellular ubiquitin-proteasome system: a budding interaction functional involvement of a novel nedd -like ubiquitin ligase on retrovirus budding pppyveptap motif is the late domain of human t-cell leukemia virus type gag and mediates its functional interaction with cellular proteins nedd and tsg overlapping motifs (ptap and ppey) within the ebola virus vp protein function independently as late budding domains: involvement of host proteins tsg and vps- ebola virus matrix protein vp interaction with human cellular factors tsg and nedd nedd regulates egress of ebola virus-like particles from host cells role of nedd and ubiquitination of rous sarcoma virus gag in budding of virus-like particles from cells isg inhibits ebola vp vlp budding in an l-domain-dependent manner by blocking nedd ligase activity the escrt pathway and hiv- budding regulation of marburg virus (marv) budding by nedd . : a different ww domain of nedd . is critical for binding to marv and ebola virus vp bimolecular complementation to visualize filovirus vp -host complexes in live mammalian cells: toward the identification of budding inhibitors ubiquitin conjugation to gag is essential for escrt-mediated hiv- budding small-molecule probes targeting the viral ppxy-host nedd interface block egress of a broad range of rna viruses quinoxaline-based inhibitors of ebola and marburg vp egress itch e ubiquitin ligase interacts with ebola virus vp to regulate budding host iqgap and ebola virus vp interactions facilitate virus-like particle egress a protein-domain microarray identifies novel protein-protein interactions towards prediction of cognate complexes between the ww domain and proline-rich ligands hspb and bag : a new chaperone complex targeting misfolded proteins to macroautophagy bag expression in glioblastoma cells promotes accumulation of ubiquitinated clients in an hsp -dependent manner chaperone-assisted proteostasis is essential for mechanotransduction in mammalian cells a role for the chaperone complex bag -hspb in actin dynamics, spindle orientation and proper chromosome segregation during mitosis bag : a multifaceted protein that regulates major cell pathways bag and friends: co-chaperones in selective autophagy during aging and disease phospholamban p. arg del cardiomyopathy is characterized by phospholamban aggregates, aggresomes and autophagic degradation rhabdovirus assembly and budding assembly and budding of negative-strand rna viruses rabies virus assembly and budding viral membrane scission escaping from the cell: assembly and budding of negative-strand rna viruses arenavirus budding: a common pathway with mechanistic differences molecular chaperone targeting and regulation by bag family proteins emerging roles of molecular chaperones and co-chaperones in selective autophagy: focus on bag proteins bag mediates chaperone-based aggresome-targeting and selective autophagy of misfolded proteins fam b, the selective autophagy receptor for endoplasmic reticulum turnover, inhibits replication of ebola virus strains makona and mayinga host cell targets for african swine fever virus analysis of hdac and bag -aggresome pathways in african swine fever viral factory formation hiv- tat protein induces glial cell autophagy through enhancement of bag protein levels bag protects bovine papillomavirus type -transformed equine fibroblasts against pro-death signals quantitative proteomics analysis reveals bag as a potential target to suppress severe acute respiratory syndrome coronavirus replication co-chaperone bag and adenovirus penton base protein partnership bag protein regulates caspase- activation in hiv- -infected human primary microglial cells epstein-barr virus nuclear antigen (ebna) a induces the expression of and interacts with a subset of chaperones and co-chaperones the co-chaperone bag regulates herpes simplex virus replication evidence for bag modulation of hiv- gene transcription bag , a host cochaperone, facilitates varicella-zoster virus replication novel partner proteins of adenovirus penton the cleavage product of amyloidbeta protein precursor sabetappalpha modulates bag -dependent aggresome formation and enhances cellular proteasomal activity in vivo interference of rous sarcoma virus budding by cis expression of a ww domain overlapping motifs (ptap and ppey) within the ebola virus vp protein function independently as late budding domains: involvement of host proteins tsg and vps- the authors wish to thank k. khalili, e. sjøttem, and s. becker for kindly providing reagents, l. king for critical reading of the manuscript, and members of the harty and freedman labs for helpful discussions. we also wish to thank g. ruthel (manager) and b.d. freedman (director) of the pennvet imaging core for assistance with microscopy studies. lastly, the authors wish to thank professor t.r. luo (guangxi university, china) for his invaluable guidance and mentoring of j.l.. key: cord- -wo anq authors: xia, hongjie; wang, peipei; wang, guang-chuan; yang, jie; sun, xianlin; wu, wenzhe; qiu, yang; shu, ting; zhao, xiaolu; yin, lei; qin, cheng-feng; hu, yuanyang; zhou, xi title: human enterovirus nonstructural protein c(atpase) functions as both an rna helicase and atp-independent rna chaperone date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: wo anq rna helicases and chaperones are the two major classes of rna remodeling proteins, which function to remodel rna structures and/or rna-protein interactions, and are required for all aspects of rna metabolism. although some virus-encoded rna helicases/chaperones have been predicted or identified, their rna remodeling activities in vitro and functions in the viral life cycle remain largely elusive. enteroviruses are a large group of positive-stranded rna viruses in the picornaviridae family, which includes numerous important human pathogens. herein, we report that the nonstructural protein c(atpase) of enterovirus (ev ), which is the major causative pathogen of hand-foot-and-mouth disease and has been regarded as the most important neurotropic enterovirus after poliovirus eradication, functions not only as an rna helicase that ′-to- ′ unwinds rna helices in an adenosine triphosphate (atp)-dependent manner, but also as an rna chaperone that destabilizes helices bidirectionally and facilitates strand annealing and complex rna structure formation independently of atp. we also determined that the helicase activity is based on the ev c(atpase) middle domain, whereas the c-terminus is indispensable for its rna chaperoning activity. by promoting rna template recycling, c(atpase) facilitated ev rna synthesis in vitro; when c(atpase) helicase activity was impaired, ev rna replication and virion production were mostly abolished in cells, indicating that c(atpase)-mediated rna remodeling plays a critical role in the enteroviral life cycle. furthermore, the rna helicase and chaperoning activities of c(atpase) are also conserved in coxsackie a virus (cav ), another important enterovirus. altogether, our findings are the first to demonstrate the rna helicase and chaperoning activities associated with enterovirus c(atpase), and our study provides both in vitro and cellular evidence for their potential roles during viral rna replication. these findings increase our understanding of enteroviruses and the two types of rna remodeling activities. for both cells and viruses, the functionality of rna molecules usually requires correct folding into proper tertiary structures and the association with correct rna-binding proteins [ , ] . to make the scenario more complex, rna molecules frequently function via transient base pairing with target rnas and/or dynamic association/dissociation with distinct rna-binding proteins [ ] . however, correct folding of rnas is quite challenging and time-consuming, because rnas can easily become trapped in thermodynamically stable intermediates that are misfolded and inactive. in response, cells and viruses encode a variety of "helper" or rna remodeling proteins, such as rna helicases and rna chaperones, that can destabilize rna-rna or rna-dna base pairing, thereby lowing the thermodynamic barrier between rna conformations and promoting the proper formation of rna tertiary structures [ ] [ ] [ ] . it is believed that rna remodeling proteins play pivotal roles in nearly all processes involving rna molecules. rna helicases are highly similar to dna helicases, contain atpase activity, and utilize the energy of atp binding and/or hydrolysis to translocate along and unwind rna duplexes. they are thought to participate in most atp-dependent remodeling of structured rnas and are classified into six superfamilies (sfs), designated sf to sf , on the basis of conserved helicase motifs [ ] . on the other hand, rna chaperones are a heterogeneous group of proteins that share no consensus sequences or motifs but are able to destabilize rna duplexes and assist in the formation of more globally stable rna structures. the major difference between rna chaperones and helicases is that rna chaperones do not require atp binding or hydrolysis for their remodeling activities [ ] . (of note, the definition of rna chaperones is sometimes blurred, as some publications have defined helicases as a subgroup of atp-dependent rna chaperones [ ] . herein, like in most publications, we define rna helicases and chaperones as two separate classes of the rna remodelers for the sake of clarity.) for viruses, particularly rna viruses, their viral rnas (vrnas) contain multiple cis-acting elements that play pivotal roles in vrna replication and translation. these highly structured rna elements require rna helicases or chaperones to aid in their proper folding and re-folding. moreover, during vrna replication, replicative intermediate double-stranded rna (dsrna) must be unwound by virus-encoded or cellular rna helicases; therefore, vrna templates can be efficiently re-utilized to synthesize more progeny vrna strands. thus far, a variety of rna viruses, such as flavivirus ns [ ] , alphavirus nsp [ ] , coronavirus nsp [ ] , and alphatetravirus hel [ ] , have been reported to encode their own rna helicases, and the majority of them belong to helicase sf and sf [ ] . enteroviruses are a large group of positive-stranded rna viruses in the picornaviridae family that contain numerous important human pathogens, including poliovirus, enterovirus (ev ), coxsackie viruses, and echoviruses. they cause approximately billion human infections annually and are responsible for a wide spectrum of diseases, ranging from relatively mild conditions, such as common cold, upper respiratory illness, and hand-foot-and-mouth disease (hfmd), to severe conditions such as aseptic meningitis, encephalitis, myocarditis, neonatal sepsis-like disease, and poliomyelitis [ ] . so far, although vaccination has almost eradicated poliomyelitis worldwide [ ] , no effective vaccine or antiviral therapy is available for many other important human enteroviruses, particularly neurotropic ev , which is the major causative pathogen for hfmd and a serious lethal threat to children. enterovirus encodes a single polyprotein that is proteolytically cleaved into separate structural and nonstructural proteins. among them, the multifunctional protein c atpase is the most conserved and complex but the least understood ( fig a) . enterovirus c atpase has been reported to participate in diverse processes, including rna binding [ , ] , rna replication [ ] [ ] [ ] , membrane anchoring and rearrangement [ , ] , autophagy inhibition [ ] , encapsidation and viral morphogenesis [ , ] , and suppression of nuclear factor kappa b activation [ ] . c atpase has long been predicted as a putative sf helicase on the basis of its aaa+ atpase activity and conserved sf signature motifs (fig b) [ , ] . similar to other well-defined sf helicases, such as simian virus (sv ) large t antigen (ltag), human papillomavirus (hpv) e , and adeno-associated virus (aav) rep, enterovirus c atpase has been reported to form homo-oligomeric ringlike structures in vitro, which is required for its atpase activity [ ] . moreover, this protein appears to associate with vrna in the vrna replication complexes in infected cells [ ] , implying that c atpase directly participates in enteroviral rna replication. however, previous attempts to determine the helicase activity associated with c atpase or other enteroviral proteins have been unsuccessful [ ] . thus, whether enteroviral or picornaviral rna replication requires the involvement of any virus-encoded rna remodeling activity is not known, and this gap hinders our understanding of this large group of medically important pathogenic viruses. interestingly, our previous study of the c atpase protein from ectropis obliqua picorna-like virus (eov; genus iflavirus, family iflaviridae, order picornavirales) revealed that eov c atpase possesses rna remodeling activity that is more like the activity of an rna chaperone since eov c atpase can destabilize rna duplexes and accelerate strand annealing in an atp-independent manner [ ] . this finding obtained from an insect picorna-like virus prompted us to determine whether enterovirus c atpase could be an rna remodeler. herein, we report that although bacterially expressed ev c atpase did not exhibit any rna remodeling activity, eukaryotically expressed ev c atpase functions not only as an rna helicase that unwinds rna helices from to in an atp-dependent manner but also as an rna chaperone that destabilizes helices from either direction and facilitates strand annealing and complex rna structure formation independently of atp. moreover, we determined the domain requirements for these two rna remodeling activities associated with c atpase and showed that protein c atpase facilitates the ev rna-dependent rna polymerase (rdrp)-mediated synthesis of vrna from vrna template in vitro. when the helicase activity of c atpase was disrupted by point mutation, the rna replication and virus production of ev were mostly abolished in cells. these data indicate that c atpase -mediated rna remodeling plays a critical role in the enteroviral life cycle, particularly in enteroviral replication. in addition, our data show that the rna helicase and chaperoning activities are also conserved in cav c atpase . to evaluate the potential helicase or rna remodeling activity of an enterovirus c atpase , we first compared the sequences and consensus motifs of ev c atpase and three other viral sf helicases: sv ltag, hpv e , and aav rep . although ev c atpase is smaller, this protein contains a core helicase region and the conserved sf signature a, b, and c motifs, similar to the three other sf helicases ( fig b) . to further analyze the potential function of c atpase , we modeled its three-dimensional ( d) structure using robetta [ ] . the computation-generated structural modeling of c atpase indicated that this protein may be comprised of three structurally independent domains: the n-terminal domain (ntd) contains amino acids (a.a.) and is predicted as a helix bundle formed by three α helices; the middle helicase core (hc) domain includes a.a. - of c atpase , and is predicted as a central five-stranded β-sheet flanked by α helices on both sides; and the c-terminal domain (ctd) includes a.a. - and contains several α helices. the three domains are linked by flexible loops (fig c) . the crystallographic structures of aav rep and rep -adp complex have been previously resolved [ , ] . our structural alignment of ev c atpase and aav rep shows that the predicted hc domain of c atpase is structurally similar with the counterpart region of rep . moreover, similarly with rep , c atpase is also predicted to contain a p-loop connecting with the central β-sheet, and the predicted atp binding site of c atpase is also exposed to the exterior and perfectly overlapped with the structurally determined atp/adp binding site of rep in the structural alignment of these two proteins ( fig d) . altogether, our sequence and structural analyses indicate that enterovirus c atpase is similar to other viral sf helicases in both conserved motifs and structures, implying that this protein should contain some rna remodeling activity. to assess the potential rna remodeling activity of enterovirus c atpase , we first expressed ev c atpase as an mbp-fusion protein in both prokaryotic (e. coli) and eukaryotic (baculovirus) systems ( fig e) . interestingly, the eukaryotically expressed mbp- c atpase had an apparently higher molecular mass than the prokaryotically expressed one (fig e, lanes vs. ). the identity and purity of the eukaryotically expressed mbp- c atpase were further confirmed via sds-page separation followed by silver staining (s fig) or in-gel digestion and high-performance liquid chromatography-tandem mass spectrometry (lc-ms/ms) analysis (s table and s text). in total, four proteins (mbp- c atpase , α-tubulin, actin and cytochrome p ) were identified with fdr value of . %. mbp- c atpase turned out to be the highest scoring protein (score: ) identified with high protein coverage of . %. the mass is similar to other viral sf helicases in both motifs and structures. (a) schematic representation of enterovirus genome and the functional motifs in c atpase , including zinc binding, oligomerization, membrane binding, and rna binding motifs. amino acid positions of each motif are numbered. (b) domain alignments of ev c atpase and sf viral helicases, including sv ltag, hpv e , and aav rep . sf helicase motifs a, b, and c are highlighted in orange. (c) the structure of ev c atpase was predicted using the hmmstr/rosetta server. the n-terminal domain (ntd), the middle helicase core (hc) domain, and the c-terminal domain (ctd) are linked by flexible loops as indicated. (d) structural alignment of ev c atpase (slate) and aav rep (yellow). the atp/adp binding site is highlighted in red. (e) mbp-fusion ev c atpase was expressed using a prokaryotic (e. coli) or eukaryotic (baculovirus) system. the purified recombinant proteins were subjected to % sds-page followed by coomassie brilliant blue r staining. spectrometry results showed that the majority ( . %) of all the spectra obtained in the purified protein sample were originated from mbp- c atpase . the quantitation information by mascot exponentially modified protein abundance index (empai) further confirmed the purity of our target protein: the empai value of mbp- c atpase is . , in comparison to the other three identified proteins including α-tubulin, actin and cytochrome p from spodoptera frugiperda sf cell with very low empai values of . , . , . , respectively. furthermore, to examine the oligomerization state of mbp- c atpase , the purified eukaryotically expressed mbp- c atpase was subjected to the size exclusion chromatography analysis using a superdex column. the major peak comprised most protein that was eluted as a molecular mass of~ kda (s a fig) , and the eluted protein was~ kda in size as determined by % sds-page and silver staining (s b fig), indicating that the eukaryotically expressed mbp- c atpase is oligomerized, consistent with previous studies of poliovirus c atpase [ ] . to evaluate the helix unwinding activity of ev c atpase , a short hexachloro fluorescein (hex)-labeled rna and a long non-labeled rna were annealed to generate a standard rna helix substrate with both and single-stranded protrusions (fig f, upper panel and s table) . the helix unwinding assay was conducted by incubating the rna helix substrate with purified mbp- c atpase , followed by gel electrophoresis. interestingly, our data showed that, although bacterially expressed mbp- c atpase did not unwind the rna helix in the presence of mgcl and atp ( fig f, lane ) , consistent with observations reported previously by another group [ ] , eukaryotically expressed mbp- c atpase efficiently unwound the rna helix substrate under the same conditions ( fig f and fig a) . of note, eov c atpase and hepatitis c virus (hcv) ns [ , ] were used as positive controls in this assay (fig a) . moreover, ev c atpase could unwind the rna duplex with matched base pairs but not the duplex with matched base pairs (s fig) , as the latter substrate might be too long to be unwound. altogether, these results indicate that ev c atpase can unwind an rna helix when the protein is eukaryotically expressed. because some rna helicases or chaperones also show unwinding activity with dna helices or rna-dna hybrids, we evaluated this possibility for ev c atpase . to this end, we constructed three different helix substrates that were dna helix ( fig b) and rna-dna hybrids with longer dna (fig c) or rna strand ( fig d) . each helix substrate was reacted with mbp- c atpase under the same conditions as illustrated in fig f. our data showed that ev c atpase could unwind all of the tested types of helix substrates (fig ) , although it unwound the dna helix less efficiently than it unwound other helices ( fig f) . thus, we conclude that ev c atpase possesses a nucleic acid helix unwinding activity for both rna and dna duplexes. ev c atpase directs both ! and ! unwinding of rna helices for helicases, the directionality of helix unwinding is a fundamental characteristic [ ] . to assess the unwinding directionality of ev c atpase , we constructed three different rna helix substrates containing a single-stranded protrusion, a single-stranded protrusion, and blunt ends, respectively (fig a, b and d ). each substrate was then incubated with c atpase for the helix unwinding assay. our results showed that ev c atpase could unwind either the -or -protruded rna helix (fig c) , and its efficiency to unwind the -protruded helix was apparently higher than its efficiency to unwind the -protruded one (fig c, lanes vs. ). moreover, c atpase could not unwind the blunt-ended helix ( fig e) . all these experiments were independently repeated several times. based on these results, we conclude that ev c atpase possesses a bidirectional unwinding activity to rna helices, while the ! unwinding is more preferred. the helix unwinding activity of c atpase can be promoted by the presence of atp for helicases, one important property is that their helix-unwinding activities require the presence of atp, and atp promotes helicase activities in a dose-dependent manner [ ] . our data showed that ev c atpase hydrolyzed atp or gtp, while utp or ctp was not efficiently hydrolyzed by this protein. to determine the effect of ntps on c atpase , we conducted a standard unwinding assay in the presence or absence of individual ntps. our results showed that the presence of atp or gtp dramatically promoted helix unwinding (fig b) . to determine whether the promoting effect of atp on helix unwinding by c atpase is dose-dependent like other helicases, we assessed the unwinding activity of mbp- c atpase in the presence of increasing atp concentrations from . to mm. our data showed that increasing atp concentrations apparently promoted helix unwinding, and the promoting effect reached a steady stage at mm atp (fig c and d ). these results indicate that the helix unwinding activity of ev c atpase needs the participation of atp or other ntps. although we determined that atp is required by ev c atpase to reach its optimal unwinding activity, very interestingly, this protein was still able to unwind a portion of rna helix in the absence of atp ( fig d) . to confirm this phenomenon and exclude the potential interference of trace amounts of atp in the reactions, we added amp-pnp, a non-hydrolyzable atp analog that blocks atpase activity, to the atp-free unwinding reaction. our data showed that although the presence of mm amp-pnp was able to abolish the unwinding activity of hcv ns , a classic rna helicase (s b fig) , the presence of or mm amp-pnp was unable to completely block c atpase to unwind a portion of the rna helix ( fig e, lanes and ) . to further exploit this issue, we sought to determine the necessity of mg + on the atpase and helix unwinding activities of ev c atpase , as these two closely coupled activities of a helicase is normally mg + -dependent [ ] . our results showed that the atpase activity of ev c atpase was strictly mg + -dependent, as this protein was almost unable to hydrolyze atp in the absence of mg + (fig f) . on the other hand, although c atpase requires the presence of mg + to reach its optimal unwinding activity, it was still able to unwind a portion of the rna helix in the absence of mg + or any divalent ions ( fig g, lane ; s d fig, lane ) , further confirming that c atpase retains partial helix unwinding activity in the absence of its atpase activity. moreover, when the mg + concentration was lower than or . mm, increasing mg + concentrations gradually promoted both the atpase and helix unwinding activities of ev (fig f and g ), whereas at higher mg + concentrations (> . mm), mg + inhibited both activities in a dose-dependent manner (fig f and h ). of note, both no protein supplementation and boiled mbp- c atpase were used as negative controls (s c fig, lanes and ) . in addition, the biochemical properties of ev c atpase , including the requirement of divalent ions (mn + , ca + , and zn + ) and ph, were also determined (s d- s f fig) . our data showed that while . mm ca + is dispensable for the helix unwinding activity of ev c atpase , . mm mn + or zn + blocked the helix unwinding by c atpase (s d fig). when the concentration of zn + is higher than . mm, the presence of zn + completely blocked the helix unwinding activity of c atpase (s e fig) . moreover, ev c atpase prefers a neutral ph as an optimal reaction condition, as this protein exhibited the highest helix unwinding activity with ph . (s f fig) . previous studies reported that the atpase activity of poliovirus c atpase can be inhibited by guanidine hydrochloride (gnhcl) [ ] , which is a potent inhibitor of enterovirus vpg-uridylation and vrna replication [ , ] . our data showed that gnhcl can also inhibit the atpase and rna helix unwinding activities of ev c atpase in a dose-dependent manner (fig i and j ). interestingly, this protein could still unwind a portion of the rna helix even at high concentrations of gnhcl, consistent with the results of amp-pnp treatment ( fig e) . altogether, our results show that ev c atpase contains an atp-dependent helicase-like activity that requires the presence of mg + , together with an atp/mg + -independent non-helicase activity that can also unwind the rna helix. ev c atpase contains both rna helicase and chaperoning activities rna remodeling proteins include two distinct classes: rna helicases and rna chaperones. their fundamental characteristics and differences are that the helix unwinding of rna helicases is atp dependent and normally has directionality, whereas that of rna chaperones does not require atp and is bidirectional [ ] . our previous results showed that in addition to its atp-dependent helicase-like activity, ev c atpase also contains an atp-independent helix unwinding activity (fig ) ; moreover, this protein can unwind rna helices bidirectionally ( fig c) . these findings suggested that ev c atpase contains both rna helicase and chaperone activities. to distinguish these two activities, we designed a set of experiments based on their fundamental difference in helix unwinding directionality and atp dependency. first, we constructed two rna helix substrates, each of which contained the same -bp double-stranded region as well as a -nt or single-stranded protrusion ( fig a) . then, each helix substrate was reacted with mbp- c atpase in the absence or presence of atp. our results showed that although the c atpase unwound the -protruded helix in an atp-dependent manner (fig b and e ), it unwound the -protruded helix exactly like an rna chaperone, which was able to unwind the helix in the absence of atp, and increasing atp concentrations could not further enhance the helix unwinding (fig c and e ). moreover, since the motif a "gks" is recognized as the ntp binding site and the core atpase site of sf helicases, we generated the atpase-defective gk aa mutant of c atpase . our data showed that the gk aa c atpase mutant was able to unwind the -protruded helix substrate in the absence or presence of atp at an efficiency similar to that of wild-type c atpase , further confirming that the unwinding of the -protruded helix by c atpase is independent of its atpase activity (fig d and e ). furthermore, the unwinding activity of c atpase on the -protruded helix could not be promoted by any individual ntp (s fig). altogether, our data indicate that the -protruded helix could be unwound via the rna chaperone activity but not the helicase activity of c atpase , whereas the -protruded helix substrate could be unwound via both activities. these findings are consistent with our previous observation that c atpase unwinds the -protruded rna helix more efficiently than the -protruded one in the presence of atp (fig c) . we conclude that ev c atpase possesses an atp-dependent rna helicase activity that unwinds rna helices with ! directionality, which is consistent with all known sf helicases [ ] , together with an atp-independent rna chaperone activity that unwinds rna helices from either direction. ev c atpase destabilizes structured rna strands and stimulates annealing rna chaperones are generally thought to destabilize misfolded rnas and assist in the formation of more globally stable rna structures. after determining that ev c atpase possesses an rna chaperone activity, we adapted a canonical assay [ ] to further verify and characterize ev c atpase . two -nt complementary rna strands, each of which formed defined stem-loop structures, were designed and constructed (fig a) . of the two, one strand was hex-labeled, and the other was not labeled as indicated. equal amounts of hex-labeled and non-labeled strands were incubated in the presence or absence of mbp- c atpase , and then gel shift assays were conducted to examine the hybridization of the two strands. of note, all the experiments were atp-free. our results showed that although minimal spontaneous hybridization could be observed in the absence of c atpase (fig b, lanes - ; fig c, lane ; and fig d, lanes - ) , the presence of ev c atpase dramatically enhanced the two strands' hybridization, as did eov c, which is a well-defined rna chaperone (fig b, lanes - ) . moreover, increasing either the amount of c atpase or its incubation time resulted in an apparent increase in strand annealing (fig c and d) . overall, our data show that c atpase can destabilize structured rna strands and accelerate the formation of more stable hybrids, fitting the characteristic of rna chaperones. the hammerhead ribozyme is a self-catalytic rna module that mediates reversible transcleavage at a specific site within an rna substrate [ ] . since the functionality of a hammerhead ribozyme strictly relies on its correct tertiary structure, hammerhead ribozymes often serve as the models for studying the structure and properties of rnas. rna chaperones are probably able to enhance the activity and turnover of ribozymes by promoting ribozyme formation, ribozyme-substrate association, and/or ribozyme dissociation from cleaved substrates. therefore, a canonical hammerhead ribozyme enhancement assay [ ] was used to further verify and characterize the rna chaperone activity of ev c atpase . in this assay, unlabeled ribozyme rna was reacted with a hex-labeled rna substrate, and the cleavage was expected to take place nts from the end (indicated by arrow), resulting in a hex-labeled -nt cleavage product that could be detected by electrophoresis (fig a) . our data showed that the presence of c atpase effectively enhanced ribozyme activity in a dose-dependent manner (fig b) , indicating that the rna chaperone activity of ev c atpase can assist in the proper formation and function of relatively complex rna structures. the ctd of c atpase is required for its rna chaperoning activity the structure of enterovirus c atpase was previously modeled to contain three domains: the n-terminal domain (ntd), helicase core, and ctd (fig c) . to assess the role of specific domains on the different activities of c atpase , a series of truncations of ev c atpase were generated and eukaryotically expressed. moreover, since the motif a "gks" and motif c "stn" are conserved in sf helicases, the gk aa and Δstn mutants of c atpase were also generated. the predicted structures of these c atpase fragments and mutagenesis sites are illustrated in s a-s c fig our data showed that the gk aa mutant of c atpase lost most atpase activity (fig b) but retained the rna chaperone activity that is insensitive to atp, as determined in these and previous experiments (figs d, c and d); the motif c deletion (Δstn) of c atpase resulted in the loss of most helix unwinding activity (s d fig). moreover, although c atpase ctd had no detectable helix unwinding activity and little atpase activity (fig a, lane ; and fig b) , the loss of ctd resulted in the complete loss of helix unwinding in the absence of atp ( fig c, lane ) , showing that the ctd is required for the rna chaperone activity of c atpase . interestingly, in the presence of atp, the helix unwinding activity of c atpase Δctd was partially restored (fig d, lane ) , indicating that the ctd plays an important but nonessential role in the helicase activity of c atpase , probably by maintaining the proper structure of the c atpase helicase core domain. after determining the rna remodeling activity of c atpase , we sought to evaluate its potential role in enteroviral rna replication. it has been proposed that during vrna replication of (+) rna viruses, replicative intermediate double-stranded rna must be unwound [ ] . to assess the potential role of c atpase in enteroviral rna replication, we first incubated purified his tagged d pol , the ev rdrp, with the (-)rna template [i.e., we transcribed in vitro the -end - nts of ev (-)rna], and with an excessive amount (at a ratio of : ) of primer rna, which was a long oligonucleotide and was pre-annealed to the template in the presence or absence of c atpase (fig a) . of note, the rdrp reactions were conducted at °c as previously described [ ] , and at this temperature, ev c atpase was still able to hydrolyze atp and unwind the rna helix, although both activities were moderately lower at °c than at °c (s a and s c fig). our data showed that the presence of c atpase significantly promoted the synthesis of (+)rna strands from the (-)rna template by d pol , and the promoting effect of c atpase was dose dependent (fig b and c; fig d, lower panel; and fig e) . interestingly, when the amount of primer rna was less than that of the (-)rna template (at a ratio of : ), the presence of increasing amounts of c atpase was unable to promote (+)rna synthesis ( fig d, upper panel; and fig e) , indicating that the major role of the c atpase helicase activity is not to directly enhance the rdrp activity of d pol or smoothen the template. of note, when the primer was absent, d pol was unable to mediate rdrp or elongation reaction in the presence or absence of c atpase (fig f, lanes and ) . in addition, the gk aa mutant of c atpase failed to promote (+)rna synthesis as expected (fig f, lanes - ; and fig g) . altogether, our results show that c atpase can facilitate the d pol -mediated production of enteroviral rna strands in vitro via promoting the efficiency to re-utilize the rna template. the rna helicase activity of c atpase is critical for the rna replication and viability of ev it has been previously reported that the gk-to-aa mutation in the motif a of poliovirus c at-pase resulted in the inhibition of virus growth [ ] . to determine whether the loss of the helicase activity of c atpase has any consequence on the viral life cycle of ev , we introduced the were measured via bio-rad quantity one software, and the relative rna production was determined by comparing the gk aa mutation, which disrupts the atpase and helicase activities, in the c atpase coding region of the infectious clone of ev . then, wt and mutant ev rna transcripts were transfected into human rhabdomyosarcoma (rd) cells. rna replication was measured via quantitative reverse transcription polymerase chain reaction (qrt-pcr) and hours posttransfection, and virus production was detected via immunofluorescent staining of ev vp in cells. strikingly, compared to wt virus, the gk aa mutation resulted in the loss of ev rna replication ( fig a) and viability (fig b) , suggesting that the gk motif and the rna helicase activity of c atpase are critical for enteroviral rna replication and viability. after determining that ev c atpase possesses both rna helicase and chaperone activities, we sought to determine whether c atpase encoded by another enterovirus exhibits similar activities. to this end, c atpase of cav was eukaryotically expressed and then subjected to the helix unwinding assays using either a -or -protruded helix substrate. exactly like ev c atpase (fig ) , cav c atpase unwound the -protruded helix in an atp-dependent manner (fig a and b) , whereas the unwinding of the -protruded helix by this protein was atp independent (fig c and d) . these data show that the atp-dependent rna helicase and atp-independent rna chaperone activities of c atpase are also conserved in cav , another enterovirus. as the most conserved nonstructural protein among all enteroviruses and even picornaviruses, c atpase has been reported to participate in diverse processes critical for the enteroviral life cycle. although this protein has long been predicted as a putative sf helicase on the basis of its aaa+ atpase activity and conserved sf motifs, its potential role in viral rna remodeling and replication remains elusive. previous studies have shown that the atpase activity of poliovirus c atpase could be inhibited by gnhcl. this compound is a potent inhibitor of enteroviral rna replication in vivo, which has been reported to inhibit poliovirus (-)-vrna synthesis and the uridylylation of the protein primer vpg [ , ] , suggesting that the atpase activity of c atpase is critical for enteroviral rna replication. here we report that protein c atpase of ev possesses a nonconventional rna remodeling activity that can function as both an atp-dependent rna helicase and an atp-independent rna chaperone and facilitates ev rdrp-mediated vrna synthesis from vrna template in vitro. the mutation of gk to aa, which disrupts c atpase helicase activity, mostly abolished ev rna replication and virus production. these data indicate that c atpase -mediated rna remodeling is pivotal for the enteroviral life cycle. moreover, our results show that the rna remodeling activities of c atpase are also conserved in cav . although c atpase contains conserved motifs typical of sf helicases, previous attempts to identify its helicase activity were unsuccessful [ ] , leading us to ask why the rna helicase/ chaperoning activity of c atpase can be detected in this study. it is noteworthy that the recombinant poliovirus c atpase protein used in the previous assay was produced in e. coil, while the ev and cav c atpase proteins used here were expressed using an eukaryotic system (baculovirus). interestingly, we found that although both the prokaryotically and eukaryotically expressed c atpase proteins possessed similar atpase activity (s fig), only the eukaryotically expressed c atpase protein possessed the rna duplex unwinding activity (fig f) . moreover, the eukaryotically expressed c atpase showed an apparently higher molecular mass than the prokaryotically expressed one (fig e) , implying that the former protein contains some posttranslational modifications that potentially play a critical role(s) in the helicase activity of c atpase . future research would uncover the potential post-translational modifications of c atpase . although they have mainly been characterized in vitro, the rna remodeling activities of virus-encoded proteins are generally believed to play critical roles in the viral life cycle [ ] . the rna genomes contain multiple cis-acting elements, including the cloverleaf, utrpoly(a), internal origin of replication (orii or cre) [ , ] , internal ribosomal entry site [ , ] , and the cloverleaf at the -end of (-)-vrna [ ] [ ] [ ] . these highly structured rna elements play indispensable roles in the replication, translation, and encapsidation of enteroviral rnas [ , , , ] and probably require the rna chaperoning activity of c atpase to facilitate their proper folding and refolding. on the other hand, during the rna replication of rna viruses, replicative intermediate dsrna must be unwound, thereby allowing the efficient recycling of vrna template for progeny vrna synthesis. it is very likely that the rna helicase activity of c atpase plays such a dsrna unwinding role during enteroviral rna replication, which is consistent with our observations that c atpase can facilitate rdrp-mediated enteroviral rna synthesis in vitro by promoting the recycling of vrna template (fig ) , and disrupting c atpase rna helicase activity in ev resulted in an almost complete loss of enteroviral rna replication and virus production in cells (fig ) . interestingly, although rna chaperones can also unwind/destabilize rna duplexes, rna chaperones are normally not considered to be involved in unwinding viral replicative dsrna, as they usually destabilize short base pairs or hairpins within structured rna elements. indeed, banerjee et al. reported that poliovirus c atpase can bind the -end cloverleaf structure of (-)-vrna [ ] , implying a role of c atpase in this structured element. in addition, our finding that disrupting c atpase helicase activity but not chaperone activity mostly abolished enteroviral rna replication and viability strongly suggests that the functions associated with rna helicase and chaperone play different roles in the same rna molecule (vrna) and cannot replace each other. we previously reported that c atpase from eov, an insect picorna-like virus belonging to the family iflaviridae, possesses an atp-independent rna chaperone activity but not helicase activity [ ] . interestingly, although eov c atpase is predicted to contain conserved sf motifs a, b, and c, the gk-to-aa mutation in eov c atpase motif a did not abolish but instead increased its atpase activity [ ] . this observation obviously contradicts the indispensable role of the motif a "gks" in the atpase and helicase activities of defined sf helicases, including ev c atpase (fig ) , confirming that eov c atpase is not a sf helicase. and the functional disparity between these two proteins is probably due to the long-time evolution and adaptation of iflavirus and enterovirus in different types of hosts, insects, and mammals. interestingly, enterovirus c atpase is not the only rna remodeling protein encoded by enteroviruses. previous studies by destefano and colleagues showed that poliovirus ab is an rna chaperone [ , ] ; moreover, our previous report confirmed that ev ab also contains in vitro rna chaperone activity [ ] . in addition, poliovirus d pol has been reported to display unwindase activity that does not require atp hydrolysis but does require an rna chain elongation reaction [ ] . it is plausible that c atpase and d pol work synergistically to unwind nascently synthesized vrna strands from template. on the other hand, the chaperone activities of c atpase and ab may function collaboratively or separately on different structured cis-acting elements of enteroviral rna. however, owing to technical limitations, it is almost infeasible to determine the exact role of rna helicase or chaperoning activity in the structures and functionalities of rna molecules in cells or in vivo. future studies may overcome these technical barriers and provide a mechanistic view of how rna remodeling proteins function in cells. rna helicases and atp-independent rna chaperones are recognized as the two distinct classes of rna remodelers. rna helicases participate in most atp-dependent rearrangement of structured rnas and rna-protein complexes [ , ] . on the other hand, rna chaperones are able to unwind rna duplexes and facilitate rna strand annealing, thereby aiding the folding or refolding of rna molecules into correct structures. the major difference between rna helicase and chaperone is that the former needs the participation of atp, but the later does not. different from previously characterized rna remodeling proteins, enteroviral c atpase can function as both a typical rna helicase and an atp-independent rna chaperone. that a single protein can contain these two different types of rna remodeling activities is intriguing. our structural analyses of protein c atpase may provide some hints. protein c atpase contains a -a.a. middle domain that includes the conserved sf signature a, b, and c motifs and is structurally similar to the helicase core domains of other sf viral helicases, indicating that the middle domain provides the basis of its rna helicase activity. on the other hand, although rna chaperones have been studied for decades, the mechanism(s) governing their atpindependent remodeling activity remains elusive. a popular model proposed to explain the mechanism is the "entropy or disorder transfer", in which certain intrinsic disordered or unstructured regions of rna chaperones can transfer their disorder or entropy to rna molecules, and such a transfer of disorder or entropy can destabilize misfolded rnas and aids rna refolding. indeed, many rna chaperones, such as hiv- vif, tat and nucleocapsid, flavivirus core protein, hantavirus n protein, and cypovirus vp , have been predicted to contain multiple intrinsic disordered regions [ , ] , providing support for this model. structure modeling using robetta indicated that the ctd of c atpase contains a highly unstructured or disordered coiled-coil region that links the helicase core domain and the ctd (fig c) . when the ctd was deleted from the protein, the truncation completely abolished the rna chaperoning activity of c atpase (fig c) . the truncation of ctd reduced but did not abolish the rna helicase activity (fig d) , suggesting that the presence of ctd is also important for the helicase activity, probably by affecting the protein's global conformation. moreover, the ctd alone does not contain any rna remodeling activity (fig a) , suggesting that the rna chaperoning function of c atpase requires the rna binding capacities of other c atpase domains. the coiled-coil regions and the middle core domains are highly conserved in enteroviruses and other picornaviruses, such as encephalomyocarditis virus (emcv) and foot-mouth-disease virus (fmdv) (s fig). on the basis of all these findings, we propose that the rna helicase function of enteroviral c atpase relies on the conserved helicase motifs and atpase activity, while specific structural characteristics, like the architecture of rna binding and intrinsic disorder regions, are the basis of its rna chaperoning function. moreover, these findings provide evidence of the correlation between intrinsic disorder and rna chaperone activity, which supports the "entropy transfer" model of rna chaperones. in conclusion, this study determined the rna helicase and chaperoning activities associated with enterovirus c atpase and provides both in vitro and cellular evidence of their potential functions in enteroviral rna replication. it also provides the first evidence that the two distinct types of rna remodeling activities, rna helicase and atp-independent rna chaperoning activities, can be integrated within one protein, introducing an extended view of rna remodeling proteins. altogether, these findings increase our understanding of enteroviruses and the two types of rna remodeling activities. the generation of pfastbac htb-mbp and pfastbac htb-mbp-ns hcv have been described previously [ ] . the cdnas for ev c atpase (genbank accession no. kc ), cav c atpase (genbank accession no. kc ) and ev c atpase fragments were amplified by polymerase chain reaction (pcr) from the plasmid containing full-length ev cdna, and cloned into the vector pfastbac htb-mbp. site-directed mutations were generated as previously described [ ] . the primers used in this study are listed in table. the resulting plasmids were subjected to bac-to-bac baculovirus system to express the recombinant proteins with an mbp fused at the n-terminal. the expression and purification of mbp alone and mbp-fusion proteins were performed as previously described [ , ] . briefly, sf cells were infected with the recombinant baculoviruses and harvested at h postinfection. cell pellets were resuspended, lysed by sonication and subject to centrifugation for min at g to remove debris. the protein in the supernatant was purified using amylase affinity chromatography (new england biolabs, ipswich, ma) according to the manufacturer's protocol, and then concentrated using amicon ultra- filters (millipore, schwalbach, germany). all proteins were quantified by the bradford method and stored at - °c in aliquots. proteins were separated on % sds-page and visualized by coomassie blue. the d structure of ev c atpase was modeled by submitting its amino acid sequence to the hmmstr/rosetta server [from robetta, university of washington (http://robetta.bakerlab. org/)] [ ] . five models were obtained, and the best one was chosen as a template based on its score, assessed by submitting it to the swiss-model server [from swiss institute of bioinformatics and the biozentrum, university of basel, switzerland (http://swissmodel.expasy.org)]. the figure of the modeled ev c atpase d structure and the structural alignment with aav rep (pdb id code u j) were drawn by pymol program . (delano scientific llc, south san francisco, ca) from coordinate file. in brief, of the two strands, one was labeled at the end with hexachloro-fluorescein (hex), and the other strand was unlabeled. hex-labeled oligonucleotide strands were purchased from takara (dalian, china). unlabeled dna strands were synthesized by invitrogen, and unlabeled rna strands were in vitro transcribed using t rna polymerase (promega, madison, wi). the in vitro transcribed rna strands were purified by poly-gel rna extraction kit (omega bio-tek, guangzhou, china) according to the manufacturer's instruction. the two strands were mixed in a proper ratio, and annealed through heating and gradually cooling as previously described [ , ] . the standard rna helix substrate with both and protrusions was annealed with rna and rna , the r à /d substrate was annealed with rna and dna , the d à /d substrate was annealed with dna and dna , the d à /r substrate was annealed with dna and rna , the -protruded rna helix was annealed with rna and rna , the -protruded rna helix was annealed with rna and rna , the blunt-ended substrate was annealed with rna and rna , and the matched bps substrate was annealed with rna and rna . all oligonucleotides used in this study are listed in s table. nucleic acid helix unwinding and strand hybridization assays the standard helix destabilizing assay was performed as previously described [ ] with minor modifications. briefly, pmol of recombinant protein and . pmol of hex-labeled helix substrate were added to a mixture containing mm hepes-koh (ph . ), . mm mgcl , and mm dithiothreitol (dtt), . % bovine serum albumin (bsa), and u rnasin (promega). after incubation at °c for min or indicated time, the reaction was terminated by adding proteinase k (final concentration of μg/μl) and ×loading buffer [ mm tris-hcl, % sds, % glycerol, and bromophenol blue (ph . )]. the mixtures were then electrophoresed on % native-page gels, followed by scanning with a typhoon imager (ge healthcare, piscataway, nj). the rna strand hybridization assay was performed as previously described [ ] . the sequences of the stem-loop-structured rna strands were indicated in fig a. the samples were also resolved on % native-page gels, followed by gel scanning a typhoon imager (ge healthcare). ntpase activities were determined via measuring the released inorganic phosphate during ntp hydrolysis using a direct colorimetric assay as previously described [ ] . all of the results given with this quantitative assay were averages of three repeated experiments. the ribozyme rna was synthesized from the in vitro transcription using t rna polymerase, and the -hex-labeled substrate rna was synthesized by takara. indicated amount of protein was added in μl reaction volumes containing . nm substrate, . nm ribozyme, mm hepes-koh (ph . ), . mm mgcl , and mm dtt, . % bsa, u rnasin. the mixtures were incubated at °c for min, and then treated by proteinase k (final concentration of μg/μl) at °c for min. the digestion reactions were precipitated with μl isopropanol and μg glycogen (- °c, min), and subjected to by centrifugation at g for min. the precipitates were dissolved in formamide and electrophoresed on % acrylamide- m urea gels, followed by scanning with a typhoon imager (ge healthcare). the rna primer and template were in vitro transcribed by t rna polymerase as described above. the primer rna strand contains the - nts of the end of ev (+)rna, and the (-)-vrna template contains the sequence complementary with the - nts of end of ev (+)rna. the transcribed products were purified and quantified as described above. then, the primer rna was pre-annealed to the template. the ev d pol gene was cloned into the vector pet b-ub plasmid, and the protein was purified as the c-terminal his× tag fusion protein via a nickel-charged histrap hp column (ge healthcare) as previously described [ ] . the rdrp reactions were carried out in a total volume of μl at °c as previously described [ ] . as previously described [ ] , the rdrp reactions were carried out in the reaction mixture containing nm d pol in mm hepes-koh (ph . ), . mm kcl, mm mgcl , mm dtt, u rnasin (promega), and . mm digoxin (dig) rna labeling mix (roche) with total volume of μl at °c. the reaction mixtures were supplemented with μl ×loading buffer ( % ×mops running buffer, % glycerol, % formamide, % formaldehyde and . % bromophenol blue), and then denatured at °c for min. the samples were electrophoresed on . % agarose-formaldehyde denaturing gels, and transferred onto n + nylon membranes (roche). the membranes were incubated with anti-dig-alkaline phosphatase antibody (roche), followed by incubation with cdp-star (roche) at °c for min. the signals were detected by radiography on x-ray film (fujifilm, tokyo, japan). all oligonucleotides used for in vitro transcription are listed in s table. cells human rhabdomyosarcoma (rd) cells were obtained from the american type culture collection (atcc), and cultured in dulbecco's modified eagle's medium (dmem, gibco) containing % fetal bovine serum (fbs, gibco), units of penicillin and mg/ml of streptomycin. the transfected or infected rd cells were maintained in dmem containing % fbs. the wild type and mutant plasmids containing full-length ev cdna were linearized with mlu i digestion. μg of each purified linear plasmid was used as the template to in vitro transcribe ev rna using the sp ribomax large scale production system (promega) according to the manufacturer's protocols. the rna transcript was transfected into %- % confluent rd cells using lipofectamine (invitrogen). - h post-transfection, the total rna from transfected rd cells was extracted using rneasy mini kit (qiagen, gmbh, germany), and stored at - °c. the amount of viral rna was quantified by using one step primescript rt-pcr kit (takara, dalian) according to the manufacturer's protocol as previous described [ ] with ev specific premiers (fwd, -ggccatttatgtgggtaactttaga- ; rev, -cgggcaatcgtgt cacaac- ) and probe ( fam-aagacagctctcgcgacttgctcgtg-bqh ). rd cells at % confluence on glass slides were transfected with indicated ev transcript. the cells were fixed at h post-transfection with pre-cooled acetone at - °c for min, followed by washing in pbs for times and incubation with mouse anti-ev vp monoclonal antibody (chemicon international, madison, wi) for min at °c. the cells were then washed in pbs and incubated with alexa fluor -labeled goat anti-mouse igg (invitrogen) for another min. cells were incubated with dapi at room temperature for min to stain the nuclei. finally, the cells were rinsed again with pbs and visualized under a fluorescent microscope (olympus, tokyo, japan). the long unwinding road of rna helicases rna helicase proteins as chaperones and remodelers rna helicases at work: binding and rearranging a guanosine-centric mechanism for rna chaperone function rna chaperones, rna annealers and rna helicases rna chaperones exist and dead box proteins get a life virus-encoded rna helicases rna remodeling by chaperones and helicases the hepatitis c virus ns protein: a model rna helicase and potential drug target ntpase and '-rna triphosphatase activities of chikungunya virus nsp protein cooperative translocation enhances the unwinding of duplex dna by sars coronavirus helicase nsp identification and characterization of rna duplex unwinding and atpase activities of an alphatetravirus superfamily helicase philadelphia: lippioncott williams & wilkonson from regional pulse vaccination to global disease eradication: insights from a mathematical model of poliomyelitis poliovirus-encoded c polypeptide specifically binds to the '-terminal sequences of viral negative-strand rna poliovirus protein c contains two regions involved in rna binding activity biochemical and genetic studies of the vpg uridylylation reaction catalyzed by the rna polymerase of poliovirus genetic and biochemical studies of poliovirus cis-acting replication element cre in relation to vpg uridylylation synchronous replication of poliovirus rna: initiation of negative-strand rna synthesis requires the guanidine-inhibited activity of protein c poliovirus c protein determinants of membrane binding and rearrangements in mammalian cells induction of membrane proliferation by poliovirus proteins c and bc foot-and-mouth disease virus nonstructural protein c interacts with beclin , modulating virus replication direct interaction between two viral proteins, the nonstructural protein c and the capsid protein vp , is required for enterovirus morphogenesis alanine scanning of poliovirus catpase reveals new genetic evidence that capsid protein/ catpase interactions are essential for morphogenesis enterovirus c protein inhibits tnf-alpha-mediated activation of nf-kappab by suppressing ikappab kinase beta phosphorylation characterization of the nucleoside triphosphatase activity of poliovirus protein c reveals a mechanism by which guanidine inhibits poliovirus replication poliovirus protein c has atpase and gtpase activities poliovirus c protein forms homo-oligomeric structures required for atpase activity reticulon binds the c protein of enterovirus and is required for viral replication the nonstructural protein c of a picorna-like virus displays nucleic acid helix destabilizing activity that can be functionally separated from its atpase activity fully automated ab initio protein structure prediction using i-sites, hmmstr and rosetta crystal structure of the sf helicase from adeno-associated virus type structure of adeno-associated virus type rep -adp complex: insight into nucleotide recognition and catalysis by superfamily helicases the hepatitis c viral ns protein is a processive dna helicase with cofactor enhanced rna unwinding structure and mechanism of helicases and nucleic acid translocases dead-box proteins as rna helicases and chaperones role of divalent metal cations in atp hydrolysis catalyzed by the hepatitis c virus ns helicase: magnesium provides a bridge for atp to fuel unwinding poliovirus '-terminal cloverleaf rna is required in cis for vpg uridylylation and the initiation of negative-strand rna synthesis conversion of vpg into vpgpupuoh before and during poliovirus negative-strand rna synthesis a cypovirus vp displays the rna chaperonelike activity that destabilizes rna helices and accelerates strand annealing the rep gene product of adeno-associated virus is a dna helicase with '-to- ' polarity poliovirus protein ab displays nucleic acid chaperone and helix-destabilizing activities ribozymes: recent advances in the development of rna tools identification of a region of hantavirus nucleocapsid protein required for rna chaperone activity hepatitis c virus subgenomic replicon requires an active ns rna helicase structural basis for active site closure by the poliovirus rna-dependent rna polymerase genetic analysis of an ntp-binding motif in poliovirus polypeptide c. virology role of rna chaperones in virus replication cis-active rna elements (cres) and picornavirus rna replication initiation of protein-primed picornavirus rna synthesis genetics of poliovirus. annual review of genetics the mechanism of translation initiation on type picornavirus iress cis-acting rna elements in human and animal plus-strand rna viruses non-template functions of viral rna in picornavirus replication '-terminal sequence in poliovirus negative-strand templates is the primary cis-acting element required for vpgpupu-primed positive-strand initiation an rna element at the '-end of the poliovirus genome functions as a general promoter for rna synthesis the twenty-nine amino acid c-terminal cytoplasmic domain of poliovirus ab is critical for nucleic acid chaperone activity the identification and characterization of nucleic acid chaperone activity of human enterovirus nonstructural protein ab rna duplex unwinding activity of poliovirus rna-dependent rna polymerase dpol atp utilization and rna conformational rearrangement by dead-box proteins atp hydrolysis is required for dead-box protein recycling but not for duplex unwinding crystal structure of the full-length japanese encephalitis virus ns reveals a conserved methyltransferase-polymerase interface characterization of a nodavirus replicase revealed a de novo initiation mechanism of rna synthesis and terminal nucleotidyltransferase activity hydrated silica exterior produced by biomimetic silicification confers viral vaccine heat-resistance ev c works as both rna helicase and chaperone plos pathogens we thank drs. peng gong and congyi zheng (wuhan, china) for providing reagents, and dr. hanzhong wang (wuhan, china) for helpful discussions. we thank ms. markeda wade (houston, texas) for professionally editing the manuscript. conceived and designed the experiments: hx xzho. performed the experiments: hx pw gcw jy xs ww yq ts. analyzed the data: hx cfq xzha ly yh xzho. contributed reagents/materials/analysis tools: cfq. wrote the paper: hx xzho. key: cord- - zlxsf z authors: foo, suan-sin; chen, weiqiang; taylor, adam; sheng, kuo-ching; yu, xing; teng, terk-shin; reading, patrick c.; blanchard, helen; garlanda, cecilia; mantovani, alberto; ng, lisa f. p.; herrero, lara j.; mahalingam, suresh title: role of pentraxin in shaping arthritogenic alphaviral disease: from enhanced viral replication to immunomodulation date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: zlxsf z the rising prevalence of arthritogenic alphavirus infections, including chikungunya virus (chikv) and ross river virus (rrv), and the lack of antiviral treatments highlight the potential threat of a global alphavirus pandemic. the immune responses underlying alphavirus virulence remain enigmatic. we found that pentraxin (ptx ) was highly expressed in chikv and rrv patients during acute disease. overt expression of ptx in chikv patients was associated with increased viral load and disease severity. ptx -deficient (ptx (-/-)) mice acutely infected with rrv exhibited delayed disease progression and rapid recovery through diminished inflammatory responses and viral replication. furthermore, binding of the n-terminal domain of ptx to rrv facilitated viral entry and replication. thus, our study demonstrates the pivotal role of ptx in shaping alphavirus-triggered immunity and disease and provides new insights into alphavirus pathogenesis. arthritogenic alphaviruses including ross river virus (rrv) and chikungunya virus (chikv) are the causative agents of the widespread arthropod-borne illnesses, ross river virus disease (rrvd) and chikungunya fever (chikf) respectively [ ] . rrv is endemic to australia, papua new guinea and south pacific islands. an average of~ , cases of rrvd endemic to australia are reported annually [ ] , and~ , individuals were infected during its first outbreak in fiji [ ] . chikv, which is closely related to rrv, has caused large sporadic outbreaks globally, with the largest recorded outbreak of up to . million cases in india [ ] . recently, , suspected and confirmed cases of chikf have been reported in the americas [ ] . in both rrvd and chikf, clinical symptoms include fever, myalgia, fatigue and maculopapular rash [ , ] . debilitating persistent polyarthritis is the clinical hallmark of alphaviral diseases, often affecting joints in the hands, wrists, elbows, knees and feet, which can persists for months to years post infection [ ] [ ] [ ] . in addition, we have recently identified severe pathological bone loss as another characteristic of alphaviral disease which may contribute to the chronic persistent arthralgia [ ] . emerging clinical evidence has demonstrated an increased tendency of chikf patients to develop ra [ ] , and rrvd patients with pre-existing arthritis such as ra have prolonged rheumatic symptoms after infection [ ] . these studies suggested a potential link between alphaviral-induced arthritis and other bone diseases, highlighting alphavirus infection as a possible predisposing risk factor for development of complicated bone disorders [ ] . the persistency of debilitating polyarthralgias has a serious impact on quality of life and the economy, with an estimated cost of million euros per year solely in the la reunion chikv outbreak [ ] . symptomatic relief is the only therapeutic option currently available, due partly to a lack of understanding of the immune responses elicited during alphaviral infection. the cellular and humoral arms of innate immunity serve as the first line of host defense against alphaviral invasion. despite the importance of the innate immune system in the defense against alphaviral infection, increasing evidence of a pathogenic role for innate mediators has also surfaced over the past few years. excessive production of soluble innate mediators such as interleukin- (il- ), granulocyte macrophage-colony stimulating factor (gm-csf), tumor necrosis factor-α (tnf-α), interferon-γ (ifn-γ), macrophage chemoattractant protein- (mcp- ) and macrophage migration inhibitory factor (mif) [ ] [ ] [ ] contributes to alphaviral disease pathogenesis. recent evidence that alphavirus-induced diseases can be exacerbated by overt expression of complement factor (c ) [ ] and mannose binding lectins (mbls) [ ] highlights the significance of the complement cascade in modulating alphaviral disease pathogenesis. long pentraxin (ptx ) is a pattern recognition molecule which belongs to the humoral arm of innate immunity. ptx has a role in all three complement pathways, enhancing the activation, inflammation and cell lysis processes [ ] . ptx can be secreted by a broad range of cell types including neutrophils [ ] , monocytes, macrophages and myeloid dcs [ ] in response to inflammatory signals such as tnf and il- [ ] . upon pathogen encounter, the release of ptx enables cells of monocyte-macrophage lineage to recognize and opsonize the pathogen, presenting it to activated phagocytic cells of the immune system for elimination [ ] . elevated expression of ptx has been implicated in many inflammatory and autoimmune diseases, including pulmonary infection [ ] , giant cell arteritis [ ] , atherosclerosis [ ] and rheumatoid arthritis [ ] . intriguingly, ptx is thought to have both protective [ , ] and pathogenic functional roles [ ] in the immune system. ptx has a variety of ligands, including complement components, microbial moieties, extracellular matrix proteins, growth factors and p-selectin [ ] . the interaction of ptx and p-selectin is involved in the regulation of inflammation and leukocyte recruitment through attenuation of polymorphonuclear leukocyte (pml, also known as neutrophils) rolling at sites of inflammation [ ] . consequently, this affects the physiological functions of pmns in pathogen defense and modulates inflammatory processes. the role of ptx in alphavirus-induced diseases has yet to be established. in this study, we identified the crucial involvement of ptx during acute alphaviral infections using specimens from chikf and rrvd patients. characterization of ptx -/mice and ptx -overexpressing hek t cells revealed pathological roles of ptx in enhancing viral infectivity during acute rrv infection, which was dependent on the binding interaction between rrv and ptx . in summary, our data demonstrated the crucial role of ptx in modulating alphavirus-induced immune responses and disease manifestation through its n-terminal interaction with the virus particles leading to enhanced viral entry and replication. elevated levels of ptx have been associated with both protective and pathogenic functions in several inflammatory diseases. to investigate the involvement of ptx during acute alphaviral infection, we analyzed pbmcs and serum from chikf and rrvd patients for levels of ptx using qrt-pcr and elisa, respectively. transcriptional expression of ptx in pbmcs collected from chikf patients was significantly higher compared to controls (fig. a) . further segregation of the chikf patient cohort based on viral load (fig. b) and disease severity ( fig. c ) [ ] revealed significantly higher transcriptional expression of ptx in patients with higher viral load and more severe disease. similarly, elisa analysis of serum specimens collected from acute rrvd patients revealed significantly higher levels of serum ptx compared to healthy controls (fig. d ). taken together, these data indicate that ptx is induced as part of the innate immune response during acute alphaviral infection and its expression is associated with viral load and disease severity. to determine the expression of ptx during alphaviral disease progression, we utilized an established mouse model of acute rrvd [ ] . rrv-infected and mock-infected mice were sacrificed at (peak viremia phase), (disease onset phase), (peak disease phase) and (recovery phase) days post infection (dpi). the serum, quadricep muscles and ankle joints were harvested for analysis. high levels of serum ptx were detected in rrv-infected mice across all time points, particularly at and dpi, in contrast to consistently low levels of ptx in serum from mock-infected mice ( fig. a) . to further investigate ptx expression at the sites of inflammation, total rna was extracted from tissues and analyzed by qrt-pcr. a high level of ptx expression was observed at dpi in the ankle joint, with levels declining as the disease progressed. in contrast, quadricep muscles showed peak ptx expression at dpi, a time that correlated with the peak of disease ( fig. b) . ihc was also performed in quadriceps harvested from rrv-and mock-infected mice at dpi (fig. c ). pronounced tissue damage was observed in the striated muscle fibers, which was associated with the presence of inflammatory infiltrates. increased ptx expression was observed in the inflammatory infiltrates of quadricep muscles at peak disease (fig. c ). ptx is secreted by a vast array of cell types. to identify the source(s) of ptx production during acute rrv infection, we harvested splenocytes from mock-and rrv-infected mice at dpi for flow cytometry analysis. total leukocytes (cd + ) demonstrated significant elevation of intracellular ptx after rrv infection. further segregation of the total leukocytes into various cellular subsets revealed ptx induction after rrv infection in only subsets of cellsneutrophils (cd b + ly c int ) and inflammatory monocytes (cd b hi ly c hi ). no induction of ptx was observed in nk cells (nk . + cd -), t cells (cd + cd -) and b cells (cd -cd + ) (fig. d ). ptx expression is elevated in chikf and rrvd patients. expression profile of ptx in pbmcs of (a) chikf patients (n = ) or healthy controls (n = ) were analyzed by qrt-pcr. data were normalized to gapdh and shown as fold expression relative to healthy controls. the chikf patient cohort was separated into (b) viral load groups: high viral load (hvl; n = ) and low viral load (lvl; n = ), and (c), disease severity group: severe (n = ) vs mild (n = ). (d) serum from rrvd patients (n = ) or healthy controls (n = ) were analyzed by elisa for ptx levels. data are presented as mean ± sem. *p < . , **p < . and ***p < . , mann-whitney u test. ptx expression is up-regulated following rrv infection in murine model. (a) -day-old c bl/ wt mice (n = - per group) were subcutaneously injected with pfu of rrv or pbs (mock). mice were sacrificed at , , and dpi. serum, quadriceps and ankle joints were harvested. ptx expression in serum of rrv-or mock-infected mice was determined by elisa. (b) transcriptional profile of ptx in quadriceps and ankle joint harvested from rrv-or mock-infected mice at various time points were determined by qrt-pcr. data were normalized to hprt and shown as fold expression relative to mock-infected. *p < . , **p < . , ***p < . , two-way anova, bonferroni post-test. data are presented as mean ± sem and are representative of independent experiments. (c) histology of rrv-induced inflammation in quadriceps of wt mice was analyzed by ihc staining with anti-ptx antibody at dpi. arrows indicate abundance of inflammatory infiltrates. images were taken at × magnification. scale bar, μm. (d) -dayold c bl/ wt (n = - per group) mice were infected subcutaneously with pfu rrv. spleens were harvested at dpi and were characterized and quantified by flow cytometry using the markers as described in materials and methods to determine mean fluorescence intensity (mfi) of ptx expression in total leukocytes, inflammatory monocytes, neutrophils, nk cells, t cells and b cells. data are presented as mean ± sem. **p < . , ***p < . , two-way anova, bonferroni post-test. high expression of ptx during inflammatory diseases has been associated with differential effects [ ] . to determine the role of ptx in rrv disease, ptx -/and wild-type (wt) c bl/ mice were infected with pfu rrv and monitored for the development of rrvd clinical signs for up to dpi. disease onset in rrv-infected wt mice occurred at dpi, with ruffled fur and very mild hind limb weakness (clinical score ), while in ptx -/mice disease onset was significantly delayed commencing at dpi. rrv-infected ptx -/mice also demonstrated milder disease signs between to dpi, compared to the rrv-infected wt mice (fig. a) . in contrast, there was no significant difference in clinical presentation between ptx -/and wt mice during peak disease (from to dpi). from dpi, ptx -/mice showed faster disease recovery than wt mice and by dpi regained full function of hindlimbs. in contrast, wt mice continued to display signs of hindlimb weakness until dpi. to examine the role of ptx in modulating rrv replication in vivo, viral titre was determined in serum, ankle joints and quadricep muscles harvested at and dpi. as seen in fig. b , viral titres in the serum and ankle joints of rrv-infected ptx -/mice were significantly reduced compared to wt mice at dpi. there were no significant differences between ptx -/and wt mice in viral titres recovered from the quadricep muscles. at dpi, viral titres recovered from the ankle joints of rrv-infected ptx -/mice were also lower than in wt mice. titres in serum and quadricep muscles from both ptx -/and wt mice were below the level of detection at this time (fig. c ). to confirm these observations, viral load quantification in ankle joints and quadricep muscles were performed using qrt-pcr. consistent with previous results, higher viral load was detected in the ankle joints of wt mice at and dpi (s a fig.) , whereas no difference in viral load was detected between rrv-infected wt and ptx -/mice in the quadricep muscles (s b fig.) . collectively, our data indicate that ptx deficiency delays the development of rrv clinical signs in infected mice during early infection and assists in rapid recovery in the latter stages of disease. additionally, the absence of ptx also reduced the level of viremia and viral load in the ankle joints of rrv-infected mice. we next sought to determine the effects of ptx on the expression of inflammatory mediators ifn-Ɣ, tnf-α, il- and inos in the early and late phases of rrvd. the quadricep muscles were collected from rrv-infected ptx -/and wt mice at early ( dpi) and peak ( dpi) rrv disease. at dpi, ifn-Ɣ (fig. a ), tnf-α (fig. b ), il- ( fig. c ) and inos (fig. d ) levels were significantly reduced in rrv-infected ptx -/mice. however, at dpi, ifn-Ɣ, tnf-α, il- and inos levels were significantly upregulated in rrv-infected ptx -/mice compared to wt animals. collectively, these data demonstrate that the absence of ptx results in delayed inflammatory responses in quadricep muscles of rrv-infected mice, as well as enhanced production of these immune mediators in the latter stages of infection. having demonstrated the effect of ptx on the induction of soluble inflammatory mediators during acute rrv infection, we next investigated the effect of ptx on leukocyte recruitment during in vivo infection. as shown in fig. c , localized cellular infiltration in quadricep muscles of rrv-infected mice occurs at peak disease ( dpi). to examine the effect of ptx on cellular recruitment during early rrv infection, mice were inoculated via the peritoneal route with rrv. at hpi, flow cytometry analysis of peritoneal lavages revealed significantly increased numbers of neutrophils and inflammatory monocytes in the peritoneal cavity of rrvinfected ptx -/mice compared to wt mice (fig. a ). this early influx of neutrophils and inflammatory monocytes coincides with the chemotactic responses observed in the quadricep muscles of ptx -/mice. among the cytokines investigated, ccl and mif were higher in quadriceps of rrv-infected ptx -/mice at dpi compared to wt mice, but not during peak disease (s a, b fig.) . no significant difference in chemotactic responses of ccl (s c fig.) , cxcl (s d fig.) and cxcl (s e fig.) was observed between the ptx -/and wt mice at and dpi. to investigate the effects of ptx deficiency on cellular infiltrates during peak rrv disease, mice were infected subcutaneously with pfu rrv and the quadricep muscles examined at dpi. previously we have shown that inflammatory monocytes and nk cells are the major cells recruited into muscles during localized inflammation [ ] . as seen in fig. b , the number of inflammatory monocytes was significantly reduced in ptx -/mice compared to wt controls. infiltration of nk cells, however, was not affected by deficiency of ptx . together, these were determined by qrt-pcr in the quadriceps at early rrv disease ( dpi) and peak rrv disease ( dpi). data were normalized to hprt and shown as fold expression relative to wt. data are presented as mean ± sem. *p < . , ***p < . , student unpaired t-test. week old c bl/ wt and ptx -/-(n = per group) mice were infected intraperitoneally with pfu rrv. peritoneal lavage harvested at hpi was characterized and quantified by flow cytometry using the markers as described in materials and methods to determine percentages of neutrophils and inflammatory monocytes. data are presented as mean ± sem. ***p < . , student unpaired t-test. (b) -day-old c bl/ wt and ptx -/-(n = - per group) mice were infected subcutaneously with pfu rrv. leukocytes were results suggest that acute production of ptx dampens early recruitment of neutrophils and inflammatory monocytes, but enhances the egress of inflammatory monocytes in the latter stages of infection. we next determined the direct effect of ptx on the rrv infection process using hek t cells overexpressing ptx . hek t cells were transiently transfected with a plasmid expressing ptx for h and approximately μg/ml of ptx could be detected in supernatants using elisa at this time. in vector-transfected hek t cells, ptx could not be detected regardless of rrv infection (s fig.) . overexpression of ptx in hek t cells resulted in a significant increase in viral titres recovered from supernatants of rrv-infected cells, compared to cells transfected with control vector, when infected with moi . , . and (fig. a, s a fig.) . this data suggests a direct effect of ptx in enhancing rrv replication. to support that the presence of ptx enhanced viral titres, supernatants from vector-and ptx -overexpressing hek t cells were harvested at h post transfection and incubated with untransfected hek t cells. in the presence of rrv, untransfected hek t cells treated with supernatant from ptx -overexpressing hek t cells supported significantly increased virus production compared to cells treated with supernatants from vector-treated control cells (fig. b ). these data confirmed that the presence of ptx is crucial for enhancing virus production. to confirm that the results of enhanced virus production was due to ptx enhancing rrv replication, hek t cells transiently transfected with vector or hptx plasmids were harvested at hour post transfection (hpt) ( to further characterize the effect of ptx during alphaviral infection, we examined the potential of ptx to directly interact with the virus and enhance viral entry. we quantified the viral load in ptx -overexpressing hek t cells at early time points following a one-hour virus adsorption step. typically, alphavirus particles attach to and enter cells during the adsorption phase of infection ( hpi), with the replication of alphavirus genome commencing to hpi [ ] . therefore, following an hour of virus adsorption, the detection of viral antigens present at hpi is indicative of binding and entry, and hpi is indicative of the synthesis of new virus particles. detection of intracellular viral antigens in rrv-infected ptx -overexpressing hek t cells revealed a significant increase in the number of rrv antigen positive cells at and hpi compared to vector-transfected cells (fig. c) , indicating that ptx facilitates viral entry. this result was further confirmed with qrt-pcr viral load analysis, which detected increased viral load within ptx -expressing cells at , , , , and hpi, compared to vector control (s b fig.) . at hpi, the first round of virus replication was observed when a sudden spike in viral load was detected (s b fig.) . interestingly, in conjunction with increased viral entry in the rrv-infected ptx -overexpressing cells, we also observed a significant increase in intracellular ptx expression, compared to the mocked-infected controls (fig. d, e) . isolated from the quadriceps harvested at dpi. cells were characterized and quantified by flow cytometry using the markers as described in materials and methods. total numbers of inflammatory monocytes and nk cells are shown. data are presented as mean ± sem. *p < . **p < . , student unpaired ttest. doi: . /journal.ppat. .g furthermore, flow cytometry analysis showed up to % of rrv + cells were ptx + , suggesting the co-localization of rrv with ptx during acute infection (s fig.) . similar results were obtained for chikv infection of ptx -expressing hek t cells. enhanced viral titres were recovered from the supernatant of ptx -expressing chikv-infected cells when compared to vector controls (s a fig.) . further evaluation of chikv-infected cells at and hpi demonstrated significant increase in viral entry in ptx -expressing cells in conjunction with increased intracellular ptx expression (s b, c fig.) . to demonstrate that the effect of ptx on enhancing rrv entry and replication contributed to the increased level of virus detected in the in vivo studies, we performed rrv infection on primary fibroblasts isolated from tails of ptx -/and wt c bl/ mice. at hpi, rrv infection of wt fibroblasts resulted in significant up-regulation of ptx mrna expression compared to mock-infected wt fibroblasts (fig. a) . moreover, viral titres in supernatants from wt fibroblasts were significantly enhanced compared to fibroblasts from ptx -/mice (fig. b ). to further demonstrate the importance of ptx in enhancing rrv replication, transcriptional profiles of ptx in mock-and rrv-infected fibroblasts were determined by qrt-pcr. data were normalized to hprt and shown as fold expression relative to mock-infected cells. (b) primary tail fibroblasts isolated from wt and ptx -/mice were infected with rrv at moi for hours. supernatants were harvested and rrv titres determined by plaque assay. (c) primary tail fibroblasts from ptx -/mice were infected with rrv recombinant mouse ptx was pre-incubated with rrv prior to infection of ptx -/primary fibroblast cultures. virus titres recovered from supernatants of ptx -rrv complex-infected ptx -/fibroblasts at hpi were significantly enhanced compared to rrv-infected ptx -/fibroblasts (control) (fig. c) . furthermore, the effects of ptx deficiency on viral entry into primary fibroblasts during the early stages of infection were examined. consistent with our earlier findings, significantly lower viral load was detected in ptx -/primary fibroblasts compared to wt after rrv infection at both and hpi (fig. d) . similarly, rrv infection of wt fibroblasts led to increased ptx expression compared to mock-infected controls at and hpi (fig. e ). immunofluorescence staining of the wt fibroblasts also revealed more intense expression of ptx , particularly within the cytoplasm, after rrv infection at and hpi (fig. f) . collectively, these data demonstrate that ptx promotes viral entry and replication at the early stages of rrv infection ( and hpi) within host cells. previous studies have demonstrated that ptx binds to a range of microbes, including viruses. for cytomegalovirus [ ] and influenza virus [ ] , recognition by ptx was shown to neutralize virus infectivity. to test whether ptx can bind to rrv, a microtitre plate-binding assay was performed. microtitre wells coated with rrv were incubated with increasing concentrations of recombinant mouse ptx (rmptx ) and rrv-ptx binding was determined. as seen in fig. a , ptx bound to rrv in a dose-dependent manner. similarly, a microtitre plate binding assay performed on chikv also demonstrated that ptx bound to chikv dose-dependently (s d fig.) . next, we examined whether ptx colocalizes with rrv during infection. during rrv infection of ptx -overexpressing hek t cells, rrv colocalized with ptx in the cytoplasm at hpi (fig. b) . similarly, rrv infection of hela cells, which are highly permissive to rrv infection and express endogenous ptx (s a fig.) , demonstrated clear evidence of ptx colocalization with rrv in the cytoplasm during infection (s b fig.) . these data show that during acute rrv infection, ptx forms a complex with rrv and colocalizes in the cytoplasm of the host cells, which may facilitate viral entry and replication processes. to confirm that the enhanced infectivity observed during acute rrv infection is specific to ptx and not to other acute phase immune proteins, a separate experiment was performed using another acute phase protein-mbl. as previously reported, serum mbl expression was significantly elevated in patients suffering from acute rrvd when compared to healthy controls (fig. a) [ ] . in the acute rrvd mouse model, elevated serum mbl-c was seen at both and dpi (fig. b) . using a microtitre binding assay, a clear dose-dependent binding interaction between rrv and mbl-c was observed (fig. c) . next, we infected c c cells (fig. d) with either complexed ptx -rrv or mbl-rrv in order to identify the specificity of acute phase immune proteins in enhancing rrv infectivity. enhanced infectivity was ( pfu rrv) and pre-bound ptx -rrv complex ( μg/ml of mouse recombinant ptx + pfu rrv) for hours. supernatants were harvested and rrv titres determined by plaque assay. (d) primary tail fibroblasts from wt and ptx -/mice were infected with rrv at moi and harvested at and hpi for viral load analysis to assess viral entry, using viral load qrt-pcr with specific probe and primers against rrv nsp rna, where total rrv nsp copy number was calculated and expressed as a percentage relative to wt infected controls, and (e) assessed for intracellular ptx expression using flow cytometry analysis. data (n = ) are presented as mean ± sem of percent relative to wt and are representative of independent experiments. *p < . , **p < . , two-way anova, bonferroni post-test. (f) primary tail fibroblasts from wt mice were infected with rrv at moi , harvested at and hpi and stained for ptx (green) and dapi (blue). images are representative of independent experiments. magnification, × . scale bar, μm. doi: . /journal.ppat. .g observed in cells infected with ptx -rrv complex at , and hpi; however, no significant difference in infectivity was observed between rrv-or mbl-c-rrv complex-infected cells (fig. e ). to determine the functional domain that is crucial for its functionality, we first examined the binding efficiency of recombinant human ptx (rhptx ) n-and c-terminal fragments (fig. a) to rrv. full-length rhptx bound to rrv in a dose-dependent manner (fig. b ) and the majority of binding activity could be mapped to the n-terminal domain. removal of the n-terminal domain led to a significant reduction in rrv binding (fig. c ). , or (c) μg/ml of human recombinant fl-, n-term-and c-term-ptx , were added to rrv-coated plates for hours at °c, followed by binding to biotin-conjugated anti-ptx antibody for additional hours at °c. optical density at nm was read using horseradish peroxidase substrate kit. data are expressed as mean ± sem of percent binding relative to fl-hptx (n = ). (d) ptx -rrv complex-infected hek t cells were harvested at and hpi. virus entry was quantified by flow cytometry using anti-alphavirus antibody. data (n = ) are presented as mean ± sem and are representative of independent experiments. **p < . , ***p < . , one-way anova, bonferroni's post-test. (e) hek t cells were infected with rrv ( pfu rrv) and pre-bound ptx -rrv complex ( μg/ml of human recombinant fl-, n-term-or c-term-ptx + pfu rrv) for hours. supernatant was harvested and rrv titres was determined by plaque assay on vero cells. we next compared n-and c-terminal domains of rhptx for their ability to facilitate rrv entry and replication. briefly, rrv was pre-incubated with full-length rhptx , n-terminal-rhptx , or c-terminal-rhptx and these mixtures were then added to hek t cells. examination of viral entry at and hpi revealed that n-terminal-rhptx was approximately % less efficient in its ability to facilitate rrv entry, compared to full-length-rhptx . in contrast, removal of the n-terminal led to a complete ablation of ptx -enhanced infection (fig. d) . despite retaining approximately % of its ability to facilitate viral entry, infection of cells with rrv-n-terminal-rhptx complex led to a reduced ability to enhance viral replication, compared to full-length rhptx . however, higher viral titre was still recovered from cells infected with rrv-n-terminal-rhptx complex when compared to control infected with only rrv. no difference in viral titre was observed in cells infected with rrv-c-terminal-rhptx complex (fig. e) . taken together, these data indicate that the n-terminal domain of ptx is responsible for the binding interaction with rrv and its functionality in facilitating viral entry. robust innate immune responses serve as the first line of host defense against alphavirus invasion. however, dysregulation of innate responses can also promote pathogenicity and disease. consistent with this, we have previously identified overt expression of pro-inflammatory cytokines [ , ] and complement components [ ] as pathogenic events in alphaviral diseases. in the current study we sought to determine the role of ptx , an acute phase protein associated with activation of the complement cascade [ ] , in the pathogenesis of alphaviral disease. during the acute phase of alphaviral infection, ptx was highly induced in serum and pbmcs of rrvd and chikf patients, respectively. notably, the magnitude of ptx induction in chikf patients was dependent on viral load and disease severity. similar observations have been reported for the short pentraxin c-reactive protein (crp), which is a common laboratory marker for diagnosis of alphaviral infection [ , ] . previously, chow and colleagues reported that elevated expression of crp was associated in chikf patients with high viral load and severe disease [ ] . in addition to elevated ptx expression in alphavirus-infected patients, we also report abundant expression of ptx in serum and spleen of rrv-infected mice at the early stage of infection. during peak disease, ptx expression was also observed within the cellular infiltrates and further characterization identified inflammatory monocytes and neutrophils as the cellular sources of ptx during acute rrv infection. these findings indicate ptx is induced in response to alphaviral infections in humans and in mice. elevated serum ptx expression has been observed in patients suffering from several arthritic conditions, including rheumatoid arthritis ( . ± . ng/ml), psoriatic arthritis ( . ± . ng/ml), polymyalgia rheumatic ( . ± . ng/ml), ankylosing spondylitis ( . ± . ng/ml) as well as other diseases such as giant cell arteritis ( . ± . ng/ml) and systemic lupus erythematosus ( . ± . ng/ml) [ ] . herein, the strong induction of ptx in rrvd (serum ptx : . ± . ng/ml) and chikf patients suggests that ptx may also be included as a laboratory marker of acute alphaviral infection. dual roles of ptx have been reported in several pathogen-induced inflammatory diseases. overexpression of ptx has protective effector function during bacterial infection with aspergillus fumigatus [ , ] , pseudomonas aeruginosa [ ] and uropathogenic escherichia coli [ ] , as well as viral infections such as murine cytomegalovirus [ ] and influenza virus [ ] . nevertheless, ptx expression has also been associated with exacerbated inflammatory responses and disease outcomes in intestinal ischemia-reperfusion injury [ ] and pulmonary infection with klebsiella pneumonia [ ] . as ptx expression was associated with disease severity during acute alphaviral infections, we utilized an established rrvd mouse model [ ] to examine the role of ptx during alphavirus infection. deficiency of ptx was associated with delayed disease onset. while ptx -/mice displayed similar clinical manifestations at peak of disease, these mice recovered more rapidly than wt animals. it has previously been reported that pro-inflammatory cytokines, including ifn-Ɣ, tnf-α and il- , and massive cellular infiltration contribute to inflammatory disease during alphaviral infections [ ] . indeed, delayed ifn-Ɣ, tnf-α and il- responses were observed in quadricep muscles of ptx -/mice during the peak of rrvd. in addition, ptx -/mice showed diminished infiltration of inflammatory monocytes to the quadricep muscles during peak disease. indeed, ptx has been shown to regulate leukocyte recruitment through interaction with p-selectin, leading to attenuation of cellular recruitment [ ] . using a peritoneal exudate model, we demonstrated increased recruitment of neutrophils and inflammatory monocytes in ptx -/mice during early stages of infection. this observation may be associated with early upregulation of ccl and mif, which are crucial for the recruitment of rrv-induced cellular infiltration [ , ] during early infection. ptx has been shown to bind apoptotic cells promoting deposition of complement components c and c q [ ] . previously, it has been reported that c deposition during rrv infection contributes to the destruction of skeletal muscle tissues [ ] . hence, it is likely that the absence of ptx in our current study ameliorates complement-induced damage of muscle tissues in rrv-infected mice. furthermore, we observed higher induction of inos in quadricep muscles of ptx -/mice at peak rrvd. inos expression was recently shown to be pivotal in mediating skeletal muscle regeneration after acute damage [ ] . these observations suggest ptx plays an immunomodulatory role during alphaviral infection. moreover, the diminished infiltration of inflammatory monocytes and higher expression of inos during peak rrvd may contribute to rapid recovery from disease in the ptx -/mice. collectively, these data identify ptx as a pathogenic factor that shapes the progression of alphaviral disease through modulation of rrv-induced immune responses. ptx is a pattern recognition molecule that interacts with viruses such as murine cytomegalovirus [ ] and influenza virus [ ] , through which it can act to inhibit infection of target cells. in our study, in vitro and in vivo approaches were used to demonstrate that ptx promotes rrv infection and replication in host cells. alphaviruses gain entry into host cells through receptor-mediated endocytosis, although the exact cell surface receptors involved remain poorly defined [ ] . herein, we demonstrate that both rrv and chikv can bind to ptx . rrv and chikv infection of ptx -expressing hek t cells led to enhanced viral entry and replication. in addition, treatment of ptx -/primary fibroblasts with rptx also resulted in enhanced viral replication during early rrv infection, likely due to the formation of ptx -rrv complex which enhances early viral entry events and replication. these data suggest that the extracellular interaction between ptx and rrv was involved in facilitating viral entry into host cells. the aggregates formed between rrv and ptx may promote more efficient multivalent binding to cell surface receptor/s for rrv, thereby promoting enhanced receptor-mediated endocytosis and viral entry. alternatively, ptx may opsonize rrv and promote its uptake via putative (at this stage unknown) cell surface receptors for ptx . in addition to demonstrating the potential of ptx enhancing rrv entry into cells, we also report that the distribution of intracellular ptx was altered during rrv infection. intracellular ptx migrates from perinuclear space to cytoplasm during infection and ptx co-localized with rrv in the cytoplasmic space suggests the possibility of intracellular associations between ptx and rrv. these interactions may further promote productive viral infection, perhaps by enhancing genomic replication. indeed, we demonstrated that cells co-transfected with ptx and rrv, and harvested prior to the release of new virions had elevated levels of intracellular virus antigen. this result further supports the hypothesis that intracellular associations of ptx and rrv may promote viral replication processes. moreover, the presence of ptx was crucial for enhanced viral replication during rrv infection of wt mice and ptx -overexpressing hek t cells. together, this study shows that ptx -rrv interaction gives rise to pathogenic effect, enhancing viral entry and replication, in contrast to previous studies using other viruses such as murine cytomegalovirus [ ] and influenza virus [ ] , where ptx binding was associated with virus neutralization, thereby contributing to a protective host response. ptx is a structurally complex multimeric protein, comprising a highly conserved c-terminal domain shared across all members of the pentraxin family and a unique n-terminal domain whose structure is poorly characterized. we showed that the n-terminal domain is crucial for ptx binding to rrv and ptx -mediated enhancement of rrv infection. however, removal of the c-terminal domain did affect the ability of the n-terminal domain of ptx to modulate viral replication, resulting in only partial enhancement of viral replication compared to full-length ptx . previous studies have reported the importance of an intact quaternary structure in order for ptx to retain its binding and biological efficacies [ ] . therefore, full-length ptx with intact quaternary structure would be necessary to retain its biological role of enhancing rrv replication. taken together, the data presented in this study provides the first evidence of a role for ptx in enhancing rrv uptake and replication during early alphaviral infection. ptx has previously been associated with protective functions against a number of viruses, including influenza virus [ ] , human/murine cytomegalovirus [ ] and coronavirus murine hepatitis virus [ ] , in contrast to the pathogenic role identified in the current study. our findings demonstrate a previously undescribed pivotal role of ptx in shaping alphaviral disease progression through immunomodulation and facilitating viral infection and replication processes during the acute phase of infection. in conclusion, our findings provide new insight into the role of ptx in acute alphaviral infection. the newly identified role of ptx in enhancing rrv infection and replication also sheds light on the poorly defined route of alphavirus entry into host cells. given the diverse functional roles of ptx as well as its ability to bind to a variety of immune factors, further study is required to define the exact ptx -triggered immune pathways induced in alphaviral-induced arthritic diseases. identification of such pathways will be an important step towards the future development of therapeutic interventions. animal experiments were approved by the animal ethics committee of griffith university (bdd/ / /aec). all procedures involving animals conformed to the national health and medical research council australian code of practice for the care and use of animals for scientific purposes th edition . chikv human pbmc samples were collected from patients that were admitted to the communicable disease centre at tan tock seng hospital during the singapore chikf outbreak. all patients were diagnosed with chikf and blood were collected at the acute phase (median of days after illness onset) of infection [ ] , with written informed consent obtained from all participants. the study was approved by the national healthcare group's domain-specific ethics review board (dsrb reference no. b/ / ). all rrv human serum samples had been submitted to the centre for infectious diseases and microbiology laboratory services (cidmls), westmead hospital for diagnostic testing and laboratory investigation of rrv with written and oral informed patient consent. serum from healthy individuals was provided by australian red cross with written and oral informed consent, approved by griffith university human research ethics committee (bdd/ / /hrec). no new human samples were collected as part of this study. serum samples were de-identified before being used in the research project. pbmc specimens of patients were classified into viral load (high viral load, hvl; n = and low viral load, lvl; n = ) and disease severity (severe illness; n = and mild illness; n = ) groups, as described previously [ ] . briefly, the hvl and lvl groups had mean viral loads of . × pfu/ml and . × pfu/ml respectively, while severe illness were defined as having a temperature of higher than . °c, pulse rate more than beats/min or platelet count less than × cells/l. serum specimens were collected from acute cases of rrv-induced polyarthritis patients in australia. pbmcs and serum specimens isolated from healthy volunteers were used as controls. all specimens were stored at - °c until use. stocks of the wt t strain of rrv were generated from the full-length t cdna clone, kindly provided by richard kuhn, purdue university, west lafayette, in. the chikv variant expressing mcherry (chikv-mcherry) was constructed using a full-length infectious cdna clone of the la reunion chikv isolate lr -opy as described previously [ ] . cell culture, proteins and transfection hek t, hela and c c cells were cultured in dmem supplemented with % fbs. primary fibroblasts were isolated from tails of wt and ptx -/mice using a previously described protocol [ ] and cultured in dmem supplemented with % fcs. transient transfection of ptx plasmids [ ] was performed using lipofectamine (invitrogen) following manufacturer's instructions. electroporation of rrv t infectious plasmid clone [ ] was performed using eppendorf eporator. recombinant n-terminal and c-terminal ptx proteins were purified as described in [ ] . recombinant mouse and human ptx , and mouse mbl-c were purchased from r&d. hek t cells and primary tail fibroblasts were plated at a density of . × per well on well plates overnight, prior to infection with rrv or chikv (moi ) for h at °c in humidified co incubator. virus overlay was removed and ml of pre-warmed growth medium was added to the monolayer of cells, marking the hour post infection (hpi). cells were incubated at °c in humidified co incubator and were harvested accordingly. all titrations were performed by plaque assay on vero cells as described previously [ ] . microtiter plates (sarstedt) were coated overnight at °c with . m carbonate-bicarbonate coating buffer alone or containing either pfu rrv or chikv (uv-inactivated for min). non-specific binding sites were blocked by % bsa in pbs for h at room temperature. recombinant ptx or mbl-c binding to virus was performed by incubating recombinant proteins on virus-coated microtitre plate for h at °c. biotin-conjugated anti-ptx or anti-mbl-c detection antibody (r&d) was added and incubated at room temperature for h. the optical density at nm was read using the streptavidin conjugated to horseradish-peroxidase (hrp) substrate (r&d). total rna extraction was performed using trizol reagent (life technologies) following manufacturer's instructions. quantification of total rna was measured by nanodrop spectrophotometer (thermo scientific). extracted total rna ( ng/μl) was reverse-transcribed using an oligo (dt) primer and m-mlv reverse transcriptase (sigma aldrich) according to the manufacturer's instructions. standard curve was generated using serial dilutions of rrv t infectious plasmid dna as described previously [ ] . quantification of viral load was performed using ssoadvanced universal probes supermix (bio-rad) in . μl reaction volume to detect nsp region rna, using specific probe ( -attaagagtgtagccatcc- ') and primers (forward: '-ccgtggcgggtattatcaat- '; reverse: '-aacactcccgtcgacaacaga- ') [ ] . reactions were performed using bio-rad cfx touch real-time pcr detection system on -well plates. cycler conditions were as follows: (i) pcr initial activation step: °c for min, cycle and (ii) -step cycling: °c for sec, followed by °c for sec, cycles. standard curve was plotted and copy numbers of amplified products were interpolated from standard curve using prism graphpad software to determine viral load. transfected hek t cells were seeded on poly-l-lysine-coated coverslips for staining. cells were fixed with % paraformaldehyde (pfa), permeabilized in pbs containing . % triton x- , and blocked with % goat serum in pbs. cells were incubated with rat monoclonal anti-ptx (mnb , abcam) or mouse monoclonal anti-alphavirus ( , santa cruz) primary antibody in pbs, followed by goat anti-rat af (invitrogen) or goat anti-mouse af (invitrogen) secondary antibody. cells were washed, mounted, and examined with a confocal laserscanning microscope (fluoview fv , olympus) at x magnification. images were collected and processed using fv -asw software. elisas were performed using duoset elisa development kit (r&d systems) following manufacturer's instructions. to analyze ptx intracellular expression, transfected hek t cells were fixed with % pfa and permeabilized with . % saponin (sigma aldrich) in pbs. indirect intracellular staining was performed with rat anti-ptx (mnb , abcam) primary antibody, followed by af conjugated anti-rat (life technologies) secondary antibody. to identify the various cell populations present in splenocytes, peritoneal lavage and quadriceps harvested from mice, cells were first incubated with anti-mouse cd / cd (fc block, bd pharmingen) and stained with the following antibodies: apc-conjugated anti-mouse gr , pe-conjugated anti-mouse f / , fitc-conjugated anti-mouse cd b, apc-conjugated anti-mouse ly c, apc-conjugated anti-mouse cd , fitc-conjugated anti-mouse cd , pe-conjugated anti-mouse cd , or pe-cy -conjugated anti-mouse nk . (bd pharmingen). for detection of alphavirus antigens, indirect intracellular staining was performed using mouse monoclonal anti-alphavirus ( , santa cruz) primary antibody, followed by af -conjugated anti-mouse (life technologies) secondary antibody. data acquisition was performed using cyanadp (beckman coulter), and analysis was done by kaluza flow analysis software (beckman coulter). animal studies [ ] [ ] [ ] week-old c bl/ male and female mice, of equal distribution, were inoculated intraperitoneally with pfu rrv in μl of pbs, to study the early effect of ptx deficiency on recruitment of neutrophils and inflammatory monocytes. peritoneal lavage was harvested at hpi with ml of ice-cold pbs. for the acute rrv mouse model, -day-old c bl/ male and female mice, of equal distribution, were inoculated subcutaneously in the thorax below the right forelimb with pfu rrv in μl. mock-infected mice were inoculated with pbs diluent alone. mice were weighed and scored for disease signs every h. mice were assessed based on animal strength and hindleg paralysis, as outlined previously [ ] , using the following scale: , no disease signs; , ruffled fur; , very mild hindlimb weakness; , mild hindlimb weakness; , moderate hindlimb weakness; , severe hindlimb weakness, , complete loss of hindlimb function; and , moribund. mice were euthanized, quadriceps and ankle joints were removed and homogenized using qiagen tissuelyser ii then centrifuged at , × g, min, °c. blood was collected via cardiac puncture. serum was isolated by centrifugation at , × g, min, °c. for analysis of infiltrating inflammatory cells by flow cytometry, mice were sacrificed and perfused with pbs at dpi. quadricep muscles were harvested, weighed, minced, and digested with dmem containing % fbs, mg/ml of collagenase iv (roche) and mg/ml of dnase i (roche), for h at °c. cells were strained through a μm strainer (bd biosciences) and washed with dmem containing % fbs and viable cells were counted by trypan blue exclusion. for histology, quadricep muscles harvested were fixed in % pfa, followed by paraffinembedding. five-micrometer sections were prepared. ihc was performed on dewaxed, rehydrated, μm paraffin-embedded tissue sections. sections were incubated with % goat serum (gibco) in % bsa/pbs for min. primary antibody staining was performed using rat antimouse ptx (mnb , abcam) in pbs, incubated overnight, at °c, in humidified chamber. tissue sections were washed in pbs for three times at min intervals. secondary antibody staining was performed using hrp-conjugated anti-rat igg b (serotec) incubated for min, room temperature, in a humidified chamber. colour was developed with , '-diaminobenzidine (dab) peroxidase substrate kit (vector laboratories), according to manufacturer's instructions and counter-stained with hematoxylin (vector laboratories). all statistical analyses were performed using prism . (graph-pad software). analysis of ptx expression profiles in comparison between healthy and rrvd or chikf patients, hvl and lvl chikf patients' groups, and severe and mild illness chikf patients' groups was done using mann-whitney u test. comparisons of ptx expression among different time points post infection in wt mice, ptx expression in mock-and rrv-infected mouse splenocytes, clinical scoring of between ptx -/and wt mice, viral replication and viral entry among rrv-infected hek t cells and fibroblasts, were performed using two-way anova with bonferroni post-test. comparisons of viral replication and viral entry among rrv-, fl-ptx , n-term-ptx and c-term-ptx -rrv infected groups were analyzed using one-way anova with bonferroni post-test. analyses of all other experimental groups were performed using student unpaired t-test. p values less than . were considered statistically significant. supporting information s fig. ptx deficiency leads to reduced viral load in ankle joints. -day-old c bl/ wt and ptx -/mice were infected subcutaneously with pfu rrv at the thorax region. viral load in ankle joint and quadriceps of rrv-infected wt and ptx -/mice (n = - per group) at (a) and (b) dpi were determined using taqman qrt-pcr with specific probe and primers against rrv nsp rna. data are presented as mean ± sem. Ãp < . , student unpaired t-test. hek t cells were transfected with human ptx or vector plasmid for h before rrv infection. transfected hek t cells were harvested at and hpi to assess for intracellular rrv and ptx expression using flow cytometry analysis. (tif) s fig. ptx binds to chikv and enhances viral entry and replication. (a) hek t cells were transfected with human ptx or vector plasmid for h before chikv infection at moi for h. supernatants were harvested and chikv titres were determined by plaque assay. data are presented as mean ± sem. ÃÃp < . , student unpaired t-test. transfected hek t cells were harvested at and hpi, (b) to assess intracellular chikv expression by flow cytometry using anti-alphavirus antibody for detection of viral entry, and (c) to assess intracellular ptx expression using flow cytometry analysis. data (n = ) are presented as mean ± sem and are representative of independent experiments. Ãp < . ÃÃp < . , ÃÃÃp < . , two-way anova, bonferroni post-test. (d) different concentrations of mouse recombinant ptx were added to chikv-coated plate for hours at °c, followed by binding to biotin-conjugated anti-ptx antibody for an additional hours at °c. optical density at nm was read using horseradish peroxidase substrate kit. chikungunya fever in travelers: clinical presentation and course climate variability and ross river virus infections in an epidemic of ross river virus infection in fiji, arthritogenic alphaviruses-an overview number of reported cases of chikungunya fever in the americas, by country or territory ross river virus: ecology and distribution chikungunya: a re-emerging virus biology and pathogenesis of chikungunya virus arthritogenic alphaviral infection perturbs osteoblast function and triggers pathologic bone loss a report of cases of rheumatoid arthritis following chikungunya fever. a mean follow-up of two years natural history of ross river virus-induced epidemic polyarthritis chikungunya virus and arthritic disease: mechanism and potential risk factor for sever disease chikungunya virus-associated long-term arthralgia: a -month prospective longitudinal study persistent arthralgia induced by chikungunya virus infection is associated with interleukin- and granulocyte macrophage colony-stimulating factor host-protective effect of circulating pentraxin (ptx ) and complex formation with neutrophil extracellular traps critical role for macrophage migration inhibitory factor (mif) in ross river virus-induced arthritis and myositis complement contributes to inflammatory tissue destruction in a mouse model of ross river virus-induced disease mannose binding lectin is required for alphavirus-induced arthritis/myositis an integrated view of humoral innate immunity: pentraxins as a paradigm the humoral pattern recognition receptor ptx is stored in neutrophil granules and localizes in extracellular traps inducible expression of ptx , a new member of the pentraxin family, in human mononuclear phagocytes tsg- , a tumor necrosis factor-and il- -inducible protein, is a novel member of the pentaxin family of acute phase proteins the long pentraxin ptx as a prototypic humoral pattern recognition receptor: interplay with cellular innate immunity long pentraxin in pulmonary infection and acute lung injury selective upregulation of the soluble pattern recognition receptor ptx and of vegf in giant cell arteritis: relevance for recent optic nerve ischemia production of the long pentraxin ptx in advanced atherosclerotic plaques expression and production of the long pentraxin ptx in rheumatoid arthritis (ra) pentraxin protects from mcmv infection and reactivation through tlr sensing pathways leading to irf activation antiviral activity of the long chain pentraxin ptx against influenza viruses the long pentraxin ptx is crucial for tissue inflammation after intestinal ischemia and reperfusion in mice regulation of leukocyte recruitment by the long pentraxin ptx characterization of ross river virus tropism and virus-induced inflammation in a mouse model of viral arthritis and myositis the long pentraxin ptx : a paradigm for humoral pattern recognition molecules macrophage migration inhibitory factor receptor cd mediates alphavirus-induced arthritis and myositis in murine models of alphavirus infection alphavirus nsp functions to form replication complexes transcribing negative-strand rna macrophage-derived proinflammatory factors contribute to the development of arthritis and myositis after infection with an arthrogenic alphavirus il- beta, il- , and rantes as biomarkers of chikungunya severity ptx as a paradigm for the interaction of pentraxins with the complement system ross river virus disease in a traveler to australia chikungunya virus aches and pains: an emerging challenge increased levels of serum pentraxin , a novel cardiovascular biomarker, in patients with inflammatory rheumatic disease non-redundant role of the long pentraxin ptx in anti-fungal innate immune response the therapeutic potential of the humoral pattern recognition molecule ptx in chronic lung infection caused by pseudomonas aeruginosa the humoral pattern recognition molecule ptx is a key component of innate immunity against urinary tract infection increased mortality and inflammation in tumor necrosis factor-stimulated gene- transgenic mice after ischemia and reperfusion injury dual function of the long pentraxin ptx in resistance against pulmonary infection with klebsiella pneumoniae in transgenic mice bindarit, an inhibitor of monocyte chemotactic proteins (mcps) synthesis, protects against bone loss induced by chikungunya virus infection biochemical and functional characterization of the interaction between pentraxin and c q requirement of inducible nitric oxide synthase for skeletal muscle regeneration after acute damage the genetics of alphaviruses structural characterization of ptx disulfide bond network and its multimeric status in cumulus matrix organization protective effects of long pentraxin ptx on lung injury in a severe acute respiratory syndrome model in mice chikungunya fever in singapore: acute clinical and laboratory features, and factors associated with persistent arthralgia infectious clones of chikungunya virus (la reunion isolate) for vector competence studies fibroblast cell lines from young adult mice of long-lived mutant strains are resistant to multiple forms of stress multimer formation and ligand recognition by the long pentraxin ptx . similarities and differences with the short pentraxins creactive protein and serum amyloid p component identification of an antiangiogenic fgf -binding site in the n terminus of the soluble pattern recognition receptor ptx macrophage-induced muscle pathology results in morbidity and mortality for ross river virus-infected mice ross river virus envelope glycans contribute to type i interferon production in myeloid dendritic cells we acknowledge angela chow, yee-sin leo and all the staff at the communicable diseases centre, tan tock seng hospital, involved in chikf patient recruitment, study coordination, and data entry. we thank linda hueston (centre for infectious diseases and microbiology laboratory services, pathology west-icpmr westmead) for providing serum specimens of rrv patients. key: cord- - l ydspz authors: webb, l. g.; veloz, j.; pintado-silva, j.; zhu, t.; rangel, m. v.; mutetwa, t.; zhang, l.; bernal-rubio, d.; figueroa, d.; carrau, l.; fenutria, r.; potla, u.; reid, st. p.; yount, j. s.; stapleford, k. a.; aguirre, s.; fernandez-sesma, a. title: chikungunya virus antagonizes cgas-sting mediated type-i interferon responses by degrading cgas date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: l ydspz chikungunya virus (chikv) is a mosquito-borne alphavirus known to cause epidemics resulting in predominantly symptomatic infections, which in rare cases cause long term debilitating arthritis and arthralgia. significant progress has been made in understanding the roles of canonical rna sensing pathways in the host recognition of chikv; however, less is known regarding antagonism of chikv by cytosolic dna sensing pathways like that of cyclic gmp-amp synthase (cgas) and stimulator of interferon genes (sting). with the use of cgas or sting null cells we demonstrate that the pathway restricts chikv replication in fibroblasts and immune cells. we show that dna accumulates in the cytoplasm of infected cells and that chikv blocks dna dependent ifn-β transcription. this antagonism of dna sensing is via an early autophagy-mediated degradation of cgas and expression of the chikv capsid protein is sufficient to induce cgas degradation. furthermore, we identify an interaction of chikv nsp with sting and map the interaction to residues in the cytosolic loop of the adaptor protein. this interaction stabilizes the viral protein and increases the level of palmitoylated nsp in cells. together, this work supports previous publications highlighting the relevance of the cgas-sting pathway in the early detection of (+)ssrna viruses and provides direct evidence that chikv interacts with and antagonizes cgas-sting signaling. introduction chikv is an arbovirus belonging to the genus alphavirus (family: togaviridae) which is transmitted primarily by mosquitos of the aedes spp [ , ] . approximately - % of chikv infections are symptomatic and the hallmark of infection is an acute febrile illness with a sudden high-grade fever as well as debilitating arthritis and arthralgia which can persist, in some infected individuals, for months to years after clearing the infection [ ] [ ] [ ] [ ] . since its first characterization in , chikv has been noted to cause sporadic and explosive outbreaks resulting in a potentially debilitating range of inflammatory diseases [ ] [ ] [ ] . between - , chikv was responsible for an outbreak on the island of la reunion where more than one third of the island's population was infected in the span of a few months [ ] . this outbreak then spread to india, where an estimated . million infections occurred, southeast asia, and ultimately to italy where the first subtropical autochthonous transmission was established [ , ] . the chikv genome is a positive-sense single stranded rna (+ssrna) with a type , ' methyl-guanosine cap and poly(a) tail, which encodes six structural and four non-structural proteins. after entry and uncoating of the viral nucleic acid, translation of the non-structural proteins occurs. two polyproteins are produced: either p or, if read-through of an opal stop codon takes place, p [ ] [ ] [ ] . non-structural protein (nsp ) of chikv has been shown to anchor viral replication complexes to cytosolic membranes through both an amphipathic helix as well as through palmitoylation of three cystine (cys) residues - [ , ] . mutations of these residues have been shown for both chikv and other alphaviruses to severely hamper viral replication kinetics and decrease pathogenicity in mouse models [ , ] . viral structural proteins are then translated from the s rna and processed threaded into the endoplasmic reticulum (er) with exception of the capsid protein which remains in the cytosol prior to encapsidation of the viral genomic rna [ ] . innate immune defenses are at the forefront of restricting viral pathogens. successful replication and subsequent release of viral progeny is largely dependent upon the ability of a virus to evade or inhibit both detection and activation of cellular antiviral responses. chikv is no exception, the type-i interferon (ifn) response has been demonstrated to be key in controlling chikv infection [ ] [ ] [ ] [ ] [ ] [ ] and primary sensing of the virus is via the pattern recognition receptor (prr) retinoic acid-inducible gene i (rig-i)-mitochondrial antiviral signaling protein (mavs) axis [ , ] . alternatively to pathogen associated molecular patterns (pamps), cell intrinsic molecules released as a result of cellular stress, termed danger associated molecular patterns (damps), are also potent inducers of innate immune responses [ , ] which lead to the induction of type-i ifns. type-i ifns are a family of cytokines, composed of both ifn alpha (ifna) and ifnß, which are induced though prr signaling [ ] . these cytokines signal in an autocrine and paracrine manner, and are critical in inducing an antiviral state in uninfected cells proximal to infected cells [ ] . antiviral states are induced by transcription and translation of hundreds of interferon stimulated genes (isgs) as a result of type-i ifn signaling [ ] . previous work has shown that chikv nsp inhibits ifn mediated signaling [ ] [ ] [ ] downstream of type-i ifn production and can inhibit rna pol ii dependent transcription [ ] . meshram et al. identified that mutations within nsp alter the production of ifn beta (ifnß) [ ] . to date, however, no function has been identified for any chikv proteins, in restricting the induction of type-i ifn due to dna stimuli. while the canonical view of prr mediated antiviral responses associates rna sensing prrs with rna based pathogens and dna sensing prrs with dna based pathogens, these systems do not exist in isolation. recent work has demonstrated a role for cytosolic dna innate immune sensors in initiating and responding to rna viral infections and it has been shown that there is a crosstalk present between rig-i like receptor (rlr) signaling and dna signaling through the adaptor protein, stimulator of interferon genes (sting) [ , ] . specifically, the cyclic gmp-amp synthase (cgas)-sting pathway has been shown to restrict both human flaviviruses and coronaviruses and the viruses in-turn have mechanisms to inhibit the pathway [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . antiviral responses are activated when cgas binds to double stranded dna or dna-rna hybrids and synthesizes the secondary messenger molecule, '- ' cyclic-guanosine monophosphate (gmp)-adenosine monophosphate (amp), (cyclic gmp-amp or cgamp) which then bind to sting [ , ] . upon cgamp binding, sting dimerizes, translocates to the trans-golgi-network (tgn), where it associates with tank binding kinase (tbk ), which ultimately results in phosphorylation of interferon regulatory factor (irf ) and induction of type-i ifn transcription [ , , , ] . interestingly, activation of the cgas-sting pathway with chemical activators or via overexpression has been shown to restrict alphaviruses, including chikv [ , , ] ; however, no studies have identified direct antagonism of the cgas-sting pathway by chikv. given the importance of type-i ifn in restricting chikv, as well as recent work highlighting the relevance of the cgas-sting pathway in restricting rna viruses, we asked whether chikv was able to antagonize the cgas-sting pathway. chikv has been shown to replicate well in primary human foreskin fibroblast (hff- ) cells and fibroblasts are thought to be primary sites of viral amplification upon infection [ ] . to better understand the innate immune response induced in primary human cells by chikv / , hff- s were infected either with chikv / or the paramyxovirus newcastle disease virus (ndv). ndv, like chikv, is sensed via rlrs [ ] ; however, ndv is an avian pathogen with limited ability to antagonize the induction of the innate immune response in human cells [ , ] . chikv / replicated to high titers in hff- s (fig a) but had a notably muted induction of ifn-ß, isg , and tnfa transcripts at hpi when compared to ndv ( fig b- d ). ndv replication was measured in hff- s and peaked by hpi (fig e) . to better understand if chikv / had similar replication in hff- s to other chikv isolates, it's replication and transcriptional profile were compared to a chikv isolate of the indian ocean lineage (chikv-iol). chikv / replicated better in hff- s when compared to chik-v-iol but did not induce largely different innate immune responses at the transcriptional level (s fig). and supernatants were plaqued on bhks at indicated timepoints. (b-d) rt-qpcr from chikv growth curve in (a) for specified genes (ifnb, isg , and tnfa) relative to rps at hpi. data representative of three independent experiments. data represented as means ± sd (n = ). statistical analysis was performed with by a one-way anova with tukey's multiple comparisons. (e) ndv viral rna relative to rps from infected hff- s (moi = . ) at indicated timepoints. data represented as means ± sd (n = ). (f) diagram of coinfection experiments. briefly, hff- s were infected with either mock, uv-inactivated chikv / (uvc), or chikv / at an moi of . . infections were allowed to proceed for hrs in order to allow for viral protein expression. (g & i) hpi, cells were then treated with mock, mva at an moi of . , or e. coli dna ( ug/ . x cells). and hrs postsecondary treatment, cell lysates were collected. ifnß transcripts were quantified via rt-qpcr for cells stimulated with mva or e. coli dna, respectively, while quantification of isg transcripts was performed as previously stated (g & i). transcripts are represented as "fold over respective mock" e.g. uvc ➔ mva condition was normalized to uvc ➔ mock condition to determine the relative gene induction resultant from secondary treatment. to investigate the ability of chikv to block dna sensing, hff- cells were infected first with infectious or uv-inactivated chikv (uvc) and subsequently infected with either modified vaccinia ankara virus (mva), a dna virus known to be sensed via cgas [ ] , or as an alternative source of cgas ligand, transfected with e. coli dna, respectively ( fig f) . surprisingly, chikv drastically reduced ifn-ß transcription induced by both a dna virus (mva) and a direct cgas agonist (e. coli dna) (fig g & i) . a slight reduction in ifn-ß transcripts was observed in the uvc condition (fig g & i) . this minor inhibition could be mediated by an intrinsic component of the viral nucleocapsid, playing a role in reducing cgas-sting signaling. importantly, the initial infections did not alter the ability of mva to replicate in these cells at hpi, demonstrating that the reduction in innate immune transcripts observed in fig g & i is a result of chikv-mediated inhibition and not due to a reduction in mva replication ( fig h) . the role of individual chikv non-structural proteins in potential inhibition of type-i ifn production was tested using a previously established type-i ifn reporter system [ ] . these reporter cells are hek- t cells that are stably transduced to express firefly luciferase under the control of an interferon beta (ifnβ) promoter and are deficient for both cgas and sting ( t-ifnβ-ffluc) (s a fig). the ability of chikv nsps to inhibit dna mediated induction of the ifnβ promotor was assessed by co-expressing the non-structural proteins of chikv, ross thailand isolate (chikv-rt), with cgas and sting. hrs post transfection, firefly luciferase activity was quantified as a proxy for activity of the ifnβ promoter. inhibition of dna dependent ifnβ promoter activity was observed for nsps , to test for chikv nsp mediated degradation of sting, nsps of chikv-rt were co-expressed with sting. when sting was co-expressed with the four individual chikv-rt nsps no degradation or cleavage of sting was observed, though a clear cleavage of sting was seen in the positive control, sting co-expressed with denv ns b [ ] (s d fig). additionally, nsps - of chikv-rt were individually expressed with cgas to look for potential interactions or degradation of the protein. immunoprecipitation of cgas pulled down nsp of chikv-rt; however, no degradation of cgas was observed in any of the co-expression conditions (s e fig). next, we validated the interaction of chikv / nsp with cgas in the context of infection by over-expression of cgas in hek- t for hrs followed by infection with chikv / and subsequent cgas immunoprecipitation. interestingly, no cgas was observed in the input sample of the infected condition with chikv / (s f fig, lane ) . however, when the immunoprecipitation for cgas was performed, we detected a reduced presence of cgas, even less than in the positive control, denv ns b , which has already been shown to interact with and degrade cgas[ ] (s f fig). to further confirm the interaction between nsp and cgas, hek- ts were transfected with chikv-rt nsp and seeded on glass-bottom plates for imaging by immunofluorescence. a clear co-localization of cgas with nsp was observed in cells which expressed both proteins (s g fig). the lack of cgas degradation seen in the exogenous expression system when compared to the infected cells shows that although there is a clear interaction of cgas with nsp , the degradation of cgas observed is not mediated by the non-structural proteins of chikv, but must require other viral or host factors. representative of two independent experiments (n = ). data are represented by means ± sd (n = ). statistical analysis was done with student's t tests. statistical significance represented as follows: ns = not significant, � = p< . , �� = p< . , ��� = p< . , ���� = p< . . https://doi.org/ . /journal.ppat. .g chikungunya virus inhibits dna sensing by degrading cgas during viral infection, stresses placed upon cells can lead to the release of dna into the cytoplasm of infected cells, thus leading to the activation of cytosolic dna sensors, such as cgas [ , ] . we previously detected such cytosolic dna in denv infected cells via immunofluorescence [ ] . to test if chikv infection results in the appearance of cytoplasmic dna in infected cells, hff- s were infected with chikv / at low or high multiplicity of infection (moi) for hrs. the cells were then probed with antibodies against nsp (viral infection), an anti-dna antibody, clone [ ] [ ] [ ] [ ] , which binds both double stranded and single stranded dna (dsdna/ssdna), and dapi which binds dsdna. during chikv infection we observed a distinct accumulation pattern of dna puncta which were absent in the mock infected condition (fig a) . furthermore, the presence of dna puncta increased in conjunction with the increase in moi (fig a) . high resolution confocal microscopy of infected cells illustrated the presence of this dna to be in the cytoplasm (fig b ( d view inset) ). wild type murine embryonic fibroblasts (mefs) have been shown to be permissive to chikv replication [ ] . we obtained wt and sting null, goldenticket (gt) mefs [ ] (fig a) and infected them with chikv / in order to understand the restriction imposed by sting on chikv. by hpi there was a log increase in infectious particle release from the gt mefs when compared to wt (fig b) . viral transcripts were also significantly lower in wt mefs when compared to their sting null counterparts ( fig c) indicating that murine sting serves as a potent restriction factor for chikv replication and infectious particle release. next, commercially available raw . cells, a murine macrophage cell line, deficient for cgas or sting, were infected with chikv / . infectious virus release was quantified from the supernatant of infected cells and revealed a sharp increase in chikv replication from both the cgas and sting null cells when compared to their wt counterpart ( fig d) . taken together, these data support previous reports that the cgas-sting pathway restricts the replication of chikv [ , , ] and provides direct evidence that the pathway poses a significant restriction upon viral replication. to determine the integrity of the cgas during chikv infection, whole cell lysates were collected from hff- s at , , and hpi. lysates were analyzed via sds-page and endogenous cgas and sting were detected via western blotting (wb). we observed a drastic decrease in cgas expression as early as hpi, before even viral non-structural proteins were detectable via wb in infected cells, while sting expression was not altered (fig a) . because chikv can inhibit cellular transcription, the reduction in cgas expression over time in chikv infected cells could also be due to reduced cgas transcripts, ultimately leading to reduced newly translated cgas. mrna levels of cgas were quantified and there were no differences in chikv infected cells when compared to mock or ndv at hpi (fig b) indicating that a decrease in cgas transcripts is not responsible for the rapid loss of cgas expression following chikv infection. alternatively, cgas levels could be reduced via global translational inhibition by the viral protein nsp [ ], resulting in a reduction in cgas protein levels through normal homeostatic protein recycling. to assess if the half-life of cgas was shorter than the time in which it takes chikungunya virus inhibits dna sensing by degrading cgas chikungunya virus inhibits dna sensing by degrading cgas chikv infection to result in a loss of cgas expression, a puromycin pulse-chase was performed. this technique has been shown to be highly sensitive in detecting a snapshot of active translation in cells [ ] . hff- s were pretreated with mock or cycloheximide (chx) for hrs to block cellular translation and then "pulsed" for min with puromycin to label newly synthesized proteins after which cells were washed and re-fed with complete media for min. we observed a clear reduction of puromycin incorporation in cells which were treated with chx for hrs, demonstrating an inhibition of new protein synthesis in treated cells. although translation had been halted in chx treated cells for hrs, there was no change in cgas expression, demonstrating that the half-life of cgas in hff- s is longer than hrs ( fig c) . in order to assess whether viral induced translational shutoff coincides with loss of cgas expression, chikv-mediated translational inhibition was determined by the previously described puromycin pulse-chase method ( fig d) . in accordance with a previous publication [ ] , chikv mediated translational shutoff was not detectable at or hpi while there was a significant reduction in cgas expression as early as hpi, when compared to mock (fig d) , demonstrating that the loss of cgas expression observed in infected cells is not due to chikv mediated translational inhibition. interestingly, there is a noticeable increase in translation in chikungunya virus inhibits dna sensing by degrading cgas both mock and infected cells by hpi suggesting that the stress produced during the process of infection (mock or chikv) in hff- s stimulates increased translation at early timepoints ( fig d, lanes and ) . as we observed a slight decrease in dna stimuli-dependent type-i ifn transcripts in the uvc conditions (fig g & i ) and because the decrease in cgas occurs very early during the infection, we hypothesized that the viral factor responsible for cgas degradation is a component of the virion. upon viral membrane fusion in endosomes, the viral nucleocapsid is exposed in the cytoplasm which is followed by disassembly of the nucleocapsid resulting in the release of the viral genome along with capsid into the cytoplasm of infected cells. to determine if chikv capsid was sufficient to result in a degradation of cgas, the viral protein was coexpressed with cgas in increasing amounts. a dose dependent cgas degradation was observed when co-expressed with capsid and this reduction was specific, as no reduction in cgas expression was observed when the innate immune sensor was co-expressed with another viral protein, nsp ( fig e) . capsid co-expression was also sufficient to inhibit cgas-sting mediated induction of a type-i ifn reporter, indicating a functional inhibition of the innate immune sensing pathway (fig f & g) . these data indicate an immediate restriction of the cgas-sting pathway by chikv via degradation of cgas. this degradation is independent of viral transcriptional and translational shutoff and is mediated by the viral capsid protein. during infection, chikv is known to induce autophagy which has a proviral effect on replication [ ] . previous work from our group demonstrated an autophagy-mediated degradation of cgas during denv infection, in order to prevent the activation of the cgas-sting pathway by mis-localized mtdna [ ] . we hypothesized that the cgas degradation observed in chikv infection might also occur via autophagy. to test this, hek- ts were transfected with cgas before treatment with a commonly used chemical inhibitor of autophagy, -methyladenine ( -ma)[ , [ ] [ ] [ ] and later infected with chikv / . treatment with -ma was able to rescue expression of cgas in infected cells, indicating a role for autophagy in cgas degradation during chikv infection (fig a) . confirmation that autophagy participates in cgas degradation during chikv infection was assayed via knockdown of autophagy related protein (atg ), a critical component in the formation of phagophores [ ] . knockdown of atg was able to rescue cgas expression in infected cells, but did not have an appreciable effect in non-infected cells (fig b) . importantly, knockdown of atg did not result in an accumulation of the lower molecular weight band of lc , demonstrating a functional inhibition of the process of autophagy in the siatg conditions ( fig b) . upon quantitative analysis of protein expression, atg knockdown did increase expression of cgas in mock-infected cells by . fold, when compared to a non-targeting control, indicating that atg participates in regulating homeostatic levels of cgas expression (fig c) . in chikv infected conditions, however, there was a striking . fold representative of two independent experiments). (d) hff- s were infected with mock or chikv at an moi of . . min prior to indicated timepoints, cells were pulsed with puromycin as described in (c). lysates were collected and proteins were detected as previously stated (data representative of two independent experiments). (e) hek- ts were transfected with indicated constructs at indicated plasmid amounts. cells were allowed to rest for hr post transfection before lysis and sds-page/immunoblotting analysis (data are representative of three independent experiments). (f) t-ifnb-ffluc cells were transfected with empty vector (vec), cgas and sting in conjunction with vec, or cgas and sting with the capsid of chikv / . cells were allowed to rest for hrs before lysis for collection of protein or quantification of luminescence. data are representative of six independent experiments. data represented as fold induction over vector alone. data are represented by means ± sd (n = ). statistical analysis was done with student's t tests ( �� = p< . )). (g) protein input for ifn reporter assay in (f). https://doi.org/ . /journal.ppat. .g chikungunya virus inhibits dna sensing by degrading cgas increase in cgas expression due to atg knockdown, confirming that autophagy modulates cgas expression during chikv infection (fig c) . reduction in atg expression when compared to respective non-targeting controls were . % (mock) and . % (chikv), respectively ( fig d) . no difference in viral infectious particle release was observed between siatg or sictl, indicating that the increase in cgas expression was not due to reduced viral replication ( fig e) . these data demonstrate a critical role of autophagy in chikv dependent cgas degradation and expand our previous work with denv, suggesting a conserved virushost interplay across different class iv rna viruses. interactions of chikv nsps with sting were tested by immunoprecipitation against the flag tag from total cell lysates and visualized via wb. a clear interaction of chikv nsp with sting was noted in an overexpression system ( fig a) . next, the nsp interaction was tested in the context of chikv / infection. once again, the interaction of nsp with sting was observed, indicating that this interaction occurs in the context of viral infection (fig b) . to better characterize the interaction between nsp and sting, deletion mutants of sting were generated (fig c) . sequential deletions of sting were made from the c-terminus of the protein to delete functional domains, but so as not to disrupt the insertion of the protein in the er membrane or affect subcellular localization. sting Δ eliminates the c-terminal domain (ctd) of sting, which includes a critical interaction region for tank binding kinase interaction [ , ] . the Δ mutant deletes the functional cyclic gmp-amp (cgamp) binding ability of the protein and the dimerization domain (dd), while the Δ mutant leaves only the transmembrane spanning portion of sting [ ] . chikv nsp was then coexpressed with the deletion mutants of sting and an immunoprecipitation was performed ( fig d) . each mutant co-precipitated nsp , indicating that interaction of nsp with sting only requires the transmembrane spanning domains of the innate immune signaling protein. given the predicted structure of sting as a four-pass transmembrane protein there are only two regions of sting exposed to the cytosol with which nsp could be interacting, the first aa at the n-term or a cytosolic loop region spanning approximately amino acids - [ , ] . deletion mutants of sting were generated lacking the first aa at the n-term or which had small deletions in the cytosolic loop of the protein, either aa - or aa - , respectively (fig e) . immunofluorescence of hek- ts transfected with sting internal deletion variants represented in e showed no significant changes in subcellular localization of the proteins (s fig). co-expression of nsp with a Δ mutant of sting illustrated that the n-terminal tail of sting is not critical for interaction, but rather that the nsp -sting interaction is mediated by the aa cytosolic loop of sting (fig f) . interestingly, a loss of nsp s interaction with sting resulted in decreased expression of the viral protein (fig f, lane ) . alternatively, co-expression of nsp with full length sting significantly and specifically increased nsp expression (s fig). recently it has been demonstrated that murine sting is palmitoylated at cysteine residues and and that this modification is critical in activation of the antiviral response against dna viruses [ ] . these residues are conserved between human and murine sting and are in the cytosolic loop region which nsp of chikv is interacting with. additionally, it has been represented as means ± sd. (b-e) data representative of two independent experiments. statistical analysis was done with student's t test. https://doi.org/ . /journal.ppat. .g chikungunya virus inhibits dna sensing by degrading cgas chikungunya virus inhibits dna sensing by degrading cgas demonstrated that nsp of chikv is palmitoylated and inhibition of this post-translational modification has myriad effects ranging from altering replication kinetics to pathogenesis in mice [ ] [ ] [ ] [ ] ] . because of this association we hypothesized that nsp s interaction with sting could result in increased levels of palmitoylated nsp and this could serve as a mechanism by which sting stabilize nsp expression. interestingly, sting mediated nsp stabilization was independent of palmitoylation as demonstrated by transfecting nsp with sting both in the presence and absence of a global palmitoylation inhibitor, -bromopalmitate ( fig g) , although there was an increase in the levels of palmitoylated nsp when the protein was co-expressed with sting ( fig h) . the described sting-nsp interaction, also resulted in a significant inhibition of the ifnβ promotor activation by cgas-sting overexpression (s b fig) . these data provide the first description of chikv interaction with sting. furthermore, we mapped the interaction to a aa cytosolic loop of sting and demonstrates that this interaction could have a pro-viral role with respect to increasing the amount of nsp in infected cells in a sting dependent manner, while downregulating the ifnβ production pathway. cgas and sting have been previously implicated in restricting the replication of alphaviruses [ , , ] . in mef and raw . cells deficient for cgas or sting, we observed that the individual proteins in this dna-sensing pathway serve to functionally restrict chikv replication and infectious particle release. these data are in accordance with previous publications which have implicated cgas and sting as restriction factors for chikv replication [ , , ] and provide further evidence that this pathway is important in inhibiting not only dna viruses but also different class iv rna viruses. similar to what our group has described during denv infection[ ], we also observed the production of distinct puncta of extranuclear dna upon chikv infection. as defined subcellular compartmentalization of dna is fundamental to the normal lifecycle of eukaryotic cells the presence of dna in the cellular cytoplasm results in a strong type-i ifn response mainly mediated by cgas-sting [ , ] . the identity of the mis-localized dna in chikv infected cells has yet to be understood, but its presence in the cytoplasm by definition serves as a danger associated molecular pattern (damp)[ , , ] . when the cgas-sting pathway was stimulated with either a dna virus or e. coli dna, we observed a replication dependent inhibition of ifnß transcripts. interestingly, there was a slight reduction in ifnß transcripts when the cells were treated with a uv-inactivated chikv (uvc). this could be because uvc treatment resulted in the cells being refractory to infection with mva; however, we did not observe a reduction in mva replication measured by quantitative pcr. another possibility was that a viral factor intrinsic to the nucleocapsid was responsible for the reduction in cgas-sting dependent signaling observed. indeed, exogenous expression of chikv capsid was sufficient to reduce cgas expression and was able to significantly inhibit cgas-sting mediated induction of a type-i ifn reporter. immunoblotting of infected primary human chikv-rt nsp and the different sting mutants indicated in (c) and cells were lysed hrs post transfection. an immunoprecipitation was performed against the flag epitope and protein samples were then analyzed via sds-page and immunoblotting performed as described previously (data representative of two independent experiments). (e) schematic of sting inserted in the er membrane highlighting regions deleted which are located in cytosolic facing domains (schematic representative of poor artistic skill). (f) t cells were transfected with internal deletion sting constructs and nsp . cells were lysed hpt and an anti-flag ip was performed followed by sds-page and immunoblotting (data representative of three independent experiments). (g) hek- t cells were transfected cells with indicated constructs overnight followed by treatment with um -bp for h. cells were lysed and analyzed via western blotting. (h) hek- t cells were transfected with indicated constructs overnight and treated for h with um alk- palmitoylation chemical reporter reagent prior to cell lysis. immunoprecipitation was performed against the ha epitope followed by click chemistry reaction with azido-rhodamine for visualization of protein palmitoylation via fluorescence gel scanning. (g & h) data representative of two independent experiments. https://doi.org/ . /journal.ppat. .g chikungunya virus inhibits dna sensing by degrading cgas fibroblasts revealed that endogenous cgas is degraded as early as hrs post infection and that this degradation is independent of global transcriptional or translational repression induced by chikv. the ability of a viral structural protein to reduce cgas expression explains the rapid loss of cgas protein levels at time-points prior to a full replication cycle of the virus with de novo production of viral proteins. furthermore, capsid mediated degradation of cgas explains the slight reduction in cgas-sting signaling observed when cells were treated with uvc prior to stimulation with mva or e. coli dna. here, no data is presented identifying antagonism of cgas-sting signaling at the level of sting, however, given the current view of cgas dependent sting signaling, the degradation of cgas by chikv, by definition, leads to an inhibition of cgas-sting signaling. further studies must be performed to understand if during chikv infection, hallmarks of sting activation are present including phosphorylation of serine , interactions with tbk , or a subcellular re-localization of sting to the trans-golgi-network (tgn). the role of autophagy during chikv infection is cell type specific and has been demonstrated to have both pro and antiviral effects [ , , , ] . in addition to canonical antiviral signaling, other antiviral functions of sting have been documented including sensing of viral fusion [ , ] and translational repression [ ] . research regarding the function of these alternative sting-dependent pathways during chikv infection is critical in developing a more complete understanding of the interplay of chikv with the cgas-sting pathway. interestingly, nsps - of chikv were also able to inhibit cgas-sting mediated induction of a type-i ifn promotor, suggesting that multiple chikv proteins may work individually or in concert to antagonize cgas-sting signaling during viral infection. antagonism mediated by the non-structural proteins was not due to any direct effects on the levels of cgas or sting expression indicating that they do not modulate the pathway via alteration of cgas or sting protein levels. with these data, no direct mechanistic conclusions can currently be drawn as to inhibition of cgas-sting signaling by nsps - of chikv. they do however provide insight that degradation of cgas may only be part of a repertoire of methods by which chikv antagonizes cytosolic dna sensing. we observed an interaction of nsp with cgas, however, nsp did not significantly alter the activity of a type-i ifn reporter system. interestingly, for sindbis virus, another alphavirus, nsp has been shown to be degraded during infection via the proteasome by the n-end rule pathway during infection [ ] . thus, degradation of cgas could be mediated partially by nsp , but only when the host protein is degraded by the proteasome in the context of infection. this potential alternative protein degradation mechanism would explain why there is no total cgas recovery during autophagy inhibition in chikv infection. additionally, for other alphaviruses it has been demonstrated that nsp interacts with all three of the other non-structural proteins [ ] [ ] [ ] and the viral rdrp, nsp is the first protein to be cleaved from the polyprotein nsp - upon chikv infection. this early cleavage event in conjunction with replicative intermediates produced immediately upon infection could synergistically result in antagonism of cgas dependent signaling, but only in the context of infection. by using two complementary methods to inhibit autophagic flux, a chemical inhibitor, -ma, and sirna knockdown of atg , a critical factor in initiation of autophagosomes, we observed a significant recovery of cgas expression in chikv infected cells. specifically, when cgas levels were normalized to their respective non-targeting controls and atg expression was taken into account, there was an . fold increase in cgas expression in chikv-infected conditions versus a . fold increase in mock-infected cells. importantly, a reduction in the amount of lc ii was observed in knockdowns of atg when compared to the non-targeting controls, indicating the knockdowns were functionally reducing autophagy. interestingly, it has been reported that there is an atg /atg independent form of autophagy in murine embryonic fibroblasts (mefs) [ ] . this atg /atg independent macro-autophagy did not result in the accumulation of lc ii in cells. because lc ii was observed in the atg knockdowns, atg dependent autophagy was reduced but we cannot rule out whether or not atg independent autophagy, plays a role in cgas degradation during chikv infection. furthermore, it has been demonstrated that during infection, chikv capsid interacts with selective autophagy mediator p and this interaction results in capsid degradation via autophagy [ ] . another study demonstrated that p can interact with and modulate cgas expression via autophagic degradation [ ] . it is thus possible that the degradation of cgas by chikv capsid is mediated via an intermediary interaction between cgas-p -and -capsid which results in both being degraded during infection. chikv nsp was found to interact with sting. nsp is the only non-structural protein of chikv which is anchored to cellular membranes [ ] [ ] [ ] and sting is a transmembrane protein which has been shown to localize to the endoplasmic reticulum (er), mitochondrial associated membranes (mam), and cytoplasmic vesicles [ , ] . it is possible that the interaction between nsp and sting is enhanced because of their proximity due to membrane association. the interaction between nsp and sting was mapped to a amino acid cytosolic loop region of the protein. it is possible that through this interaction, nsp is disrupting dimerization of sting and thus dampening antiviral responses. however, the cytosolic loop domain of sting has not been shown to function in dimerization, so that possibility is unlikely. further work must be done to understand the residues required for sting interaction from the molecular perspective of nsp as well as if there are differences between human and murine sting with respect to the nsp interaction as previously reported for denv ns b [ ] . it has been reported that sting interacts with both rig-i and mavs and that sting null cells have reduced type-i ifn production when stimulated with vesicular stomatitis virus (vsv) or sendai virus (sev) [ , , ] . the nsp -sting interaction, could thus be affecting rig-i-like receptor (rlr) signaling by disrupting the innate immune signaling complex of rig-i-mavs-sting, resulting in an inhibition of rlr mediated type-i ifn induction during chikv infection. alternatively, this interaction could serve to inhibit another reported antiviral role of sting: translational repression during rna virus infection [ ] . furthermore, no research has directly addressed whether or not chikv is able to inhibit rlr mediated antiviral sensing or signaling. for a complete understanding of chikv mediated antagonism of innate immunity, it will be critical to study potential viral antagonism of rlr sensing as well as the role of sting's crosstalk with these sensors. interestingly, the nsp -sting interaction serves to increase protein levels of nsp in cells specifically when compared to a gfp control. we found that the increase in nsp levels resulted in increased palmitoylated nsp in cells, although an inhibitor of palmitoylation, -bp, did not alter the increased nsp expression indicating that the process of palmitoylation was not required for the increase in nsp expression. palmitoylated nsp has been previously shown to enhance replication kinetics and pathogenesis of alphaviruses in mice [ , ] while mutant chikv viruses with non-palmitoylatable nsp s showed severely altered replication kinetics and nsp membrane association was disrupted [ ] . this provides a potential dual role for the nsp -sting interaction: simultaneously inhibiting sting mediated antiviral signaling while being stabilized, thus increasing the total levels of nsp . furthermore, chikv nsp has been identified as a druggable target [ ] . currently, there are no complete protein models of either sting or chikv nsp , hindering in silico modeling of protein-protein interactions. by determining the cytosolic loop domain of sting as being critical for the nsp -sting interaction and identifying that sting increases nsp expression, our data provide valuable information for understanding the molecular basis of interfacing between these two proteins which can aid in small molecule inhibitor screening and design. significant evidence supports that the dna sensing pathway of cgas and sting plays an important role in restricting rna virus infections and viral antagonists of cgas and sting have been identified for both flaviviruses and human coronaviruses [ , , , , , , ] . in this work we sought to understand if chikv interacted with or restricted the cgas-sting innate immune signaling pathway. taken together, these data have strong potential implications for the rational design of attenuated chikv viruses, provide information regarding nsp for small molecule inhibitor design, and identify, for the first time, direct antagonism of a cytosolic dna sensing pathway by chikv. human foreskin fibroblast (hff- ) cells were obtained through atcc (atcc scrc- ) and cultured in dulbecco's modified essential medium (dmem) supplemented with % fetal bovine serum (fbs). human embryonic kidney- t (hek- t) cells were also obtained from atcc (atcc crl- ) and were grown in dmem supplemented with % fbs, u ml - l-glutamine, u ml - penicillin/streptomycin. mosquito cells from aedes albopictus mosquitos, clone c / (obtained originally from j. munoz-jordan, cdc, puerto-rico) were maintained in rpmi medium with % fbs at ˚c. baby hamster kidney cells (bhk) were passaged in minimum essential medium (mem) alpha + glutamax-l purchased from gibco and supplemented with % fbs, u ml - penicillin/streptomycin, and mm -( -hydroxyethyl)- -piperazineethanesulfonic acid (hepes). u os cells were a gift from dr. carolyn coyne's laboratory and were maintained in media used for the hek- ts. hek- t-ifnß reporter cells, previously described [ ] were grown as stated for hek- ts. vero cells were purchased from atcc (atcc ccl- ) and were maintained in media described for hek- ts. all tissue culture reagents were purchased from invitrogen. mefs, wt and gt, were a gift from dr. jonathan miner (wustl) and were maintained in (dmem) supplemented with % fetal bovine serum (fbs). raw . cells were purchased from invitrogen and cultured according to the manufacturer's instructions. catalog numbers: wt: rawl-isg, cgas ko: rawl-kocgas, and sting ko: rawl-kostg. the chikungunya / (chikv / ) strain, originally derived from a patient isolate in thailand [ ] , used in this study was kindly provided by dr. st. patrick reid at the university of nebraska, omaha. the virus was passaged one time in vero cells and supernatants were collected, clarified, and stored at - ˚c. viral titers were determined by limiting dilution plaque assay on bhk- s as previously described for dengue virus (denv) [ ] . newcastle disease (ndvb -gfp), originally obtained from dr. adolfo garcia-sastre, were grown in nine day old embryonated chicken eggs and tittered via tcid on chicken embryonic fibroblasts (cefs) as previously described [ , ] . vaccinia virus aliquots were a kind gift from dr. nacho mena, of the gracia-sastre laboratory. dengue virus serotype (denv- ) strain was grown for six days as previously described in c / cells [ ] . chikv indian ocean lineage (chikv iol) was generated from the la reunion (strain - ) infectious clone (coffey et al, ). to generate infectious virus, the infectious clone plasmids were linearized overnight with noti, purified, and used for in vitro transcription with the sp mmachine kit (ambion). in vitro transcribed rna was phenol:chloroform extracted, ethanol precipitated, aliquoted at μg/μl and stored at - ˚c. μg of in vitro transcribed rna was electroporated into bhk cells and virus was harvested at hours post electroporation. working virus stocks chikungunya virus inhibits dna sensing by degrading cgas were generated by passaging the virus in bhk cells for hours. viral titers were determined by plaque assay on vero cells. uv-c bulb was placed inches from the ml viral aliquots in -well culture dishes (corning) with the lid removed. plates were placed on a magnetic stir plate and sterile magnetic stir bars were placed in each well. uv-inactivation was allowed to proceed for min at room temperature. viral aliquots were then stored at ˚c for future use. uv-c bulb used: g t . w. the wavelength of light was nm. the ability of different chikv nsps to inhibit the induction of the ifnß reporter was assessed in hek- t cells stably expressing firefly luciferase under the control of an ifn-ß promotor ( t-ifnß), previously described [ ] . , t-ifnß cells were transiently reverse transfected using lipofectamine (thermo) with ng total dna per well of different constructs expressing either human cgas-pcmv (origene), human sting-(pcdna), denv ns b -(pcaggs), chikv gfp-nsps plasmids, (kindly provided by dr. subhash g vasudevan (duke-nus graduate medical school, singapore), chikv capsid (ptr- ) or pcaggs with no coding insert (empty vector (ev)), in -well plates, using lipofectamine reagent (invitrogen) per the manufacturer's protocol. hours post transfection, ifn-ß promotor induction was measured using the neolite luminescence reporter gene assay system (perkinelmer) per manufacturer's protocol. western blots were performed as described in "immunoblot analysis" section. primary human foreskin fibroblasts (hff- ) were seeded in -well plates at a density of . x cells/well. hours after seeding, cells were treated with either mock (dmem), ndvb -gfp at an moi of . , or chikv, either chikv iol or / at an moi of . . infections were allowed to proceed for hr in a total volume of μl of sera-free dmem. after hr, infection media was removed and cells were re-fed with ml of hff- media (dmem with % fbs). , , , and hours post infection supernatants were collected and quick-frozen in dry ice/ethanol then stored at - ˚c. at the selected time-points, rna from cells was collected according to the manufacturers' protocol using the quick-rna mini-prep (zymogen) and stored at - ˚c. protein lysates were collected by re-suspending cells in ripa lysis buffer (sigma aldrich) and were subsequently stored at - ˚c. prior to infection, . x hff- s were seeded in -well culture dishes (corning). cells were then treated with mock, chikv / or uv-inactivated chikv / (uvc) at an moi of . . after primary treatment, cells were allowed to rest for hrs. the secondary treatments were administered hrs post primary treatment. cells were infected with either mock or modified vaccinia ankara (mva) at an moi of . to induce the rlr or cgas-sting pathways and rna was collected and hrs post-secondary treatment. alternatively, cgas-sting induction was performed via mock or e. coli dna ( ug/well) (invivogen) transfection using lipofectamine according to manufacturer's instructions. rna was collected at and hrs post transfection. all rna collections were performed according to the manufacturer's protocol using the quick-rna mini-prep (zymogen). determination of induction of innate chikungunya virus inhibits dna sensing by degrading cgas immune signaling gene transcripts was determined by normalizing all conditions to rps and then determining fold induction over respective mock e.g. induction of isg transcripts as a result of mva infection was determined by comparing uvc ➔ mva over uvc➔mock (primary ➔ secondary) conditions. rna from cells was extracted using quick-rna mini-prep (zymogen) according to the manufacturers protocol (including the in-column dnase treatment). concentration of ribonucleic acid was determined via spectrophotometer at nm. rna was then stored at - ˚c until rt reaction. rt reaction was done with the iscript cdna synthesis kit (bio-rad) utilizing random hexamer priming, according to the manufacturer's instructions with - ng of total rna. rt-qpcr was used to quantify relative levels of gene expression in infected and uninfected cells and was performed using the iq sybr green supermix (biorad) according to the manufacturer's instructions. the biorad c thermal cycler was used with the following pcr profile: ˚c for min followed by cycles of ˚c for s then ˚c for s. quantification of gene expression was performed based on ct values of a given gene normalized to the housekeeping gene, rps , gapdh, or both where indicated. cellular lysates were obtained by incubating cells in ripa lysis buffer (sigma aldrich) supplemented with edta-free, complete ultra tablets, mini (roche) for min on ice. quantification of protein in cellular lysates was performed via colorimetric bradford assay (bio-rad) utilizing bovine serum albumin (bsa) for generation of a standard curve. cellular lysates were re-suspended, in a : ratio, in x laemmli sample buffer (bio-rad) supplemented with -mercaptoethanol and boiled at ˚c for min in a heating block (fisher scientific). all samples were then loaded on polyacrylamide-sds gels and the denatured proteins were separated by electrophoresis via conventional methods. protein was then transferred to nitrocellulose membranes (bio-rad). blots were blocked with phosphate buffered saline (pbs) with % milk for one hour at room temperature. antibodies used: cgas (d d g), sting (d p f), rig-i (d h ), atg (d b ) (cell signaling technology) at a : dilution, anti-flag (f ), anti-ß-actin (a ), and anti haemagglutinin (ha) (h ) (sigma aldrich) at a : dilution, anti-gfp (ma - ) (invitrogen), anti-chikungunya virus clone a (mabf ), anti-puromycin clone d (mabe ) (emd millipore). antibodies against chikv / were kind gifts from drs. stapleford and reid. secondary antibodies against mouse (na v) and rabbit (na v) (ge healthcare). detection of immunocomplexes were performed using supersignal chemilumisescence system (thermo). densitometry analysis was performed using imagej software. t cells were seeded on -well glass-bottomed plates (mattek) which had been pre-coated with . % polylysine for hr at rt or hff- s were seeded directly on the glass-bottomed plates without pretreatment. hrs post transfection or infection, cells were fixed at rt with . % formaldehyde then permeabilized with . % triton-x before blocking with % bsa in pbs for hr at rt. cells were incubated overnight at ˚c with primary antibodies: (h : sigma chikungunya virus inhibits dna sensing by degrading cgas aldrich), anti-ssdna (mab : emd millipore), anti-nsp (provided by dr. stapleford), anti-flag (f : sigma), or anti-calnexin (ma - : invitrogen). cells were then incubated with for hr at rt with alexa fluor-conjugated anti-mouse , anti-rabbit (life technologies), μg ml - dapi (invitrogen) or phalloidin (a : invitrogen) as indicated. confocal imaging was performed using a zeiss lsm with airyscan. images were collected at bits and a resolution of x pixels. d images were created via reconstruction of z-stacks in zen blue software. deletions of functional regions of sting were generated and cloned into the ptr- mammalian expression vector using ecori and bamhi restriction sites utilizing the in-fusion hd cloning kit (clontech). immunoprecipitation was performed using the ezview red anti-flag m affinity gel (sigma aldrich). briefly, affinity gel was washed x in lysis buffer (ripa lysis buffer (thermo) supplemented with complete mini edta-free protease inhibitor (roche). whole cell lysates were mixed with the affinity gel and incubated for hr at ˚c, rotating. after incubation, affinity gel was washed x for min each in lysis buffer. following washes, the affinity gel was resuspended in μl x laemmli buffer (bio-rad) with -mercaptoethanol (sigma aldrich) and boiled for min at ˚c. samples were then stored at - ˚c until immunoblot analysis. chikungunya virus inhibits dna sensing by degrading cgas studies of protein palmitoylation were performed according to detailed published protocols [ ] [ ] [ ] . in brief, transfected cells were treated for h with the alk- ( um) chemical reporter of protein palmitoylation. proteins of interest were immunoprecipitated from cell lysates and reacted via the copper(i)-catalized azide alkyne cycloaddition reaction ("click chemistry") with azido-rhodamine (kindly provided by dr. howard hang of the rockefeller university) for visualization of protein acylation via fluorescence gel scanning on a typhoon (amersham) fluorescence imager. western blotting of the samples provided controls for loading and sample inputs. for inhibition of protein palmitoylation, transfected cells were treated for h with um -bromopalmitate (sigma). unpaired, two-tailed, student's t-test was used for direct comparisons while one way or two way anovas with tukey's multiple comparisons we used for viral growth curves or multiple comparisons. specific analysis used for respective figures are listed in the figure legends. p-values were determined to be significant when p < . . relevant p-value cutoffs used are listed in figure legends. no samples were excluded when analyzing these data. cells were allowed to rest for hrs before lysis for collection of protein or quantification of luminescence. (c) input protein expression for reporter experiment (b) was visualized via sds-page followed by immunoblotting. data representative of four independent experiments. data are represented by means ± sd (n = ), fold induction over mock. statistical analysis was done with student's t tests ( � = p< . , �� = p< . , ��� = p< . ). (d) hek- t cells were transfected with indicated constructs and cells were lysed hpt. denv- ns b served as a positive control for sting cleavage/degradation while the catalytically inactive ns b s a was used as a negative control. gfp tagged chikv-rt nsp constructs were used to test for degradation or cleavage of sting. protein lysates were analyzed via sds-page and subsequent immunoblotting. data representative of one independent experiment. (e) hek- t cells were transfected with indicated constructs (nsps - -ha chikv-rt) and cells were allowed to rest for hrs before lysis. lysates were subjected to immunoprecipitation against a flag epitope and proteins were visualized via sds-page and immunoblotting. data representative of three independent experiments. (f) indicated constructs were expressed in t cells and cells were allowed to rest for hrs. after resting, cells were infected with either mock or chikv / (moi = . ). hpi cells were lysed and an immunoprecipitation preformed against a flag epitope. protein interaction was analyzed via sds-page followed by immunoblotting. data representative of two independent experiments. chikungunya virus inhibits dna sensing by degrading cgas togaviridae: the viruses and their replication. fields virology high level of vector competence of aedes aegypti and aedes albopictus from ten american countries as a crucial factor in the spread of chikungunya virus - pmid: chikungunya fever: epidemiology, clinical syndrome, pathogenesis and therapy persistent arthralgia associated with chikungunya virus: a study of adult patients on reunion island a report of cases of rheumatoid arthritis following chikungunya fever. a mean follow-up of two years chikungunya virus and arthritic disease an epidemic of virus disease in southern province reemergence of chikungunya virus an epidemic of virus disease in southern province, tanganyika territory, in - . i. clinical features a major epidemic of chikungunya virus infection on reunion island the signal for translational readthrough of a uga codon in sindbis virus rna involves a single cytidine residue immediately downstream of the termination codon fatty acid synthase promotes the palmitoylation of chikungunya virus nsp role of the amphipathic peptide of semliki forest virus replicase protein nsp in membrane association and virus replication effects of palmitoylation of replicase protein nsp on alphavirus infection mutations at the palmitoylation site of non-structural protein nsp of semliki forest virus attenuate virus replication and cause accumulation of compensatory mutations replication cycle of chikungunya: a re-emerging arbovirus emerging alphaviruses are sensitive to cellular states induced by a novel small-molecule agonist of the sting pathway inhibition of dengue and chikungunya virus infections by rig-i-mediated type i interferon-independent stimulation of the innate antiviral response characterization of a novel human-specific sting agonist that elicits antiviral activity against emerging alphaviruses type i ifn controls chikungunya virus via its action on nonhematopoietic cells pan-viral specificity of ifn-induced genes reveals new roles for cgas in innate immunity chikungunya virus induces ips- -dependent innate immune activation and protein kinase r-independent translational shutoff cytosolic sensing of viruses sensing of rna viruses: a review of innate immune receptors involved in recognizing rna virus invasion type i interferons in infectious disease interferon-stimulated genes: a complex web of host defenses chikungunya virus nonstructural protein inhibits type i/ii interferon-stimulated jak-stat signaling activation of sting requires palmitoylation at the golgi characterization of reemerging chikungunya virus influenza virus evades innate and adaptive immunity via the ns protein newcastle disease virus v protein is a determinant of host range restriction modified vaccinia virus ankara triggers type-i ifn production in murine conventional dendritic cells via a cgas/sting-mediated cytosolic dna-sensing pathway collateral damage during dengue virus infection: making sense of dna by cgas chikungunya virus-induced autophagy delays caspase-dependent cell death the n-ethyl-n-nitrosourea-induced goldenticket mouse mutant reveals an essential function of sting in the in vivo interferon response to listeria monocytogenes and cyclic dinucleotides sunset, a nonradioactive method to monitor protein synthesis chikungunya triggers an autophagic process which promotes viral replication the autophagic inhibitor -methyladenine potently stimulates pka-dependent lipolysis in adipocytes systemic application of -methyladenine markedly inhibited atherosclerotic lesion in apoe(-/-) mice by modulating autophagy, foam cell formation and immune-negative molecules dual role of -methyladenine in modulation of autophagy via different temporal patterns of inhibition on class i and iii phosphoinositide -kinase apg p/cvt p: a novel proteinactivating enzyme essential for autophagy structural analysis of the sting adaptor protein reveals a hydrophobic dimer interface and mode of cyclic di-gmp binding molecular evolutionary and structural analysis of the cytosolic dna sensor cgas and sting plos pathogens chikungunya virus inhibits dna sensing by degrading cgas the effects of palmitoylation on membrane association of semliki forest virus rna capping enzyme inhibition of virus multiplication by foreign nucleic acid recognition of cytosolic dna activates an irf -dependent innate immune response structural mechanism of cytosolic dna sensing by cgas the cgas-cgamp-sting pathway of cytosolic dna sensing and signaling species-specific impact of the autophagy machinery on chikungunya virus infection identification of a candidate therapeutic autophagy-inducing peptide virus-cell fusion as a trigger of innate immunity dependent on the adaptor sting influenza a virus targets a cgas-independent sting pathway that controls enveloped rna viruses sindbis virus rna polymerase is degraded by the n-end rule pathway modification of asn of nsp suppresses a sindbis virus nsp minus-strand polymerase mutant requirement for the amino-terminal domain of sindbis virus nsp during virus infection suppressor mutations that allow sindbis virus rna polymerase to function with nonaromatic amino acids at the n-terminus: evidence for interaction between nsp and nsp in minus-strand rna synthesis discovery of atg /atg -independent alternative macroautophagy trim inhibits cgas degradation mediated by selective autophagy receptor p to promote innate immune responses semliki forest virus mrna capping enzyme requires association with anionic membrane phospholipids for activity chikungunya virus nsp interacts directly with nsp and modulates its atpase activity membrane binding mechanism of an rna virus-capping enzyme plos pathogens chikungunya virus inhibits dna sensing by degrading cgas the ubiquitin ligase rnf regulates antiviral responses by mediating degradation of the adaptor protein mita the adaptor protein mita links virus-sensing receptors to irf transcription factor activation the viral capping enzyme nsp : a novel target for the inhibition of chikungunya virus infection sars coronavirus papain-like protease inhibits the type i interferon signaling pathway through interaction with the sting-traf -tbk complex hepatitis c virus ns b blocks the interaction of sting and tbk to evade host innate immunity inhibition of the type i interferon response in human dendritic cells by dengue virus infection requires a catalytically active ns b complex development of an attenuated strain of chikungunya virus for use in vaccine production infection of human cells by dengue virus is modulated by different cell types and viral strains dengue virus inhibits the production of type i interferon in primary human dendritic cells newcastle disease virus (ndv)-based assay demonstrates interferon-antagonist activity for the ndv v protein and the nipah virus v, w, and c proteins modulation of dengue virus infection in human cells by alpha, beta, and gamma interferons palmitoylome profiling reveals s-palmitoylation-dependent antiviral activity of ifitm chemoproteomics reveals toll-like receptor fatty acylation the palmitoyltransferase zdhhc enhances interferon-induced transmembrane protein (ifitm ) palmitoylation and antiviral activity the authors would like to thank dusan bogunovic, florian krammer, and jean lim for their valuable input on the direction of the project as part of lgw phd advisory committee. we thank paula lopez-monteagudo for her invaluable technical advice and camaraderie as a lab mate. we also thank jonathan miner, carolyn coyne, subhash g vasudevan, adolfo garcia-sastre and nacho mena for providing reagents. key: cord- -ydijp b authors: hufsky, franziska; ibrahim, bashar; beer, martin; deng, li; mercier, philippe le; mcmahon, dino p.; palmarini, massimo; thiel, volker; marz, manja title: virologists—heroes need weapons date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: ydijp b nan virologists. you might know a couple of them, but unless you are a virologist yourself, the probability that you have collaborated with one in the past is low. the community is relatively small, but they pack a heavy punch and are expected to play a leading role in the research into pathogens that lies ahead. you may ask why we think virologists are our future. suffice it to say that it is not just because they have invented technologies that belong to the space age, including use of viruses as vehicles to shuttle genes into cells [ ] , organic nanoparticles with specific tools attached to their surfaces to get inside target cells [ ] , and using genetically modified viruses as therapies to fight against cancer [ ] . did you know that virologists currently only know of about , viral species but that more than , mammal-associated viruses [ ] are thought to await discovery? just think about the viruses hidden in the arctic ice [ ] or in the insects and other animals from once cut-off regions in the world, which now face everincreasing human exposure [ ] . but a heroic (as well as an apocalyptic) role for virologists may also be on the horizon, as the adoption of phage therapy may, in the future, be used to control harmful bacteria when antibiotics fail [ ] . bioinformaticians. you may know a couple of them, but usually you "just" need one to do proper math for your -omics experiments. nowadays, nearly everyone in the life sciences has used blast [ ] at least once, or made an alignment, or asked a bioinformatician to analyze high-throughput sequencing data. of course, bioinformaticians do more than straightforward tasks. software is based on algorithms and on data structures. biological data are intrinsically complicated, and it is demanding to find appropriate data structures to process them efficiently [ ] . bioinformaticians routinely have to develop tailored, study-specific algorithms and tools used by a wide variety of scientists, including biochemists, biologists, geneticists, and molecular life scientists; but we rarely find virus-specific tools used by virologists. why is this? it is possibly not the problem of virologists but of bioinformaticians themselves. although it was a virus that was the first organism to have its genome completely sequenced [ ] , bioinformaticians quickly focused attention on larger organisms, including humans, mice, plants, fungi, and bacteria. perhaps there was no time left to take care of viruses? it also may be that, to a bioinformatician, a virus may appear uninteresting, at least at first. for example, the most dangerous human pathogenic (mostly rna) viruses are short, usually contain only a singledigit number of genes that lack introns, contain only mononucleotide or dinucleotide repeats, and very few regulatory elements [ ] . but astonishingly, we now know that the human genome consists of %- % virus-derived sequences (depending on how this is measured: % can be directly traced back to viruses, whereas a figure of % includes lines and sines that are thought to be of viral origin [ ] ). viruses have therefore played a very large part in shaping the evolution of the human genome as well as the genomes of organisms from all domains of life. furthermore, we can observe evolution in viruses in realtime thanks to their extraordinary mutation rates, rapid replication cycles, large population sizes, and immense recombination potential [ ] ; this means that we are able to observe molecular evolution in a matter of days! can you imagine that nearly all existing bioinformatical tools are not specifically designed for the context of viruses? we argue that without bioinformaticians, virologists are fighting with one arm tied behind their backs. clearly virologists are well positioned to tackle some of the major disease threats that will inevitably face humans in the decades to come-but they are currently not the best equipped. in order to give virologists weapons, i.e., bioinformatic tools, we recently founded the european virus bioinformatics center (evbc). as alluded to above, this center was developed based on our belief that we lack bioinformatical tools specifically designed for virology. we urgently need to develop algorithms for multiple genome alignments. it sounds trivial, but we still have no tool that can align, for example, , full-length coronaviruses (approximately , nt). we cannot visualize such alignments that are clearly needed to rapidly identify mutational hotspots and compensatory mutations. can you believe that there is not even a unified database for viruses? we only have good databases for influenza (epiflu [ ] ), hiv [ ] , and human pathogenic viruses (vipr [ ] ). there is the possibility to archive viral sequences in ncbi, but virologists use this facility only rarely because of its limited utility. for instance, submitters are asked to commit the name of a chromosome, which clearly is not applicable to viruses. these examples emphasize that we need to bring virologists and bioinformaticians together. again, this seems obvious and, perhaps, trivial. but in reality, they speak different languages. we urgently need young scientists that understand both virology and bioinformatics so that they can bridge the gap between these two complementary yet different disciplines. the evbc aims to develop bioinformatical tools for nearly all areas: ( ) for detection of viruses, e.g., from high-throughput sequencing data; ( ) virus assembly; ( ) quasispecies reconstruction; ( ) intraviral interactions; ( ) virus entry, i.e., protein-protein interaction; ( ) virus -host interactions; ( ) phylogeny/cophylogeny; and ( ) therapy. for the latter two areas, we have already established tools for hiv and influenza a, but a large number of outstanding research questions remain. finally, the evbc will initiate and coordinate ring trials, undergraduate courses, graduate summer schools, and courses for principal investigators. interested? take a look here: evbc.uni-jena.de. viral vectors for gene therapy: the art of turning infectious agents into vehicles of therapeutics nanoparticles and their applications in cell and molecular biology oncolytic virotherapy for cancer treatment: challenges and solutions. the journal of gene medicine a strategy to estimate unknown viral diversity in mammals biodiversity and distribution of polar freshwater dna viruses global transport networks and infectious disease spread natural alternatives to in-feed antibiotics in pig production: can immunomodulators play a role? animal: an international journal of animal bioscience gapped blast and psi-blast: a new generation of protein database search programs big biological data: challenges and opportunities nucleotide sequence of bacteriophage phi x dna challenges in rna virus bioinformatics endogenous retroviruses in the human genome sequence real-time evolution of zika virus disease outbreak global initiative on sharing all influenza data-from vision to reality. euro surveillance: bulletin europeen sur les maladies transmissibles = european communicable disease bulletin improving hiv proteome annotation: new features of bioafrica hiv proteomics resource. database: the journal of biological databases and curation vipr: an open bioinformatics database and analysis resource for virology research key: cord- -u xryoo authors: mingorance, lidia; castro, victoria; Ávila-pérez, ginés; calvo, gema; rodriguez, maría josefa; carrascosa, josé l.; pérez-del-pulgar, sofía; forns, xavier; gastaminza, pablo title: host phosphatidic acid phosphatase lipin is rate limiting for functional hepatitis c virus replicase complex formation date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: u xryoo hepatitis c virus (hcv) infection constitutes a significant health burden worldwide, because it is a major etiologic agent of chronic liver disease, cirrhosis and hepatocellular carcinoma. hcv replication cycle is closely tied to lipid metabolism and infection by this virus causes profound changes in host lipid homeostasis. we focused our attention on a phosphatidate phosphate (pap) enzyme family (the lipin family), which mediate the conversion of phosphatidate to diacylglycerol in the cytoplasm, playing a key role in triglyceride biosynthesis and in phospholipid homeostasis. lipins may also translocate to the nucleus to act as transcriptional regulators of genes involved in lipid metabolism. the best-characterized member of this family is lipin , which cooperates with lipin to maintain glycerophospholipid homeostasis in the liver. lipin -deficient cell lines were generated by rnai to study the role of this protein in different steps of hcv replication cycle. using surrogate models that recapitulate different aspects of hcv infection, we concluded that lipin is rate limiting for the generation of functional replicase complexes, in a step downstream primary translation that leads to early hcv rna replication. infection studies in lipin -deficient cells overexpressing wild type or phosphatase-defective lipin proteins suggest that lipin phosphatase activity is required to support hcv infection. finally, ultrastructural and biochemical analyses in replication-independent models suggest that lipin may facilitate the generation of the membranous compartment that contains functional hcv replicase complexes. a a a a a millions of humans are chronically infected by hepatitis c virus (hcv) worldwide [ ] . chronic hcv infection is a major biomedical problem as it causes liver inflammation and fibrosis, which can lead to severe liver disease, such as cirrhosis and hepatocellular carcinoma [ , ] . there is no vaccine against hcv and, although blood-screening tests and other prophylactic measures have reduced the dissemination of this pathogen, a number of newly acquired infections still occur associated with risk behavior or with unknown origin [ , ] . however, chronic hcv infection can be successfully eradicated from chronically infected individuals through specific direct-acting antiviral (daa) combination therapies, virtually in all treated patients [ ] . since these specific treatments have only been in place recently, there are no sufficient clinical data on the long-term benefit of these treatments in relieving the severity of advanced liver disease [ , ] . hcv is a hepacivirus (flaviviridae) with a positive sense, single-strand rna genome that encodes a single open reading frame (orf) flanked by untranslated regions (utr), which are essential for viral polyprotein translation and viral genome replication. hcv orf is co-and post-translationally processed by cellular and viral proteases to produce ten major proteins. these have been functionally classified in a replication module, that includes the minimal viral components of the rna replicase (ns , ns a, ns b, ns a and ns b) and an assembly module, which comprises the major structural components of enveloped hcv virions, the capsid protein (core) and the envelope glycoprotein complex formed by e and e heterodimers; as well as the polypeptides p and ns , which are not structural components of virions but contribute to infectious particle assembly in a concerted action with the viral replicase [ ] . hcv utilizes key aspects of cellular lipid metabolism for essentially every aspect of the virus replication cycle and strongly interferes with host cell lipid homeostasis [ ] [ ] [ ] . in fact, chronic hcv patients display high rates of liver steatosis, severity of which inversely correlates with serum liver derived-lipoprotein [ ] . thus, although host immune response remains a major component in hcv pathogenesis, direct interference of hcv infection with hepatocyte lipid metabolism may contribute to overall disease progression [ ] . identified a limiting role of lipin pap activity in the generation of hcv replicase complexes. defective replicase assembly leads to strong inhibition of hcv propagation in lipin -deficient cells but not that of another (+) strand rna virus, indicating a specific role for lipin in hcv infection. given the relevance of lipin for lipid metabolism and its steatogenic potential, we set out to independently verify data from published differential transcriptomic profile studies that suggested that hcv infection may alter lpin mrna abundance in cell culture [ , ] . a specific and statistically significant lpin mrna induction (fig a) was observed in huh- cells after single cycle infection experiments [multiplicity of infection (moi) ] with a cell cultureadapted genotype a hcv (d ) variant [ ] at the peak of the infection ( and hours post-infection) and as compared with mock-infected cells (fig b) . lpin mrna induction was prevented when infected cells were treated with μm sofosbuvir (fig c) , an hcv rna polymerase inhibitor [ ] that reduced viral rna accumulation by more than two orders of magnitude (fig d) , indicating that active hcv replication is required to induce lpin mrna accumulation. lpin mrna has been shown to be upregulated by mechanisms that involve induction of reactive oxygen species (ros), as treatment of the cells with ros-scavenger molecule n-acetylcysteine (nac) is capable of preventing lpin mrna induction under glucose deprivation [ ] or during h o treatment [ ] . given that transcriptional activation of a subset of lipogenic genes during hcv infection is also prevented by addition of antioxidants [ ] , we sought to determine if lpin mrna induction by hcv infection is mediated by ros production and therefore dampened by nac treatment. lpin mrna induction was prevented in the presence of the antioxidant (fig e) , despite comparable hcv rna accumulation in mock-treated and nac-treated hcv-infected cells (fig f) , suggesting that virus replicationinduced ros production is required to induce lpin mrna accumulation. western-blot analysis confirmed that the observed transcriptional change leads to a correlative protein accumulation (fig g and h) ; reinforcing the notion that acute hcv infection alters lipin expression. in order to determine if lipin subcellular localization was altered during hcv infection, we performed confocal microscopy studies in control and hcv-infected huh- cells. lipin staining was observed as cytoplasmic punctated structures both in control and hcv-infected cells. to study if lipin signal colocalized with viral antigens, we performed double staining with antibodies against lipin and double-stranded rna (dsrna) or replicase subunits ns and ns a. none of the viral antigens strictly colocalized with lipin (pearson´s< . ) (s a fig). however, mander´s coefficients indicate that the majority of lipin overlapped with a small fraction of ns and ns a (s c fig). in contrast to lipin , lipin signal did not overlap with that of viral proteins ns and ns a (s b fig). our results indicate that no major lipin rearrangements are observed after hcv infection and that only a minor fraction of ns proteins colocalize with lipin . to study if lipin plays any role in hcv infection, lipin -deficient cells were generated by transducing human hepatoma (huh- ) cells with lentiviral vectors expressing shrnas targeting lpin mrna or a control vector expressing an irrelevant shrna. lipin expression silencing was verified by western-blot, typically days post-transduction (fig a) . a partial ( %; shlpin - ) and a more profound (> %; shlpin - ) reduction in lipin accumulation was observed after transduction with specific shrnas as compared with the control (fig b) . . rnas from mock-infected cells and hcv infected. impact of sofosbuvir ( μm; daa) (c, d) or n-acetylcysteine ( mm; nac) treatment (e, f) on hcv rna accumulation and lpin mrna levels. data are shown as average and standard deviation of two independent infection experiments performed in triplicate (n = ). g-protein samples of infected and control cells collected at hours post-infection were subjected to western-blot analysis to determine lipin and ns levels, using beta-actin as loading control h-quantitation of the relative lipin protein levels in mock and hcv-infected cells (n = ). statistical significance was determined using student´s t-test ( à p< . ; Ãà p< . ). https://doi.org/ . /journal.ppat. .g huh- cells were transduced with lentiviral vectors expressing control or lpin -specific shrnas. seven days after transduction samples of the cells were collected for western-blot analysis using antibodies against lipin and lipin and actin as loading control. parallel cultures were subjected to an mtt assay to determine their viability as described in the methods section. (a) representative western-blot showing cellular lipin and lipin protein expression levels and a loading control (actin). (b) average expression values for lipin and lipin as determined by western-blot and cell viability as determined by an mtt assay. data are shown as average and sd of six independent transduction experiments (n = ). (c) huh- cells were transduced with lentiviral vectors expressing control or lpin -specific shrnas. at day post-transduction, cells were infected at moi . with hcv d virus. samples of cell supernatants were collected at days and post-infection to determine the extracellular infectivity titer. average and sd of the infectivity titers at day and of two independent infections performed in triplicate (n = ). (d) huh- . cells were transduced with lentiviral vectors expressing control or lpin -specific shrnas. silenced cells were infected days after transduction at moi . with genotype a hcv (tncc) in the presence or absence of made ( μm; shcontrol+daa). intracellular hcv rna was determined by rt-qpcr hours post-infection. data are shown as average and sd of three experiments performed in triplicate (n = ). statistical significance was determined using student´s t-test ( à p< . ; Ãà p< . ). as expected, lipin and lipin expression is inversely correlated in lipin shrna-expressing cells (fig b) . the viability of lipin-deficient cells, as determined by mtt assay [ ] was comparable to that of control cells (fig b) . these results illustrate that lipin shrna-expressing cells respond homeostatically to functionally compensate partial (shlpin - ) and more pronounced (shlpin - ) loss of lipin (fig b) . control and lipin -deficient cells were infected with a genotype a d at moi . to study viral spread by determining extracellular infectivity titers at different times post infection. fig c shows limited propagation of this virus in lipin -deficient cells, with a statistically significant reduction of up to two (shlpin - ) and three orders of magnitude (shlpin - ) in viral titer between the control and lipin -deficient cell lines at day post-infection. these results indicate that lipin is required for efficient hcv propagation and that homeostatic lipin accumulation (fig b) is not sufficient to support efficient hcv infection. since important differences in the interaction of different hcv genotypes with cellular lipid metabolism have previously been described [ ] , we set out to determine if the observations made with a jfh -derived virus (genotype a) were extensive to other hcv genotypes. we performed low multiplicity infections (moi . ) with genotype a tncc virus strain in lipin -deficient huh- . cell lines, which are susceptible to infection by this recombinant virus [ ] . first, we verified that huh- . . also display a significant reduction in genotype a infection efficiency when lipin is silenced (s b fig) . in order to determine tncc infection efficiency, intracellular hcv rna accumulation was determined hours post-inoculation in control and lipin -deficient huh- . cells. viral rna detected under these conditions reflects the ability of genotype a to infect and replicate viral rna in the different cell lines, as treatment of control cells with an hcv polymerase inhibitor ´-c-methyladenosine ( made; μm) [ ] reduced viral rna content by two orders of magnitude ( fig d) . lipin silencing consistently and significantly reduced tncc replication by approximately -fold both in shlpin - and shlpin - cell lines (fig d) , indicating that lipin is also limiting for genotype a hcv infection. to determine the specificity of these observations, identical lipin -deficient and control huh- cultures were inoculated at moi . with a human alpha-coronavirus cov- e bearing a gfp reporter gene (hcov- e-gfp) [ ] . inoculation of control and lipin -deficient cells with this virus resulted in comparable progeny virus production, as determined by infectivity titration in cell supernatants hours post-infection (s fig) . these results suggest that lipin is not rate limiting for cov- e-gfp infection and that lipin expression is particularly limiting for hcv. to determine which aspects of the hcv replication cycle are limited by lipin silencing, single cycle infection experiments were conducted by inoculating control and lipin -deficient cell cultures at moi with genotype a d virus. infection efficiency was measured by titration of progeny virus infectivity present in the supernatant of infected cells and intracellular hcv rna accumulation at and hours post-infection. infection of lipin -deficient cells resulted in a significant reduction of progeny infectious virus production in shlpin - and shlpin - cells as compared with the titers observed in the supernatants of control cells ( fig a) , reinforcing the notion that lipin silencing interferes with hcv infection. reduced virus production is likely due to parallel reduction of intracellular hcv rna levels observed in lipin -deficient cells as compared with the control cell line (fig b) . this reduction was observed at all time points, except for that at hours, indicating that the size of the inoculum and initial virus adsorption is comparable among the different cell lines (fig b) . thus, lipin silencing suppresses hcv infection by interfering with a step of the hcv lifecycle preceding intracellular hcv rna accumulation. next, we set out to determine if lipin silencing has any impact on persistent hcv infections to verify if lipin is also limiting for late aspects of the virus lifecycle. persistently infected cells continuously replicate viral rna, express viral antigens and secrete infectious virions. thus, it is a valuable system to measure steady-state hcv rna replication as well as infectious particle assembly and secretion. persistently infected cultures were transduced with the lentiviral vectors described above to produce persistently infected, lipin -deficient cells (s fig) . shown as genome equivalents per microgram of total rna ge/μg). panels c, d-persistently infected cultures were generated by inoculation with jfh- virus at moi . . once cultures reached > % of hcv-positive cells, they were transduced with lentiviral vectors expressing control, hcv rna-targeting or lpin -specific shrnas. at day post-transduction, cells were split and samples of the cells and supernatants were collected hours later to determine infectious virus production rate by infectivity titration hcv (c) and rna levels by rt-qpcr (d). all data are shown as mean and sd of independent experiments performed in triplicate (n = ). statistical significance was determined using student´s t-test ( à p< . ; Ãà p< . ). analysis of extracellular infectivity titers revealed that infectivity titers in lipin -deficient and control cells were comparable, with the exception of a marginal reduction in shlpin - expressing cells, indicating that lipin silencing does not strongly interfere with infectious virus production ( fig c) . intracellular hcv rna levels in lipin -deficient cells were comparable to that of the control cells (fig d) , indicating that lipin expression is not rate limiting for hcv rna replication once infection has been established. taken together, the results shown above indicate that lipin is only limiting at early steps of hcv infection leading to viral rna accumulation. as an independent verification of the hypothesis that lipin is limiting for early aspects of hcv infection, we used a single cycle surrogate infection model based on the production of hcv virions bearing a defective reporter genome encapsidated by trans-complementation (hcv tcp ). hcv tcp are capable of producing abortive single-cycle infections, efficiency of which is proportional to the luciferase activity found in the target cells [ ] . as expected from the results shown in fig , infection of lipin -deficient cells with hcv tcp resulted in a % reduction in the reporter luciferase activity in shlpin - cells and % in shlpin - determined at hours post-infection, proportionally to the degree of silencing in these cells ( fig a and b ). these results underscore the role that lipin plays at early steps of hcv infection and indicate that either viral entry or a step leading to efficient hcv rna replication is impaired in these cells. lpin- mrna is alternatively spliced in human liver to produce isoforms α and β, which may differ in catalytic activity, subcellular localization and gene expression regulation [ ] . to determine the relative contribution of these isoforms to hcv infection, isoform-specific shrnas were generated and used to transduce huh- cells, as described above. lpin α-specific shrna (shlpin - ) reduced total lipin protein by %, while lpin β-specific shrna (shlpin - ) reduced total lipin expression by only % (fig a) . infection of control and lipin isoform-deficient cells with hcv tcp revealed that lipin α silencing resulted in a strong ( %) reduction in hcv infection while lipin β silencing resulted in a milder ( %) but significant reduction in hcv infection efficiency ( fig b) . these results indicate that both isoforms alpha and beta are limiting for hcv infection and suggest that the total amount of lipin present in the cell determines hcv infection efficiency. taken together, the results obtained with four different shrnas indicate that total lipin expression levels strongly correlate with hcv tcp infection efficiency (fig c) , underscoring a role for this host protein in early aspects of hcv infection. reduced hcv rna accumulation in a single cycle infection (fig ) as well as reduced hcv tcp infection (fig ) may be due to a defect in entry of incoming virions. hcv e e -pseudotyped retroviral vectors bearing a luciferase gene (hcv pp ) were used to measure viral entry because they constitute a sound model to study viral adsorption, receptor-mediated internalization and e e -mediated fusion in endosomes [ ] . to assess the specificity of these observations, parallel cultures were inoculated with vsv-g pseudotyped retroviral vectors (vsv pp ). control and lipin -deficient cell lines were infected with hcv pp (genotype a; jfh- strain) and vsvpp. as a positive control of inhibition of hcv entry, we used hydroxyzine ( μm), which efficiently blocks hcv infection by interfering with viral entry [ ] . as expected, hydroxyzine selectively inhibited hcv pp infection, as shown by reduced luciferase levels hours postinoculation only in hcv pp -infected cells ( fig a) . interestingly, lipin -deficient cells (shlpin - and shlpin - ) were fully susceptible to hcv pp and vsv pp infection, as comparable luciferase activity levels were found in all cell lines hours post-inoculation ( fig a) . these results indicate that lipin is not rate limiting for receptor binding, particle internalization or e e -mediated endosomal fusion, which are steps recapitulated in this model [ ] . based on the data presented thus far, we hypothesized that lipin is limiting for a step in the hcv lifecycle downstream of hcv entry, leading to hcv rna accumulation. to verify the hypothesis that lipin silencing causes strong reduction in initial hcv rna accumulation by interfering with a step downstream of viral entry, we bypassed this step of the viral replication cycle by transfecting a subgenomic hcv rna replicon bearing a reporter luciferase gene into control and lipin -deficient cells. first, we evaluated hcv-ires driven primary translation of incoming genomes by transfection of a replication-deficient mutant replicon that bears an inactivation mutation in the catalytic site of ns b rna polymerase [ ] . luciferase activity measured at hours post-transfection was not reduced in any of the cell lines, indicating that transfection efficiency and hcv ires-dependent primary translation was not significantly affected by lipin silencing (fig b) . a significant increase in renilla luciferase activity was observed in shlpin - cells both when transfecting a replicon ( fig b) or a plasmid expressing renilla luciferase under a minimal rna polymerase ii promoter (s a fig), suggesting that the increase in luciferase activity observed in these cells is not related with hcv. hcv rna replication was evaluated by measuring accumulation of a reporter luciferase gene at and hours post-transfection of a replication competent hcv subgenomic replicon. under these experimental conditions luciferase accumulation at hours also represents hcv ires-driven primary translation of the input rna, while reporter luciferase activity at hours depends on effective hcv rna replication. in contrast to primary translation, hcv rna replication inferred by luciferase activity at hours was strongly reduced in lipin -deficient cells ( % in shlpin - and % in shlpin - cells) (fig c) , indicating that initiation of hcv replication is dependent on normal lipin expression. this reduction was not due to a non-specific defect in luciferase expression, as co-transfection of a plasmid expressing renilla luciferase lead to comparable luciferase accumulation in all cell lines, discarding the possibility that death of transfected cells or other spurious effects are responsible for the reduced luciferase activity accumulation (s a fig) . similar experiments were conducted in atg b-deficient cells, as this host factor was shown to be limiting for primary hcv translation [ ] . our studies confirmed that, while partial atg b silencing (s b fig overall, these data indicate that hcv rna replication is not initiated efficiently in lipin -deficient cells and that blockade occurs at a step downstream translation of incoming hcv genomes. in order to determine if any of the known functions ascribed to lipin is required for hcv infection, we tested the ability to restore hcv infection susceptibility of silencing-resistant wild-type (wt) or mutant lipin versions bearing a mutation in the catalytic site responsible for its phosphatase activity (dxdxt) and a mutant in the lxxil motif, which is inactive both for transcriptional activation as well as for phosphatase activity [ ] . control and lipin -deficient cells were transfected with wt lipin beta cdna as well as with dxdxt and lxxil mutants. comparable overexpression levels of the wt and mutant proteins was obtained in each cell line, although overexpressed lipin levels were consistently higher in lipin -deficient cells (fig a) . cells were subsequently inoculated with hcv d at moi and relative infection efficiency was calculated by determining extracellular infectivity titers hours post-infection. overexpression of wt lipin did not significantly alter susceptibility to hcv infection in control cells, although we observed a small but consistent reduction in extracellular infectivity titers when overexpressing wt and mutant lipin constructs (s a fig using relative infection efficiency as readout of this set of experiments, we could clearly observe a statistically significant increase ( -fold) in the relative infection susceptibility in cells overexpressing wt lipin cdna as compared with mock-transfected cells or cells expressing similar or higher levels of the mutants (fig a) , suggesting that wt lipin modestly, though significantly, rescues hcv infection while phosphatase (dxdxt) and transcriptional coactivation (lxxil) mutants do not ( fig b) . these results suggest that lipin transcriptional coactivation capacity is not sufficient to support hcv infection while lipin phosphatase activity is essential. however, given that lxxil mutant is deficient both in transcriptional co-activation and pap activity [ ], we cannot determine if transcriptional co-activation by lipin is also required to support hcv infection. the data described above suggest a role for lipin phosphatase activity in a step of hcv replication cycle between translation of the viral genome and formation of functional replicase complexes. data regarding primary translation where inferred from a surrogate model of translation based on a reporter luciferase gene ( fig b) . to address if indeed viral polyprotein is properly processed and inserted into detergent-resistant microdomains to form the characteristic membranous ultrastructures bearing the viral replicase, we used a replication-independent surrogate model of polyprotein expression. this system is based on a vector encoding the portion of the viral polyprotein corresponding to the replicase (ns -ns b) under the transcriptional control of the t polymerase and the translational control of encephalomyocarditis virus (emcv) ires (ptm-ns / b) [ ] . replication-independent polyprotein overexpression control and silenced cells were inoculated with hcv e e (hcv pp ) or vsv-g-pseudotyped retroviral vectors (vsv pp ) days post-transduction. control cells treated with hydroxyzine ( μm) were used as positive inhibition control. luciferase activity was determined in the different cell lines at hours post-inoculation. hdx, hydroxyzine pamoate ( μm). panels b, c-control and lipin -deficient cells were transfected with a replication-deficient mutant (b) or replication competent subgenomic hcv replicon bearing a luciferase gene (c). luciferase activity was determined in the different cell lines at hours post-transfection for both replicons and hours post-transfection for the replication-competent replicon rna. data are expressed as average and sd of three independent experiments performed in triplicate (n = ). statistical significance was determined using student´s t-test ( à p< . ; Ãà p< . ). https://doi.org/ . /journal.ppat. .g systems enable assessment of polyprotein processing as well as studying the formation of virus-derived membranous structures [ , ] . control and lipin -deficient cells were infected with a recombinant vaccinia virus expressing t rna polymerase (vact ) and subsequently transfected with the plasmid ptm-ns / b to enable viral replicase expression. sixteen hours post-transfection, cells were processed for lipin is required for hcv replicase formation western-blot using anti-ns . accumulation of ns is comparable in control and both lipin deficient cells, underscoring the notion that lipin is not limiting for polyprotein translation and processing (fig a) . similarly, ns and ns a expression and subcellular distribution was similar in all cell lines (fig b) . these results suggest that there are no major differences in accumulation of viral proteins in lipin -deficient cells and that a step downstream is affected in these cells. transmission electron microscopy (tem) of ultrathin cell sections of cells expressing hcv replicase components shows the expected accumulation of a mixture of characteristic doublemembrane vesicles (dmv) as well as multiple membrane vesicles (mmv) (fig d, e and f) that were not found in mock-transfected cells (fig c) , as reported in previous studies using similar systems [ ] . individual vesicle diameter displays heterogeneous size distribution in which the predominant population is distributed between - nm (median nm; average ± sd: ± nm, n = ) with larger vesicles being less predominant (fig g and s fig) . this size distribution is compatible with that observed similar replication-deficient systems and during hcv infection. treatment of these cells with nm daclatasvir (dctv), resulted in a strong reduction in the number of vesicles per section area (s b fig) , without significantly altering the size distribution of the remaining vesicles (fig g) , as reported by berger et al. [ ] . similarly, the diameter distribution of the vesicles found in lipin -deficient cells was comparable to that in control cells ( fig g) . however, hcv-induced structures were significantly less abundant hours post-transfection in lipin -deficient cells than in controls cells (s c fig) . this reduced abundance is illustrated by a significant reduction in the fraction of cells displaying vesicular structures in lipin -deficient cell cultures (fig h) despite comparable transfection efficiency and viral protein expression levels, indicating that lipin may be required in a critical step leading to formation of the hcv-induced vesicular compartment. to validate the tem results independently, we set out to establish a biochemical assay to evaluate replication-independent replicase complex formation. one of the characteristics of the hcv replicase complexes is that they are located in detergent-resistant membranes (drm) [ , ] . in this sense, ns proteins are associated with replicase complexes that co-sediment with drm markers such as caveolin- or sigma- receptor (sigmar ) in low-density fractions in isopycnic gradient ultracentrifugation experiments [ , ] . control and lipin deficient cells were infected with vact and subsequently transfected with limiting doses of the plasmid ptm-ns / b [ ] . parallel samples were treated with dctv ( nm). sixteen hours post-transfection, cell lysates were generated and subjected to equilibrium ultracentrifugation in - % sucrose gradients. gradient fractions were collected and subjected to western-blot analysis to determine the impact of lipin silencing on ns , sigmar and actin sedimentation profiles. fig a shows how, as previously shown for replicon and jfh -infected cells, ns can be detected in drm fractions (fractions - ), as determined by the presence of sigmar in those fractions (fractions - ; s a fig), although in this experimental system, unlike during viral infection [ ] , most ns co-sediments together with solubilized proteins, as shown for actin (fractions - ; s a fig) . drm-associated ns is reduced in dctv-treated and lipin -deficient cells, while total ns expression remains unchanged (fig a) . four independent experiments were performed in which relative-drm associated ns , normalized to that found in solubilized fractions, was calculated for each experimental condition (fig b) . lipin -deficient cells display a consistent and statistically significant reduction in the drmassociated, but not total ns abundance (fig a and b; shlpin - and shlpin - ) , similar to that observed in control cells in the presence of dctv (figs a and b; shcontrol+dctv) , consistent with the tem data (figs and s ) and the notion that ns in drm fractions may reflect the abundance of replicase complexes formed in these cells. this reduction is not due to an overall reduction in cellular drm abundance in lipin -deficient cells, as lipin -deficient cells display similar sigmar distribution pattern as the control cells (s fig). overall, tem data and drm floatation assays strongly suggest that lipin is rate limiting for the generation of replicase complexes from fully processed polyprotein subunits. hepatitis c virus replication cycle is tightly linked to host cell lipid metabolism and interference with cellular lipid homeostasis contributes to viral pathogenesis [ ] . one of the most evident consequences of this interference is the high prevalence of liver steatosis among chronically infected patients [ , ] . this clinical manifestation of the infection has been linked to, among others, chronic er stress, mitochondrial dysfunction and metabolite depletion induced by hcv infection, which result in the activation of persistent homeostatic adaptation of the cellular lipid metabolism to permit cell survival, at the cost of pathogenic metabolic alterations [ , [ ] [ ] [ ] [ ] . among the different regulatory networks that have been shown to be stimulated during hcv infection, pparα [ ] , pgc- α [ ] , hif- [ ] and srebp [ , ] have also been shown to regulate transcription of lpin mrna [ , , , ] . thus, it is likely that stimulation of one or several of these regulatory networks by hcv infection results in the lpin mrna transcriptional activation observed in this ( fig a) and other studies [ , ] . importantly, prevention of lpin mrna accumulation with nac ( fig e) did not significantly interfere with hcv rna replication (fig f) , suggesting that enhanced lpin mrna accumulation is not required for efficient hcv infection. we favor the hypothesis that ros induced by hcv protein accumulation actively participates in lpin induction, as treatment with the antioxidant nac prevented lpin mrna accumulation, similar to what has been shown for other srebp-regulated genes during hcv infection [ ] . accumulation of lpin mrna during hcv infection results in concomitant protein accumulation (fig g and h) . however, post-translational mechanisms such as phosphorylation, acetylation or sumoylation regulate lipin protein stability, membrane association as well as subcellular localization thus influencing the activity of lipin as pa-phosphatase and as transcriptional coactivator [ ] . hence, it is difficult to predict the implications of lipin protein accumulation during hcv infection. thus, future studies on the interference of hcv infection with cellular lipin functions will be required to determine its role in hcv-related pathogenesis, particularly in its contribution to steatosis. lipin is required for hcv replicase formation the data presented in this study provide evidence that lipin is rate limiting for hcv infection at an early step of the infection leading to formation of membranous hcv replicase complexes, downstream of viral polyprotein expression and processing. we provide evidence for reduced accumulation of viral rna during single cycle infection experiments (fig d) , that is reminiscent of a faulty initiation of viral replication, as suggested by reduced replication of a transfected subgenomic replicon in lipin -deficient cells (fig d) . data obtained in replication-independent polyprotein expression models suggest that generation of the membranous compartment that contains functional replicase complexes is severely limited in lipin -deficient cells, as suggested by a significant reduction of the fraction of cells where these structures could be visualized by tem (figs and s ). this hypothesis is further supported by a significant reduction in drm-associated ns proteins in lipin -deficient cells (fig ) , which may reflect limitations in the association of viral replicase subunits with cholesterol and sphingolipid-rich membranes in lipin -deficient cells [ , , ] . four different shrnas targeting lpin mrna decrease susceptibility to hcv infection proportionally to their ability to reduce total lipin protein accumulation (fig ) . these results, together with the cdna rescue experiments (fig ) , strongly reduce the possibility of observing rnai-associated off-target phenomena. interestingly, homeostatic accumulation of lipin protein in lipin -deficient cells (fig a and b) is not sufficient to compensate for lipin loss to support efficient hcv infection. the notion that, despite being capable of mutually compensating basic liver functions [ , ], lipin and lipin play non-redundant functions in the liver has previously been proposed [ , , ] . lipin is tightly regulated at many different levels and its activity accommodates pap activity in response to different physiological situations such as fasting and insulin signaling [ ] . compelling evidence indicates that, while lipin and lipin cooperate to maintain liver lipid homeostasis, the two proteins differ in many aspects. for instance, lipin is transcriptionally induced by pgc- α and it is also an inducible amplifier of this transcriptional network [ ], whereas lipin is not [ ] . lipin is sumoylated and sumoylation regulates its nuclear localization and function, whereas lipin sumoylation could not be demonstrated, despite the presence of a canonical sumoylation motif in its primary sequence [ ] . lipin enzymatic activity is blocked by mammalian target of rapamycin (mtor)-dependent phosphorylation in response to different metabolic stimuli [ , ], whereas lipin is constitutively active even when phosphorylated [ ] . thus, lipin is considered more as a constitutive phosphatidic acid phosphatase with lower specific activity than lipin [ ] . in addition to these differential regulatory networks, it has been shown that in vitro pap activity of purified lipin and lipin is differentially influenced by the composition of the substrates (liposomes and lipid-detergent micelles) as well as the ph at which the assay is performed [ ] . this differential lipid substrate recognition may be reminiscent of the different preferential association with membranes of different subcellular compartments (s fig) [ , ] . these differences suggest that, while lipin and lipin may share some common features, they are not functionally interchangeable, particularly not in the case of hcv infection [ ] . our data support the notion that lipin silencing has a strong impact on hcv infection without affecting basic cellular functions (fig b) or significantly interfering with infection by an unrelated virus (s fig). despite great efforts and different overexpression systems, functional rescue of lipin functions by wt lipin cdna overexpression in lipin -deficient cells only lead to a small but consistent rescue of virus infection efficiency, which was only observed when overexpressing wt lipin (figs and s ) . given the multiple transcriptional, post-transcriptional and post-translational regulation levels existing for lipin expression, it is conceivable that only a fraction of the overexpressed lipin is fully competent to sustain hcv infection. moreover, high overexpression levels could only be achieved in lipin -deficient cells (fig a) , underscoring the notion that intracellular lipin levels are tightly regulated by the host. nevertheless, overexpression of a mutant lipin lacking phosphatase activity (dxdxt) or a mutant inactive as transcriptional coactivator (lxxil) were not capable of enhancing hcv infection in lipin -deficient cells as compared with the wt lipin ( fig b) . these data reveal that lipin phosphatase activity is required for lipin to support hcv infection and suggest that, while transcriptional co-activation by lipin may be important, this function is not sufficient to support hcv replication. lipins are important enzymes in the main pathway for de novo phospholipid biosynthesis by providing dag derived from the glycerol- -phosphate pathway to produce pc and pe through the kennedy pathway [ ] . production of membranous replicase compartments likely requires de novo synthesis of pc and/or pe, which are major components of biological membranes that depend on dag biosynthesis [ ] . in fact, local pc biosynthesis is required for efficient replication of (+) rna viruses and certain pc species accumulate in hcv-infected cells [ , ] . although increased lipin accumulation may be sufficient to compensate lipin silencing at the whole-cell level [ ], it is possible that acute, local demand of de novo synthesized phospholipids is required at defined suborganellar compartments during early steps of hcv infection and that lipin -deficiency shortens or alters the availability of different membrane components, demand that may not be satisfied by lipin , given the differential regulation [ ] and subcellular localization of these two proteins (s fig). remarkably, deletion of the yeast lipin homologs pah /smp (s. cerevisae) or ned (s. pombe) gene, results in deregulated proliferation of the er and nuclear envelope membranes [ , ] , with concomitant enhancement in (+) rna virus replication [ , ] . the membranous alterations and elevation of total phospholipid content observed in pah -deficient yeast have been ascribed to transcriptional activation of pah -independent alternative phospholipid biosynthetic programs due to pa accumulation [ , ] . our data in mammalian cells are more compatible with a shortage of phospholipid production, which may be at the basis of the reduced abundance viral membranous structure (figs and ) . this opposite outcomes of infection may derive from the fact that yeast use mainly pa (lipin pap substrate) as a precursor for pc biosynthesis, while mammals mainly use dag (lipin pap product) as precursor for pc biosynthesis through the kennedy pathway [ , [ ] [ ] [ ] . moreover, in contrast to yeast and lower eukaryotes, which express only one lipin gene, three different lipin genes coordinate glycerolipid homeostasis in mammals [ ] . thus, interfering with expression of one of the members of the family may not be sufficient to observe the same effects observed when deleting pah , as transcriptional and posttranslational homeostatic compensations are in place in mammals, particularly between lipin and lipin in liver tissue [ , ] . in this sense, deletion of either lipin or lipin in mouse models results in a relatively balanced liver phospholipid content while simultaneous deletion of both lipins is embryonically lethal [ ] . accordingly, only minor alterations of er membranes [ ] and no significant alterations in total pc levels [ ] have been reported in lipin -deficient mouse liver. given the fact that hcov- e is fully capable of replicating in these cells (s fig), it is unlikely that a general disruption of de novo phospholipid biosynthesis occurs in lipin -deficient cells, particularly since hcov- e infection also induces profound er membrane rearrangements required for replication [ ] , some of which are structurally similar to dmvs observed during hcv infection [ ] . thus, we favor the hypothesis that a subcellular pool of glycerophospholipids is managed by lipin in huh- cells and that lipin silencing perturbs local levels of pa and dag, limiting local availability of precursors of structural components of virus-induced membranes. alternatively, lipin deficiency may alter local amounts of important signaling molecules, in particular, that of its substrate (pa) or its product (dag). deregulation of the local pa and dag pools may cause important alterations for the host cell, as both metabolites are potent chemical messengers that regulate different aspects of cellular homeostasis [ ] [ ] [ ] . regarding pa conversion into dag by lipins, it has been shown that pah (yeast lipin homolog) phosphatase activity is critical for transforming local pools of pa into dag at the er membrane to facilitate membrane fusion events mediated by snare complexes [ , ] . mammalian lipin phosphatase activity is also critical for transforming local pools of pa that accumulate at the surface of mitochondria to promote mitochondrial fission [ ] or at the surface of endolysosomes to facilitate autophagy [ ] . thus, lipin and probably other members of the lipin family modulate different aspects of intracellular membrane signaling. given that the function of host factors known to be involved in functional hcv replicase biogenesis, like vapa, vapb and osbp [ ] are indirectly regulated by local pa/dag pools [ ] , it is tempting to propose that lipin silencing interferes with the function of one or several of these, or other yet uncharacterized cellular factors. in contrast to what has been reported for other host factors required for hcv replicase complex formation [ ] , we did not find evidence of lipin protein relocalization during hcv infection (s fig). thus, determining the precise mechanism by which lipin regulates hcv replicase formation is challenging, as association of lipin with different cell membranes is transient and highly regulated by posttranslational modifications [ ] . moreover, some of the known lipin cellular functions may be compensated by other lipins, particularly lipin in the liver. nevertheless, our data clearly indicate that lipin participates at early stages of hcv replication and that the aforementioned homeostatic compensations by other lipins in regards to cellular metabolism may constitute an advantage when considering lipin as a host target for anti-hcv therapy. hcv antiviral compounds ´-c-methyladenosine ( made), sofosbuvir and daclatasvir were obtained from boc sciences (ny, usa), selleckchem (texas, usa) and medchem express (new jersey, usa) respectively and dissolved in dmso to obtain mm stock solutions. nacetylcysteine (nac) and puromycin were obtained from sigma-aldrich (missouri, usa), dissolved in water to a final concentration of . m and mg/ml respectively. hydroxyzine pamoate was purchased from sigma-aldrich (missouri, usa) and dissolved in dmso to a final mm concentration. human hepatoma huh- and derived subclones huh- . , huh- . . (clone ) have been described [ ] [ ] [ ] and were kindly provided by dr. chisari (tsri-la jolla, ca). hek- t cells [ ] were kindly provided by dr. ortin (cnb-madrid, spain). cell cultures were maintained subconfluent in dulbecco´s modified eagle´s medium (gibco) supplemented with mm hepes (gibco), u/ml penicillin/streptomycin (gibco), μm non-essential amino acids (gibco) and % fetal bovine serum (sigma-aldrich). genotype a (jfh- strain), d adapted virus (d ) and genotype a (tncc) virus have been described elsewhere [ , , ] . tncc plasmid was kindly provided by dr. jens bukh (huidovre hospital; copenhagen, denmark). recombinant viruses were generated by electroporation of an in vitro transcribed rna as described previously [ ] , using clone cells (jfh- and d ) virus and huh- . cells for tncc. supernatants containing the infectious virus were used to inoculate naive huh- . . clone cells at low multiplicity of infection (moi . ffu/cell) to prepare working virus stocks. for tncc infections, supernatants of electroporated cells were directly used. lentivirus production. lentiviral vectors shrna expression were produced by co-transfection of plasmids pmdl-rre, pmd g and prsv-rev (a gift from didier trono-addgene plasmids # ; # ; # ), together with the genomic plasmid (mission plko-puro; sigma-aldrich) into hek- t cells [ ] . selected shrna target sequences are: cgagagaa agtggttgacata (shlpin - ); (cctcagacagaaatgcagttt) shlpin - ; cact cccagtccttccggttc (shlpin - ); cctgttccatccttcggaaag (shlpin - ). plasmids encoding a atg b shrna (atg b ) [ ] , non-targeting shrna (shcontrol) as well as an shrna targeting hcv ires (shhcv)were previously described [ ] . a lentiviral vector plasmid encoding lipin -alpha cdna was purchased from genecopeia (cat nbr: ex-t -lv ). to generate lipin beta isoform, exon sequence was inserted using neb-q mutagenesis kit (neb). similarly, silent mutations were introduced in shlpin - target sequence to obtain wt lipin beta protein expression from a cdna resistant to silencing. mutations in the conserved motifs dxdxt (didgt>eidgt) and lxxil (lghil>lghff) were also introduced in lipin beta cdna using neb-q kit, based on previously described mutations [ , ] . the sequence of the entire lpin cdnas was determined by sanger sequencing before use, to assess the introduction of only the desired mutations. coronavirus e-gfp. recombinant human coronavirus e-gfp [ ] was kindly provided by dr. volker thiel (university of bern, switzerland). virus was propagated by infection (moi . ) in huh- cells. supernatants were collected at different time points and titrated by end-point dilution and immunofluorescence microscopy. lentiviral vectors expressing control and lpin -specific shrnas were used to inoculate huh- cells. twenty-four hours later, cells were subjected to selection with . μg/ml of puromycin to assess the lowest lentivirus dose capable of conferring puromycin resistance to % of the cell population. selected cell populations were subsequently cultured in the presence of puromycin until lpin silencing was ascertained by western-blot using anti-lipin antibodies, typically at day - post lentiviral transduction, time at which all experiments were performed in the absence of puromycin. before execution of all the experiments shown in this study, lipin expression was assessed by western-blot. cell viability was determined by a thiazolyl blue tetrazolium blue (mtt) formazan formation assay [ ] . control and lipin -deficient cell lines ( . cells/well) were plated onto -well plates and were inoculated with d virus at a moi ffu/cell. samples of the cells and supernatants were collected , and hours post-infection. for multiple cycle infection experiments (moi . ), samples of the supernatants were collected at day , and post-inoculation. cells were split : in the multiple cycle infection experiments at days and to maintain the cultures subconfluent. extracellular infectivity titers were determined by endpoint dilution and infection foci counting as previously described [ ] . intracellular hcv rna was determined by reverse transcription and quantitative pcr (rt-qpcr) as previously described [ ] . total protein samples were prepared in laemmli buffer and separated using polyacrylamide denaturing gel electrophoresis (sds-page). proteins were subsequently transferred onto pvdf membranes and incubated with % milk (lipins) or % bsa in pbs- . % tween for one hour at room temperature (rt). primary antibodies against lipin (clone b- ; santa cruz), lipin (h- ; santa cruz), ns (clone e ; biofront), beta-actin (ab ; abcam) and tubulin (clone aa ; sigma-aldrich) were diluted in pbs- . % tween and incubated for hour (four hours for lipins) at rt. membranes were subsequently washed for minutes with pbs- . % tween three times. horseradish peroxidase-conjugated secondary antibodies were incubated for hour at room temperature in % milk-pbs- . % tween and subsequently washed three times for minutes at room temperature. protein bands were detected using enhanced chemoluminescence reactions and exposure to photographic films. specific bands were quantitated using the imagej software [ ] on non-saturated, scanned films. confocal microscopy was performed with a leica tcs sp laser scanning system (leica microsystems). images of × pixels at eight bit gray scale depth were acquired sequentially every . - . μm through a x/ . n.a. immersion oil lens, employing las af v . . software (leica microsystems). colocalization indexes were calculated using jacop plugin for image j [ ] from a minimum of regions of interest (roi). images were processed using imagej, were medians of pixel were obtained for the different channels, only for illustration, not for analysis. color levels, brightness and contrast were manipulated for illustration using technical and biological controls as reference. total rna extraction was performed using the gtc extraction method [ ] . purified rna ( - ng) was subjected to rt-qpcr using random hexamers and a reverse transcription kit (applied biosystems). quantitative pcr was performed using x reaction buffer from (applied biosystems) and specific oligonucleotides as previously described [ , ] . standard curves were prepared by serial dilution of a known copy number of the corresponding amplicon cloned in a plasmid vector. control and lipin -deficient huh- . cells were inoculated with tncc virus (moi . ). due to the relatively low propagation levels of the tncc virus in this experimental setup, parallel cultures were infected and treated with ´-c-methyladenosine ( made; μm) to determine the levels of non-replicative, background hcv rna. cells were incubated for hours at ˚c, time after which samples of the cells were collected to determine intracellular hcv rna levels by rt-qpcr. to establish persistently infected cell cultures huh- cells were inoculated at moi . with jfh- hcv strain as previously described [ ] . cell cultures were maintained subconfluent for two weeks, time after which infection rates reach nearly % of the cells, as assessed by immunofluorescence microscopy. at this point cells were split and transduced with the corresponding lentiviral vectors in order to generate lipin -deficient cell cultures as well as control cell lines. once silencing had been verified by western-blot, typically at day - post-transduction, cells were split and samples of the cells and supernatants were collected hours later to determine intracellular hcv rna levels by rt-qpcr and extracellular infectivity titers by end-point dilution and immunofluorescence microscopy. infectious, spread-deficient hcv particles produced by trans-complementation (hcv tcp ) have previously been described [ ] . briefly, huh- . . clone cells expressing core-e and e -ns regions from jfh- by lentiviral transduction, were electroporated with a jfh- subgenomic dicistronic replicon bearing a firefly luciferase gene with reagents kindly provided by dr. ralf bartenschlager (u. of heidelberg). supernatants containing hcv tcp were collected , and hours post-electroporation, pooled and assayed for viral infectivity. hcv tcp infection efficiency was determined by inoculating naïve huh- cells with the electroporation supernatants and measuring luciferase activity hours post-infection using a commercially available kit (promega). retroviral particle production pseudotyped with different viral envelopes has previously been described [ , ] with the materials kindly provided by dr. f. l. cosset (inserm, lyon). control and lipin-deficient cell lines were inoculated with hcv pp and vsv pp and incubated for hours, time at which total cell lysates were assayed for luciferase activity using a commercially available kit (promega). a selective hcv entry inhibitor, hydroxyzine pamoate (hdx) from sigma-aldrich (missouri, usa), was used as positive control of inhibition [ ] . a plasmid containing the sequence corresponding to a subgenomic jfh- replicon bearing a firefly luciferase reporter gene was kindly provided by dr. ralf bartenschlager (u. of heidelberg) [ ] . after digestion with the restriction enzyme mlui, the linearized plasmid was transcribed in vitro using a commercial kit (megascript t ; ambion-paisley, uk). the resulting products were digested with dnase and precipitated with licl. pelleted rna was washed with % and % ethanol, and resuspended in nuclease-free water. in vitro transcribed rna was transfected into the different cell lines together with a plasmid expressing renilla luciferase under a minimal promoter (prl-null; clontech-california, usa) using lipofectamine and the manufacturer´s recommendations (life technologies-california, usa). firefly and renilla luciferase activities were measured in the sample using a commercial kit (dual luciferase assay system; promega-wisconsin, usa) at different times post-transfection. lipin -deficient cells were generated by lentiviral transduction of shlpin - shrna. at day post-transduction, control and lipin -deficient cell populations ( x cells/m well) were transfected in suspension using lipofectamine with plasmids ( ng/m well) expressing wt lipin beta isoform shlpin _ -resistant cdna or dxdxt or lxxil motif mutants [ ] . transfected cell cultures were incubated for hours and subsequently inoculated at moi with d virus. infection efficiency was determined by measuring extracellular infectivity titers hours post-infection. parallel cultures were used to determine relative wt and mutant protein expression efficiency by western-blot. infectivity titers were measured as described above. the relative impact of cdna expression was estimated by determining the ratio between the infectivity found in lipin -deficient cells and the control cells transfected with the same plasmid. in order to average experiments with different raw infection efficiency, all the experiments were referenced to the ratio in the mock-transfected cells. control and lipin -deficient huh- cells were inoculated with cov- e (moi . ) for hours at ˚c. cells were washed twice with warm pbs and replenished with dmem- % fcs. extracellular infectivity titers were determined hours post-infection by end-point dilution and fluorescence microscopy in huh- cells. huh- cells were inoculated at moi with a recombinant vaccinia virus expressing the t phage rna polymerase (vact ) [ ] . two hours later, cells were transfected with the plasmid ptm-ns / b [ , ] (kindly provided by dr. lohmann; u. of heidelberg) and lipofectamine (thermofisher-massachussets, usa) following the manufacturer´s recommendations in terms of total dna per well (typically μg per mm dishes with . x cells/well) and % of the recommended lipofectamine:dna ratio. transfected cells were cultured in the presence of the dna replication inhibitor cytosine β-d-arabinofuranoside (arac; sigma-aldrich) for hours to prevent vact replication [ ] . when indicated, media was also supplemented with nm daclatasvir (dctv). total cell extracts were used to determine viral protein accumulation by western-blot using anti-ns antibody (clone e ; biofront) and β-actin (abcam; ab ) as loading control. for ultrastructural electron microscopy studies, control and lipin -deficient cells expressing ns - b polyprotein (see above) were cultured on glass coverslips and fixed in situ after polyprotein expression with a mixture of % paraformaldehyde (taab) and . % glutaraldehyde (taab) ( h at room temperature), post-fixed with % osmium tetroxide in pbs ( min), treated with % aqueous uranyl acetate ( min), dehydrated with increasing quantities of ethanol and embedded in epoxy resin (taab). ultrathin, -nm-thick sections were cut in parallel to the monolayer, transferred to formvar-coated em buttonhole grids and stained with aqueous uranyl acetate ( min) and lead citrate ( min). sections were visualized on a jeol jem exii electron microscope (operating at kv). quantitation of hcv-induced structures was performed as follows. to quantitate the differences in total vesicle abundance, tem sections were visually inspected under the microscope for the presence/absence of vesicular structures. the number of positive cells and total number of cells were inserted in a x contingency table to determine the statistical significance of the differences between control and lipin -deficient cells using two-tailed fisher´s exact test or two-tailed chi square test. in addition, we determined the frequency of hcvinduced vesicles in dctv-treated control cells by dividing the number of structures per inspected area and calculating the average and sd of the frequencies found in the different images. the diameters of individual vesicles were determined manually using size-calibrated images and image j software. lipin -deficient and control cells ( . x cells) were infected with vact virus (moi ) and transfected with limiting doses of ptm-ns / b plasmid (typically ng/ well), as higher plasmid doses may difficult observing the reported differences. cells were lysed by adding μl of tne ( mm tris-hcl ph . , nacl mm and edta mm) buffer containing . % triton x- and protease inhibitors (complete; roche-basel, sw). lysates were incubated for minutes on ice before clearing them by -minute centrifugation at , r.p.m. clear supernatants were mixed : with % sucrose tne solution. this mixture was applied on top of a % sucrose-tne cushion and was overlaid with % and % sucrose-tne until completing the discontinuous gradient. gradients were centrifuged for hours at , x g. fourteen fractions were collected from the top and analyzed by sds-page and western-blot using antibodies against ns (clone e ; biofront), sigmar (s- ; santa-cruz), caveolin- (epitomics; - ) and beta actin as described previously [ ] . ns signal was quantitated using imagej software and the fraction of drm-associated ns was determined as the ratio of ns signal in fractions , and to the total ns signal in the gradient. global epidemiology and burden of hcv infection and hcv-related disease immunobiology and pathogenesis of viral hepatitis liver injury and disease pathogenesis in chronic hepatitis c from non-a, non-b hepatitis to hepatitis c virus cure reversion of disease manifestations after hcv eradication risk for hepatocellular carcinoma after hepatitis c virus antiviral therapy with direct-acting antivirals: case closed? the molecular and structural basis of advanced antiviral therapy for hepatitis c virus infection unique ties between hepatitis c virus replication and intracellular lipids hepatitis c virus rna replication and assembly: living on the fat of the land the ins and outs of hepatitis c virus entry and assembly liver international: official journal of the international association for the study of the liver identification of the hepatitis c virus rna replication complex in huh- cells harboring subgenomic replicons sequential biogenesis of host cell membrane rearrangements induced by hepatitis c virus infection. cellular and molecular life sciences: cmls three-dimensional architecture and biogenesis of membrane structures associated with hepatitis c virus replication structural changes in cells imaged by soft x-ray cryo-tomography during hepatitis c virus infection hepatitis c virus rna replication occurs on a detergentresistant membrane that cofractionates with caveolin- characterization of the hepatitis c virus rna replication complex associated with lipid rafts replication of hepatitis c virus rna occurs in a membrane-bound replication complex containing nonstructural viral proteins and rna sigma- receptor regulates early steps of viral rna replication at the onset of hepatitis c virus infection hepatic-specific lipin- deficiency exacerbates experimental alcohol-induced steatohepatitis in mice lpin rs polymorphism in pediatric nonalcoholic fatty liver disease hepatitis c virus rna functionally sequesters mir- an integrated transcriptomic and meta-analysis of hepatoma cells reveals factors that influence susceptibility to hcv infection persistent hepatitis c virus infection in vitro: coevolution of virus and host sofosbuvir: a new oral once-daily agent for the treatment of hepatitis c virus infection ros-mediated p induction of lpin regulates fatty acid oxidation in response to nutritional stress induction of lipin by ros-dependent srebp- activation hepatitis c virus induces proteolytic cleavage of sterol regulatory element binding proteins and stimulates their phosphorylation via oxidative stress a rapid colorimetric assay of fungal viability with the tetrazolium salt mtt regulation of the hepatitis c virus rna replicase by endogenous lipid peroxidation highly efficient full-length hepatitis c virus genotype (strain tn) infectious culture system characterization of resistance to non-obligate chain-terminating ribonucleoside analogs that inhibit hepatitis c virus replication in vitro dendritic cell-specific antigen delivery by coronavirus vaccine vectors induces long-lasting protective antiviral and antitumor immunity efficient trans-encapsidation of hepatitis c virus rnas into infectious virus-like particles alternatively spliced lipin isoforms exhibit distinct expression pattern, subcellular localization, and role in adipogenesis infectious hepatitis c virus pseudo-particles containing functional e -e envelope protein complexes selective inhibition of hepatitis c virus infection by hydroxyzine and benztropine. antimicrob agents chemother production of infectious hepatitis c virus in tissue culture from a cloned viral genome the autophagy machinery is required to initiate hepatitis c virus replication recruitment and activation of a lipid kinase by hepatitis c virus ns a is essential for integrity of the membranous replication compartment daclatasvir-like inhibitors of ns a block early biogenesis of hepatitis c virus-induced membranous replication factories, independent of rna replication and facts fictions of hcv and comorbidities: steatosis, diabetes mellitus, and cardiovascular diseases nuclear receptors control pro-viral and antiviral metabolic responses to hepatitis c virus infection cellular stress responses in hepatitis c virus infection: mastering a twoedged sword entangled in a membranous web: er and lipid droplet reorganization during hepatitis c virus infection. current opinion in cell biology hepatitis c virus, cholesterol and lipoproteins-impact for the viral life cycle and pathogenesis of liver disease endoplasmic reticulum stress links hepatitis c virus rna replication to wild-type pgc- alpha/liver-specific pgc- alpha upregulation hepatitis c virus-linked mitochondrial dysfunction promotes hypoxia-inducible factor alpha-mediated glycolytic adaptation the hepatitis c virus-induced nlrp inflammasome activates the sterol regulatory element-binding protein (srebp) and regulates lipid metabolism hypoxia causes triglyceride accumulation by hif- -mediated stimulation of lipin expression sterol-mediated regulation of human lipin gene expression in hepatoblastoma cells lipin proteins and glycerolipid metabolism: roles at the er membrane and beyond morphological and biochemical characterization of the membranous hepatitis c virus replication compartment three mammalian lipins act as phosphatidate phosphatases with distinct tissue expression patterns temporal and spatial regulation of the phosphatidate phosphatases lipin and sumoylation regulates nuclear localization of lipin- alpha in neuronal cells phosphorylation of lipin and charge on the phosphatidic acid head group control its phosphatidic acid phosphatase activity and membrane association lipin binds phosphatidic acid by the electrostatic hydrogen bond switch mechanism independent of phosphorylation the yeast lipin smp couples phospholipid biosynthesis to nuclear membrane growth an evolutionarily conserved fission yeast protein, ned , implicated in normal nuclear morphology and chromosome stability, interacts with dis , pim /rcc and an essential nucleoporin inactivation of the host lipin gene accelerates rna virus replication through viral exploitation of the expanded endoplasmic reticulum membrane host pah p phosphatidate phosphatase limits viral replication by regulating phospholipid synthesis interactions among pathways for phosphatidylcholine metabolism, ctp synthesis and secretion through the golgi apparatus phospholipid biosynthesis in eukaryotes the fatty liver dystrophy mutant mouse: microvesicular steatosis associated with altered expression levels of peroxisome proliferator-regulated proteins inhibition of cytosolic phospholipase a alpha impairs an early step of coronavirus replication in cell culture membranous replication factories induced by plus-strand rna viruses shaping up the membrane: diacylglycerol coordinates spatial orientation of signaling diacylglycerol kinases: shaping diacylglycerol and phosphatidic acid gradients to mitochondria: signaling with phosphatidic acid phosphatidic acid sequesters sec p from cis-snare complexes to inhibit priming a common lipid links mfn-mediated mitochondrial fusion and snare-regulated exocytosis pirna-associated germline nuage formation and spermatogenesis require mitopld profusogenic mitochondrial-surface lipid signaling. developmental cell lipin- regulates autophagy clearance and intersects with statin drug effects in skeletal muscle pkd regulates membrane fission to generate tgn to cell surface transport carriers. cold spring harbor perspectives in biology growth of human hepatoma cells lines with differentiated functions in chemically defined medium highly permissive cell lines for subgenomic and genomic hepatitis c virus rna replication interferon modulation of cellular micrornas as an antiviral mechanism production of high-titer helper-free retroviruses by transient transfection the cellular functions of the yeast lipin homolog pah p are dependent on its phosphatidate phosphatase activity robust hepatitis c virus infection in vitro nih image to imagej: years of image analysis a guided tour into subcellular colocalization analysis in light microscopy single-step method of rna isolation by acid guanidinium thiocyanate-phenol-chloroform extraction expression of the splicing factor gene sfrs is reduced in human obesity and contributes to enhanced lipogenesis cytoplasmic expression system based on constitutive synthesis of bacteriophage t rna polymerase in mammalian cells transient expression of the vaccinia virus dna polymerase is an intrinsic feature of the early phase of infection and is unlinked to dna replication and late gene expression we are grateful to drs. bartenschlager key: cord- -ymkrgj h authors: moon, stephanie l.; wilusz, jeffrey title: cytoplasmic viruses: rage against the (cellular rna decay) machine date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: ymkrgj h nan as our appreciation increases for the pervasive nature of transcription in the cell, so too has our appreciation for the major role of rna decay/stability in regulating both the quantity and the quality of gene expression. as soon as viral rnas appear in the cell, they must be prepared to combat or avoid cellular rna decay pathways. this review describes the myriad ways that viruses deal with the general host rna decay machinery that is active in the cell immediately upon viral infection-turning what, at first, appears to be very hostile territory for a foreign transcript into a sort of ''promised land'' for viral gene expression. it is interesting to note that cells likely try to adapt to this viral interference with the general rna decay machinery by inducing a variety of novel rnases as part of a molecular arms race. the cellular rna decay machinery constantly monitors transcripts, from the time they are synthesized in the nucleus until the end of their lifespan in the cytoplasm. aberrant products of transcription initiation (e.g. prompts), capping, and termination are quickly degraded by nuclear rna quality control surveillance complexes. misfolded, ''mis''-translated (e.g. mrnas with a premature termination codon), and mispackaged mrnas are also quickly degraded in the cytoplasm. in addition to removing aberrant mrnas, up to % of cellular gene expression may be controlled by changes in mrna stability. when a typical cellular mrna is targeted for decay, it initially undergoes deadenylation-the removal of the poly(a) tail. the mrna is then subject to processive exonucleolytic degradation in either the - direction by the exosome or dis l , or it is marked by the lsm - /pat complex for decapping by dcp / and degraded in the - direction by xrn [ ] . when the transcripts of cytoplasmic viruses are generated, they must actively avoid or overcome the assault by these aggressive cellular mrna decay complexes in order to be translated and effectively generate virions. it should be easy for the cellular rna decay machinery to recognize these foreign transcripts-typical host mrnas, for example, are assembled into characteristic ribonucleoprotein complexes in the nucleus, but the rnas of cytoplasmic viruses never have this opportunity. in addition, some viral rnas do not have caps or poly(a) tails, and some have multiple open reading frames or long utrs, which should target them for nonsense-mediated decay. yet, the transcripts generated by cytoplasmic viruses survive and flourish in this hostile cytoplasmic environment. interestingly, viruses do more than simply mimic host mechanisms like polyadenylation, triple helix structures, and capping to protect their transcripts from host exonucleases [ ] . cytoplasmic viruses use diverse strategies to overcome the odds and trick the host into ignoring or even, preferentially, stabilizing their transcripts. such viral rna trickery is a fascinating aspect of host-virus interaction that we are just now beginning to understand. several cytoplasmic viruses directly repress key aspects of the cellular rna decay machinery to promote viral rna stability. picornaviruses use an aggressive mechanism for suppression of host rna decay factors. xrn , dcp , dcp , pan (a deadenylase), and auf (a factor that targets rnas for decay) are rapidly degraded during poliovirus or human rhinovirus infections by viral proteases and/or the host cell proteasome [ , ] . the importance of this suppression has recently been demonstrated through the negative effects that auf has on picornavirus replication [ ] . the dispersal of p-bodies (cytoplasmic aggregates of host rna decay factors) in several viral infections is also evidence of disruption of cellular rna decay activities [ ] . alternatively, arthropod-borne flaviviruses, including west nile virus (wnv), generate a large amount of a short subgenomic rna (sfrna) by stalling the xrn - exoribonuclease on pseudoknot-like structures in the viral utr [ , ] . interestingly, stalling of xrn on the viral utr also inactivates the enzyme, presumably due to its slow release from sfrna [ ] . the repression of xrn by the generation of sfrna is very important in a flavivirus infection. wnv variants that cannot effectively form sfrna show defects in viral growth in certain cell types and reduced cytopathology [ , ] . disparate rna viruses have, therefore, evolved unique mechanisms by which they disarm host rna decay pathways by inactivating or proteolytically degrading important nucleases to promote productive viral infections. paradoxically, several cytoplasmic viruses even turn a host rna decay factor into a stabilizing factor. several viruses have developed a way to steal the host lsm - complex that normally marks deadenylated transcripts for - degradation. brome mosaic virus genomic rna has internal poly(a) tracts and trnalike structures in the utr that facilitate lsm - binding to promote viral translation and replication [ ] . hepatitis c virus (hcv) rnas have similarly been shown to bind lsm - , and knockdown of the rna decay factors lsm and patl dramatically reduces hcv translation and replication [ ] . finally, many transcripts of the cytoplasmic dna orthopoxviruses have unique, nontemplated poly(a) tracts at their ends that bind lsm - and can stabilize rnas [ ] . therefore, by attracting the lsm - complex and associated factors in an unconventional fashion, viral rnas certainly have figured out a way to make the best of what would normally be a bad situation for a transcript. it has been known for some time that members of the arenaviridae, bunyaviridae, and the nuclear orthomyxoviridae families steal the capped ends of host mrnas to incorporate this cisacting stability element into their own transcripts [ ] . emerging evidence indicates that the -o-methylation of cap structures is read by innate immune interferon stimulated genes (isgs) as a way to differentiate host versus virus transcripts. cap-stealing mechanisms used by segmented rna viruses to generate their mrnas circumvent this innate detection system. furthermore, recent evidence indicates that cellular trans-acting factors that stabilize host transcripts are also purloined by thieving viral rnas. the cellular hur protein is a well-characterized shuttling factor that promotes the stability of mrnas by interacting with u-rich elements. alphaviruses contain highly conserved u-rich elements or other high-affinity hur binding sites in the utr of their rnas that bind hur during infection to promote viral rna stability and efficient virus production [ , ] . hur is not the only regulatory mrna decay factor that is commandeered by cytoplasmic rna viruses. rabies virus glycoprotein mrna and poliovirus transcripts steal host poly(c) binding protein (pcbp ), leading to increased transcript abundance and stability [ , ] . usurping pcbp may help rabies virus tightly regulate expression of its glycoprotein to avoid host immune detection as it replicates and migrates to the central nervous system during infection. viral rnas may also ''sponge'' mirnas (e.g. [ ] ) and, perhaps, cellular rna binding proteins by sequestering these cellular factors on high affinity binding sites present on viral transcripts to promote viral-specific gene expression. viral-encoded ribonucleases: if you don't like the sandbox that you are playing in, make a new one virally encoded endonucleases are important for many aspects of viral replication, including the fine-tuning of viral gene expression by rapidly depleting old viral mrnas to enhance the expression of newly transcribed mrnas [ ] . in addition, to make a cell more amenable to virus production, these virally encoded nucleases may also create a new ''sandbox'' in the cytoplasm for viral rnas by initiating the large-scale decay of cellular mrnas and dramatically altering the landscape of host gene expression. interestingly, the internal cleavage of host mrnas by disparate betacoronaviruses, influenza viruses, vaccinia viruses, and the nuclear herpesviruses may force host exoribonucleases like xrn and the exosome to divert their attention to degrading this large number of products of viral endonucleolytic decay [ ] . the host rna decay machinery may, therefore, become saturated as endonucleolytic decay products rapidly accumulate during viral infection, limiting its normal functions. thus, virus-derived nucleases may disrupt normal gene expression and rna decayrelated quality control mechanisms to help viral rnas escape detection by the cellular rna decay machinery. considering the importance of rna stability in regulating transcript abundance, the inactivation or commandeering of cellular rna decay factors by viruses is likely to significantly alter host gene expression. how might changes in host mrna stability contribute to virus-induced pathology during infection (figure )? one example of this phenomenon is that wild-type kunjin virus was significantly more pathogenic in both tissue culture and mouse models of infection than a mutant virus incapable of forming sfrna [ ] . inactivation of xrn by kunjin virus sfrna likely causes the stabilization and increase in abundance of numerous short-lived host transcripts, including chemokines, cytokines, and cell cycle regulators [ ] . dysregulation of these factors by xrn inhibition may lead to excessive inflammation, dysregulation of the immune response, and/or changes in cell growth. recent work in yeast has demonstrated the ability of xrn to enter the nucleus and influence transcription rates, thus acting as a link between rna decay and transcription [ ] . excitingly, the authors found that the exonucleolytic activity of xrn was also required for the coupling between transcription and mrna decay. could sfrnamediated inactivation of xrn cause a defect in the coordination of rna decay and transcription in the host? if so, this could dramatically alter host gene expression and directly influence pathogenesis. viral rnas have evolved a wide variety of mechanisms to successfully interface with the host rna decay machinery. in fact, some of the most important questions in this field have yet to be answered. what are the consequences of viral inactivation of decay factors like xrn in terms of disease? can viruses also influence host transcription by manipulating rna decay pathways to short-circuit feedback regulatory mechanisms? how do virusinduced changes in rna decay pathways interface with potential changes in innate immune responses? virus families often use conserved strategies to evade the cellular rna decay machinery. therefore, perhaps researchers can develop effective, broad-spectrum antivirals to disarm these strategies and destabilize viral rnas. future research in this burgeoning field will likely uncover novel mechanisms of the regulation of host and viral gene expression and facilitate new methods for treating viral diseases. regulation of cytoplasmic mrna decay conservation of a triple-helix-forming rna stability element in noncoding and genomic rnas of diverse viruses poliovirus-mediated disruption of cytoplasmic processing bodies picornavirus modification of a host mrna decay protein cellular mrna decay protein auf negatively regulates enterovirus and human rhinovirus infections diversion of stress granules and p-bodies during viral infection rna structures required for production of subgenomic flavivirus rna an rna pseudoknot is required for production of yellow fever virus subgenomic rna by the host nuclease xrn a noncoding rna produced by arthropod-borne flaviviruses inhibits the cellular exoribonuclease xrn and alters host mrna stability a highly structured, nuclease-resistant, noncoding rna produced by flaviviruses is required for pathogenicity complexes bind to specific sites in viral rna genomes and regulate their translation and replication translation and replication of hepatitis c virus genomic rna depends on ancient cellular proteins that control mrna fates lsm proteins bind and stabilize rnas containing poly(a) tracts a genome-wide rnai screen reveals that mrna decapping restricts bunyaviral replication by limiting the pools of dcp -accessible targets for capsnatching sindbis virus usurps the cellular hur protein to stabilize its transcripts and promote productive infections in mammalian and mosquito cells dephosphorylation of hur protein during alphavirus infection is associated with hur relocalization to the cytoplasm the untranslated region of the rabies virus glycoprotein mrna specifically interacts with cellular pcbp protein and promotes transcript stability poly(rc) binding proteins mediate poliovirus mrna stability down-regulation of a host microrna by a herpesvirus saimiri noncoding rna virus-encoded endonucleases: expected and novel functions a common strategy for host rna degradation by divergent viruses gene expression is circular: factors for mrna degradation also foster mrna synthesis key: cord- - oigu k authors: moser, theresa s.; schieffer, daniel; cherry, sara title: amp-activated kinase restricts rift valley fever virus infection by inhibiting fatty acid synthesis date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: oigu k the cell intrinsic innate immune responses provide a first line of defense against viral infection, and often function by targeting cellular pathways usurped by the virus during infection. in particular, many viruses manipulate cellular lipids to form complex structures required for viral replication, many of which are dependent on de novo fatty acid synthesis. we found that the energy regulator ampk, which potently inhibits fatty acid synthesis, restricts infection of the bunyavirus, rift valley fever virus (rvfv), an important re-emerging arthropod-borne human pathogen for which there are no effective vaccines or therapeutics. we show restriction of rvfv both by ampk and its upstream activator lkb , indicating an antiviral role for this signaling pathway. furthermore, we found that ampk is activated during rvfv infection, leading to the phosphorylation and inhibition of acetyl-coa carboxylase, the first rate-limiting enzyme in fatty acid synthesis. activating ampk pharmacologically both restricted infection and reduced lipid levels. this restriction could be bypassed by treatment with the fatty acid palmitate, demonstrating that ampk restricts rvfv infection through its inhibition of fatty acid biosynthesis. lastly, we found that this pathway plays a broad role in antiviral defense since additional viruses from disparate families were also restricted by ampk and lkb . therefore, ampk is an important component of the cell intrinsic immune response that restricts infection through a novel mechanism involving the inhibition of fatty acid metabolism. emerging and re-emerging arthropod-borne viral pathogens have lead to significant world-wide morbidity and mortality in humans and domestic animals, and are of medical and agricultural concern. bunyaviruses are an important group of insect-borne rna viruses that include disease causing members such as sin nombre, hantavirus, crimean-congo hemorrhagic fever virus, and rift valley fever virus (rvfv). rvfv is a mosquito borne category a agent initially endemic to sub-saharan africa. however, outbreaks of rvfv have recently occurred in egypt and the arabian peninsula, indicating the potential of this virus to spread to new geographical areas [ ] . rvfv has particular importance as an agricultural pathogen, where infection of livestock can lead to significant morbidity and mortality among young animals, and cause catastrophic abortion rates [ ] . most humans infected with rvfv develop self-limited febrile illness, although approximately - % die from the disease due to hemorrhagic symptoms [ ] [ ] [ ] [ ] . no effective vaccines or antiviral therapies have yet been developed against rvfv. all viruses undergo sequential steps to complete their replication cycles. bunyaviruses and other rna viruses compartmentalize their rna replication machinery on cellular membranes. an essential feature of these infections is the ability of viruses to rearrange and proliferate internal cellular membranes into distinct structures compartmentalizing the viral replication complex and supporting viral genome replication [ ] . depending on the virus, these membrane modifications can be derived from distinct cellular sources, including er, golgi, endosomal, and mitochondrial membranes, and may have complex biogenesis pathways derived from multiple intracellular origins [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . bunyamwera virus, a member of the bunyavirus family related to rvfv, induces the formation of a new golgi membrane-derived tubular structure with a globular head that harbors the viral replication complex [ , ] . disrupting the formation of this structure is associated with decreased levels of virus replication [ ] . while different families of viruses use membranes derived from different cellular sources, and create membranous structures with distinct morphologies, there are some similarities in these structures, suggesting that commonalities exist in the mechanisms by which disparate viruses depend upon lipid metabolism or trafficking [ ] . one clear point of overlap includes a requirement for cellular lipid biogenesis pathways and the generation of newly synthesized lipids [ ] . furthermore, enveloped viruses, which include bunyaviruses, require incorporation of cellular membranes into their lipid envelopes during virus assembly, in a process that may also involve lipid modifications [ ] . amp-activated kinase (ampk) is a heterotrimeric complex that is the core energy sensor of the cells [ ] . thus ampk activity is important for survival during periods of stress, and also has implications in type ii diabetes, obesity, metabolic syndrome, longevity, and cancer [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the ampk complex consists of a catalytic alpha subunit, and regulatory beta and gamma subunits [ ] . activation is triggered through binding of amp or adp to the bateman domains of the gamma subunit, leading to increased phosphorylation of the threonine on the alpha subunit by inducing allosteric activation and inhibiting dephosphorylation [ ] [ ] [ ] [ ] . the canonical upstream activator that catalyzes this phosphorylation event is the constitutively active tumor suppressor lkb , but additional activators such as camkkb have been identified [ ] [ ] [ ] [ ] [ ] . under conditions of energetic stress, ampk signals the cell to stop anabolic pathways and activate a catabolic state by inducing oxidative pathways that generate energy while inhibiting synthesis and growth pathways, thereby returning the cell to a state of energy homeostasis [ ] . to achieve this regulation, ampk targets a number of downstream pathways including those involved in lipid metabolism. as a potent regulator of lipid metabolism, ampk activity inhibits both sterol and fatty acid synthesis, while promoting fatty acid degradation [ ] . ampk directly phosphorylates acetyl-coa carboxylase (acc) and hmg-coa reductase (hmgcr), thereby inactivating these rate limiting enzymes in the metabolism of fatty acids and sterols respectively [ , ] . in particular, acc catalyzes the irreversible conversion of acetyl-coa to malonyl-coa, a key metabolite that plays multiple roles in fatty acid metabolism. first, malonyl-coa is the substrate for fatty acid biogenesis, which drives de novo production of the fatty acid palmitate [ ] . second, malonyl-coa is a co-substrate for chain lengthening of endogenously synthesized and dietary-derived essential fatty acids into higher polyunsaturated fatty acids [ ] . third, malonyl-coa binding inhibits carnitine palmitoyltransferase i (cpt- ), an essential factor in the transport of fatty acids to the mitochondria for beta oxidation [ ] . thus malonyl-coa production by acc promotes fatty acid synthesis, while inhibiting fatty acid oxidation. mammalian systems encode two nonredundant acc isoforms, acc and acc , which are both inactivated by ampk-mediated phosphorylation. studies suggest that malonyl-coa produced by acc is involved in fatty acid oxidation, while acc contributes to fatty acid biogenesis [ ] . therefore, activation of ampk through stress or low energy conditions induces fatty acid oxidation through acc , while inhibiting fatty acid synthesis through acc , with a net result of lipid breakdown. we found that ampk is potently antiviral against rvfv, and this restriction is dependent on the upstream activator lkb . furthermore, pharmacological activation of ampk inhibited viral infection. ampk was activated by rvfv infection, and in particular we observed striking changes in acc activity dependent on ampk, leading us to discover that ampk is antiviral through its role in fatty acid metabolism. cells lacking ampk had increased global lipid levels, while pharmacological activation of ampk led to decreased cellular lipids, consistent with ampk control of lipid availability as a restriction point for viral replication. importantly, we could bypass the antiviral effects of ampk by feeding cells palmitate, the first fatty acid produced downstream of acc. since palmitate treatment restored rvfv infection, we demonstrate that ampk specifically restricts infection through its role in inhibiting fatty acid biosynthesis. since many viruses are dependent upon fatty acid biosynthesis for their replication, we tested whether ampk restricted additional rna viruses. we found that indeed, ampk has antiviral activity against multiple arboviruses from disparate families including: the flavivirus kunjin virus, the togavirus sindbis virus, and the rhabdovirus vesicular stomatitis virus. taken together, our data suggest that ampk activation is broadly anti-viral, and may provide a novel antiviral therapeutic target. we previously reported that ampk was required for efficient vaccinia infection through its role in macropinocytosis [ ] . this led us to investigate the role of ampk in other virus infections; we were particularly interested in rvfv as it is a virus that is medically important, but little is known about the mechanisms by which it establishes a productive infection. for our studies we used the lab adapted strain mp that has amino acid differences from the wild type strain, since the wild type strain must be used in high containment facilities [ ] . in order to test the role of ampk in rvfv infection, we took advantage of mouse embryonic fibroblasts (mef) that are genetically altered and null for both of the catalytic a subunits, ampka and ampka (ampka / ampka / ) [ ] [ ] [ ] . we challenged either the ampka / ampka / mefs or their sibling control wild type mefs with rvfv and measured infection by plaque assay ( figure a ). we found an increase in titer from pfu/ml to pfu/ml, indicating a -fold increase in the number of plaques formed in ampka /ampka / mefs compared to wild type ( figure b) , concomitant with a -fold increase in average plaque area in ampka /ampka / mefs ( figure c ). moreover, rvfv infection was also increased in ampka /ampka / mefs as measured by an immunofluorescence assay that detects production of the rvfv n protein produced during viral replication ( figure d , quantified in figure e ), indicating that rvfv is able to infect and spread more efficiently in the absence of ampk. consistent with a role for ampk both in early events during viral replication and in spread as measured by plaque assay figure a ), we observed an increase in viral infection at early time points before virus spread, as well as increased spread in cells lacking ampk by monitoring the production of rvfv n protein over time by microscopy ( figure s a-b) . this increased spread, indicated by the increase in plaque size ( figure c ), as well as the immunofluorescence assay ( figure s a -b), could result from increased production of infectious virus or increased infectivity of the virions produced in cells lacking rna viruses represent an important worldwide source of infection and disease in both humans and animals. while individual viruses have unique characteristics, some stages of infection are conserved and must be completed in order to successfully infect and grow. viruses must undergo genome replication, protein synthesis, and assembly of new virus particles. in particular, numerous rna viruses manipulate cellular membranes to create new complex structures required for viral replication in a process that is often dependent on fatty acid biosynthesis. this is a process that is tightly regulated by the energy sensor ampk. we have found that energy-mediated activation of ampk restricts infection of the bunyavirus rift valley fever virus by decreasing levels of fatty acid synthesis. furthermore, several additional rna viruses from disparate families that share this dependence of membrane modification and fatty acid synthesis are also restricted by ampk. thus ampk likely represents a novel component of the cell intrinsic immune response to rna viruses, and may be a good target for the development of antiviral therapeutics against a range of medically important viruses. ampk. we measured the amount of infectious virus produced in wild type and ampka /ampka / mefs over time in a onestep growth curve. medium from infected cells was collected at various times after infection, and virus was tittered on wild type bhk cells. little virus (less than pfu/ml) was detected at - hpi, indicating that input virus was not detected in this assay ( figure f ). virus release began at hpi, where we already observed an -fold increase in titer in the ampk deficient mefs ( . pfu/ml versus . ) ( figure f ). this increase in titer was also observed at hpi. therefore, the increase in rvfv spread is likely due to increased virus production in ampka / ampka / mefs. ampk is activated through phosphorylation of a threonine residue on the catalytic alpha subunit [ ] . since ampk deficiency increased rvfv infection, we hypothesized that ampk activation would inhibit infection. therefore, we tested whether rvfv was sensitive to pharmacological treatments that activate ampk. first, we tested drugs that activate ampk by reducing the levels of cellular energy using an independent cell line, the human osteosarcoma cell line (u os). we tested the glucose analog -deoxyglucose ( dg), and the atp synthase inhibitor oligomycin, and found that both treatments significantly decreased infection by rvfv compared to vehicle controls ( figure a ). in contrast, the ampk inhibitor compound c significantly, albeit modestly, increased rvfv infection ( figure s a ). since dg and oligomycin activate ampk indirectly by reducing cellular energy levels, and thus likely have other effects that may also contribute to viral infection, we tested whether these treatments affected vaccinia virus infection, which is not restricted by ampk, but rather requires ampk, independent of the energy sensing pathway for efficient viral infection [ ] . vaccinia virus infection was not affected by these treatments ( figure b ), indicating that the compound-treated cells remain healthy enough to support viral infection, and the reduced infection levels were specific to rvfv. moreover, we found that none of these drug treatments reduced cell number by greater that %, and therefore were not cytotoxic ( figure s b ). next, we took advantage of a recently developed thienopyridone compound a that activates ampk directly, independently of the energy status of the cell [ , ] . this drug mimics both allosteric activation of ampk and inhibition of dephosphorylation without affecting binding of amp to the gamma subunit [ ] . we found that rvfv infection of u os cells was significantly reduced in the presence of this compound ( figure c ), and that both dg and a inhibit rvfv in a dose-dependent manner ( figure s a -b), indicating that ampk activation restricts rvfv infection independently of the pleiotropic effects of reduced cellular energy levels. moreover, we also found that the ampk activating drugs dg and a significantly inhibit rvfv infection in mefs ( figure d ). to determine if the effects of these drugs was specific for ampk we treated ampka /ampka / mefs with the direct ampk activator a . treatment with this drug inhibited rvfv less than fold in ampka /ampka / mefs and was not significant, whereas infection was inhibited greater than -fold in the wild type cells ( figure s a ) with no toxicity in either cell type ( figure s b ), indicating that the major action of this drug was through ampk as previously published [ , ] . taken together, these studies suggest that ampk activation has antiviral activity against rvfv in multiple cell types. since pharmacological activation of ampk restricted rvfv infection, we were interested in investigating which pathway upstream of ampk was responsible for this restriction. the classic activator of ampk is the tumor suppressor lkb , which phosphorylates ampk in response to a variety of stimuli that cause a reduction in cellular energy levels, such as glucose starvation or hypoxia [ ] . in order to determine if lkb signaling was important for ampk-mediated rvfv restriction, we tested whether lkb also restricted rvfv. we challenged mefs that are null for lkb and complemented with either vector alone (lkb / ; vec), or an lkb cdna (lkb / ; lkb ) [ ] and found increased rvfv infection in mefs lacking lkb by plaque assay ( figure a ). quantification revealed a fold increase in the number of plaques (increase in average virus titer from . to . pfu/ml in lkb null mefs) ( figure b ) and a -fold increase in plaque area in lkb / ; vec mefs compared to mefs complemented with lkb ( figure c ). moreover, we observed increased infection in the lkb / ; vec mefs compared to those complemented with lkb by immunofluorescence ( figure d , quantified in e). finally, we measured rvfv infection over time in cells lacking lkb and found increased infection in the absence of lkb at early and late times after infection, indicating increased initial infection as well as spread ( figure f ). since ampk activation downstream of lkb is dependent on a decrease in cellular energy, we measured cellular atp levels during rvfv infection using a luciferase assay. while dg significantly reduced cellular atp levels, neither a nor rvfv had any impact on atp levels as measured by this assay ( figure s ). while infection with rvfv did not induce global changes in cellular atp, this does not rule out localized changes in cellular energy that could influence ampk. in addition to lkb other upstream activators of ampk have been identified. notably, calcium-calmodulin kinase kinase (camkk) has been shown to activate ampk in response to an increase in intercellular calcium [ , , ] . since lkb did not restrict rvfv as strongly as ampk did (figure ), we investigated if other upstream activators, such as camkk could also contribute to rvfv restriction. to this end, we treated u os cells with the camkk inhibitor sto prior to infection, and found no increase in rvfv infection in response to this drug, although at very high concentrations there was a decrease in infection ( figure s c ). this decrease was likely due to additional kinases that are inhibited at these concentrations [ ] . this finding is consistent with previous reports that changes in intercellular calcium levels are not induced by rvfv infection [ ] . we next investigated if lkb and camkk function redundantly to restrict rvfv infection. we tested whether simultaneously inhibiting both lkb and camkk would lead to a greater increase in rvfv infection than lkb deficiency alone. to this end, prior to infection, we treated lkb null mefs or those complemented with lkb with sto and monitored rvfv infection. consistent with our previous findings, we observed a -fold increase in the percentage of infected cells in lkb null cells compared to those complemented with lkb ; however pretreatment with sto had no effect on infection level in either cell type ( figure g ). in contrast, and as expected, we found that pretreatment with the ampk activating compound a significantly inhibited rvfv in both lkb null and complemented mefs ( figure g ). taken together, these data suggest that lkb is the major upstream activator responsible for ampkmediated restriction of rvfv. to dissect the mechanism by which ampk restricts rvfv infection, we first determined which early step in the viral replication cycle is restricted by ampk. we observed decreased protein production, as measured by immunofluorescence ( figure d -e and figure s ) in addition to decreased production of infectious progeny virus ( figure f ) in the presence of ampk. this suggests that ampk may inhibit a step in the viral replication cycle at, or prior to, protein production. to determine if viral rna replication was affected by ampk, we monitored both viral genomic rna replication and viral mrna production in the presence or absence of ampk. we found an increase in both viral mrna (n) and genomic rna (s segment) in ampk deficient mefs both early in infection and upon virus spread ( figure a -c). at hpi, a time point prior to rvfv release, we observed a fold increase in viral mrna production in ampk deficient mefs compared to wild type, which continued to increase over time ( figure a -b). likewise, genomic rna production was increased prior to virus release and spread ( figure a and c). these data suggest that the increased n protein production observed by immunofluorescence at early time points (figure s a) may be due to increased n mrna production. next, we investigated whether entry, a step upstream of rna replication, was inhibited by ampk. first, we tested whether rvfv binding was more efficient in the absence of ampk. to this end, mefs were pre-bound with rvfv for an hour at uc, unbound virus was removed and rvfv binding was measured by quantitative rt-pcr to detect genomic rvfv s segment within virions. we observed no difference in virus binding in wild type or ampk deficient cells ( figure d ). moreover, the majority of virus was removed by trypsin treatment in both wild type and ampk deficient mefs, indicating these virions had bound to the cell surface, but not entered ( figure d ). since ampk did not impede virus binding, we next performed a time of addition assay to test whether ampk-activating drugs restricted entry. since bunyaviruses such as rvfv enter cells through a ph-dependent route of endocytosis [ ] [ ] [ ] , we used the lysosomotropic agent ammonium chloride, which raises the ph of lysosomal compartments, to define the timing of virus entry. ammonium chloride inhibited infection strongly (to % of the hpi addition) when added hour prior to infection or with infection (t = ); however, by hpi, more than % of infection had returned, indicating that the majority of rvfv had entered by this time point ( figure e ). thus we compared each treatment to the post entry level of rvfv infection (ammonium chloride added at hpi). ampk activating drugs dg, and a significantly inhibited infection when added at post entry stages ( figure e) ; however, since one of the ampk activating drugs, a , had a significantly greater impact on rvfv when added prior to or with infection, we cannot rule out that ampk also inhibits rvfv entry. taken together these data suggest that ampk restricts rvfv during initial stages of replication post entry, likely at the step of rna replication. this reduction in viral rna and protein production likely leads to a reduction in release of infectious virus and spread observed at later stages of infection. the classical cell-mediated response to viral infection is the type i interferon system [ , ] . therefore, we investigated whether ampk impacts the expression of interferon beta (ifnb) or its downstream effector - -oligoadenylate synthetase (oas ) by qrt-pcr. we found that rvfv infection induced both ifnb and oas in both wild type and ampk deficient cells although the basal levels and induction of these genes were higher in cells lacking ampk ( figure s a-b) . this result was opposite to what would have been predicted, if ifnb induction was responsible for the antiviral phenotype. in addition, we tested whether ifnb treatment induced ampk or acc phosphorylation and found that it did not ( figure s c , quantified in d). altogether, these data indicate that ampk has antiviral activity independent of the classical type i ifn response. since ampk activation has antiviral activity against rvfv, we examined whether ampk is activated by rvfv infection. to this end, we measured ampk phosphorylation at thr by immunoblot. ampk phosphorylation was increased at and hours after infection compared to uninfected controls ( figure a , quantified in figure s a ), indicating that rvfv infection induced ampk activation. furthermore, we found that uv-irradiated virus, incapable of replication ( figure s ), also induced ampk phosphorylation at and hours after treatment ( figure c ), suggesting that activation was triggered by incoming virus particles and viral replication was not required. finally, we confirmed that lkb was required for rvfv-dependent activation of ampk ( figure s ). ampk regulates several downstream pathways that could be important for viral infection, in particular protein translation and lipid synthesis [ ] . thus, we examined the activation status of two classical downstream effectors of ampk involved in translation and lipid biosynthesis which are inactivated by ampk-mediated phosphorylation [ ] . elongation factor (eef ) is an important regulator of translation elongation, and acetyl-coa carboxylase (acc) consists of two enzymes involved in fatty acid metabolism (acc and acc ) [ , ] . both eef and acc had increased levels of phosphorylation at and hours after infection with rvfv compared to uninfected controls, consistent with the activation status of ampk ( figure a , quantified in figure s b -d). little difference in total protein levels of ampk, acc or eef was observed during infection. taken together, these data suggest that rvfv infection leads to increased ampk signaling. to explore the mechanism by which ampk restricts rvfv replication, we examined the impact of ampk on translation and lipid biogenesis, both of which contribute to important steps in virus infection. in particular, ampk inhibits translation initiation by inactivating mtorc , and translation elongation by inactivating eef [ ] [ ] [ ] . inactivation of mtorc by ampk leads to decreased translation initiation as well as increased autophagy, both of which could have anti-viral effects [ ] . since ampk activation inhibits mtorc activity, we hypothesized that mtorc , and thus protein synthesis, would be overactive in ampk deficient cells, perhaps allowing for increased viral protein production and replication. we tested the requirement for mtorc signaling in rvfv infection using the mtorc inhibitor rapamycin, and found no significant difference in rvfv infection in cells treated with rapamycin compared to vehicle controls in either wild type or ampka /ampka / mefs ( figure s a ). this finding suggests that the antiviral activity of ampk is independent of mtorc signaling. furthermore, since ampk activation can increase autophagy, which has been shown to have antiviral effects in some models [ ] , we tested whether inhibition of autophagy impacted rvfv infection by plaque assay, and found no significant difference in mefs expressing a atg hairpin, which knocks down atg , compared to control mefs (figure s b-c). next we investigated whether reduced translation elongation through eef inactivation could be responsible for ampk's antiviral activity against rvfv ( figure a ). since eef is regulated by multiple upstream pathways in addition to ampk, we first determined the sensitivity of eef to ampk regulation. in wild type mefs, treatment with the ampk activating drugs dg, oligomycin, or a led to increased phosphorylation of ampk, as well as downstream effectors eef and acc ( figure b , quantified in figure s e -h), as expected. as a control, we found that ampk deficient mefs did not express phosphorylated ampk or total ampk under any treatment condition. interestingly, we observed an increase in phosphorylated eef in response to all three drugs in ampka / ampka / mefs ( figure b , quantified in figure s h ). in contrast, while we observed an increase in acc phosphorylation in response to drug treatments in wild type mefs, phosphorylated acc was undetectable in ampk deficient mefs both basally and in response to treatment with ampk activating compounds ( figure b ). these phenotypes were not due to changes in total protein levels as they remained unchanged under all treatment conditions; although the ampk deficient mefs had a slightly lower basal level of acc ( figure b ). these findings suggest signaling pathways other than ampk are important in regulating eef phosphorylation, while acc phosphorylation is exquisitely regulated by ampk. given this observation, we pursued acc as a potential regulator of antiviral defense. acc is the first rate-limiting enzyme and master regulator of fatty acid metabolism, both by inhibiting fatty acid biosynthesis and activating fatty acid catabolism through beta-oxidation [ , ] . fatty acid biosynthesis is an important component of viral infection since numerous rna viruses, including bunyaviruses, proliferate cellular membrane structures for proper formation of the viral replication complex, in addition to using cellular membranes for their lipid coats [ , , , ] . in order to assess the importance of fatty acid synthesis in rvfv infection, we tested the ability of rvfv to replicate within cells pretreated with the fatty acid synthase inhibitors. fatty acid synthase is the next enzyme in fatty acid metabolism, using the product of acc to generate palmitate, and thus is required for all fatty acid biosynthesis [ ] . we observed a -fold decrease in rvfv infection in the presence of fatty acid synthase inhibitors cerulenin and c by immunofluorescence, similar to the decrease observed in cells pretreated with the ampk activator a (figure d ), indicating that de novo fatty acid synthesis is an important step early in rvfv infection. acc is the enzyme that converts acetyl-coa into malonyl-coa, a precursor in the synthesis of palmitate, the first product of de novo fatty acid biosynthesis. since ampk activation inhibits de novo fatty acid synthesis by inactivating acc, we tested whether altered levels of ampk activation or expression affected cellular lipid levels. to this end, we stained mefs with the lipophilic bodipy fluorescent dye. we found that treatment with the ampk activator a led to a decrease in bodipy staining compared to untreated mefs ( figure e , quantified in f), consistent with decreased fatty acid synthesis during ampk activation. in contrast, mefs lacking ampk had increased bodipy staining compared to wild type cells ( figure g , quantified in h). these findings are consistent with previous reports that ampk activating drugs, such as a increase levels of beta-oxidation while decreasing fatty acid synthesis [ , , ] , and suggest that the absence of ampk leads to overproduction of cellular lipids, while ampk activation globally reduces cellular lipid levels. if ampk activation restricts rvfv infection by reducing levels of fatty acid synthesis, exogenous addition of fatty acids should restore infection. therefore, we tested whether we could bypass the requirement for ampk-regulated fatty acid synthesis by pretreating cells with palmitate, the first product of fatty acid biosynthesis. we treated u os cells with palmitate overnight, and then added a hour prior to infection with rvfv to activate ampk. after hours of infection, cells were fixed and stained for rvfv to measure percent infection in an immunofluorescence assay that monitors the initial round of infection. in cells treated with the ampk activator a alone, we found a -fold decrease in rvfv infection, consistent with our previous findings ( figure a, quantified in b) . however, addition of palmitate prior to treatment with a was able to restore infection to levels seen in untreated cells ( figure a , quantified in b). we observed a -fold increase in rvfv infection in cells treated with a and palmitate compared to those treated with a alone ( figure b ), while addition of palmitate alone had little effect on infection ( figure a -b). since chronic exposure to high concentrations of palmitate has previously been reported to inhibit ampk activation, we confirmed by immunoblot that ampk phosphorylation was not inhibited by the concentrations of palmitate used in our assay ( figure s ). together, these data suggest that ampk restricts rvfv infection primarily through inhibiting fatty acid biosynthesis. a dependence on lipid biosynthesis and virally induced membrane modifications is not unique to bunyaviruses; many rna viruses require extensive membrane modifications and proliferations to support their replication complex [ , ] . therefore, we tested whether ampk restricts additional arboviruses. to this end we tested the ability of the flavivirus kunjin virus (kunv), the togavirus sindbis virus (sinv), and the rhabdovirus vesicular stomatitis virus (vsv) to grow in wild type and ampka /ampka / mefs by immunofluorescence. kunv ( figure a figure k -l) infections were also increased in lkb / ; vec compared to mefs expressing lkb , indicating that both ampk and its canonical upstream activator lkb restrict additional arboviruses. moreover, we have found that kunv is also sensitive to the ampk activator a , and can be partially rescued by palmitate addition (figure s a-b) , although palmitate treatment itself decreased kunv infection ( figure s c ). these data suggest that ampk may restrict multiple rna viruses by limiting fatty acids. taken together our data suggest that ampk is broadly anti-viral across disparate virus families, and may represent a novel cellular target for anti-viral therapeutics. arboviruses represent a group of emerging pathogens of both medical and agricultural importance for which there are few therapies. rvfv is a particularly important member of this group that causes disease both in humans and livestock, and is considered a category a pathogen due to its high pathogenesis and potential for geographical spread. here, we identified ampk as a novel antiviral factor that restricts rvfv infection independent of the type i ifn system. this restriction is dependent on the canonical upstream activator lkb . furthermore, we found that ampk is activated by rvfv infection, and this activation restricts infection at the level of rna replication likely by reducing fatty acid biosynthesis, an essential process in rvfv infection. we extended these studies by demonstrating that additional arboviruses, known to require lipid biosynthesis, were also restricted by this pathway. since treatment with drugs that activate ampk restricted infection, this could represent a novel therapeutic strategy toward the control of many rna viruses. ampk is a central regulator of cellular energy that regulates a number of cellular pathways that could influence viral replication, including protein and lipid biosynthesis [ ] . ampk activation inhibits protein translation through two major downstream pathways. first, ampk activation inhibits translation initiation by inhibiting mtorc activity. second, ampk inhibits translation elongation through inactivation of eef . we explored these two targets as potentially regulating rvfv infection. however, we found rvfv was insensitive to treatment with the mtorc inhibitor, rapamycin, regardless of ampk status. furthermore, eef phosphorylation induced by drugs that alter the energy status of the cell was not affected in the absence of ampk, indicating additional upstream regulators are contributing to eef activity. therefore, we explored lipid biosynthesis as a potential target for ampk-dependent anti-viral activity. ampk controls fatty acid metabolism through acc, and may be the only physiologically relevant kinase that controls acc activity [ ] . this is consistent with our findings that acc phosphorylation was exquisitely dependent on ampk, in contrast to eef , which was phosphorylated during energy depletion even in the absence of ampk. acc is the enzyme responsible for the conversion of acetyl-coa to malonyl-coa [ ] . malonyl-coa production impacts lipid metabolism in at least three ways. malonyl-coa is a substrate driving de novo palmitate production, and is also important in converting simple essential fatty acids into more complex polyunsaturated fatty acids that can be used to build triglycerides and other cellular lipids [ ] . finally, malonyl-coa inhibits transport of fatty acids to the mitochondria, thus inhibiting fatty acid oxidation [ ] . in addition to its role in fatty acid metabolism, ampk is also an important regulator of hmg-coa reductase (hmgcr), the rate limiting enzyme in the synthesis of isoprenoids and sterols, including cholesterol. cholesterol is known to contribute to infection of multiple viruses, and therefore could also be targeted in ampk-mediated virus restriction. since we found that fatty acid biosynthesis was required for rvfv infection, and changes to ampk expression and activation status led to global changes in cellular lipid levels, we hypothesized that inhibiting fatty acid synthesis downstream of acc was responsible for ampk-mediated restriction of rvfv. this was supported by our finding that we could bypass the requirement for malonyl-coa production by introducing exogenous palmitate. since the addition of palmitate rescued rvfv overcoming the restriction mediated by ampk activation (figure ), the ability of ampk to inhibit fatty acid biosynthesis is likely the most important determinant of ampk-mediated rvfv restriction. palmitate is a substrate for the biosynthesis of a number of lipid moieties that could contribute to rvfv infection. palmitate undergoes chain elongation and additional modifications in the er to produce saturated fatty acids as well as triglycerides, phospholipids, and cholesterol esters [ , ] . it is also a substrate for sphingolipid biosynthesis in the golgi. sphingolipids become incorporated into cellular membranes and participate in signaling events that could contribute to rvfv infection. finally, palmitate addition is a form of post-translational modification of some proteins [ ] . there are several stages during the course of rvfv infection where cellular lipids are utilized. many rna viruses induce the formation of novel membranous structures derived from various organelles within the cell to support the viral replication complex [ ] . notably, formation of these structures is often dependent on de novo fatty acid synthesis [ ] [ ] [ ] [ ] . while rvfv-induced membrane alterations have not been well characterized, a related bunyavirus, bunyamwera virus, was reported to induce golgi-derived tubular structures with globular heads in association with the viral replication complex, suggesting that other bunyaviruses could likewise induce membrane changes [ , ] . in addition to rna replication, enveloped viruses bud from cellular membranes, thereby incorporating those lipids into the viral particle [ ] . rvfv assembly occurs on golgi membranes, with virus particles ultimately budding into the golgi for transport and release at the plasma membrane [ ] . cellular lipids derived from de novo palmitate production downstream of acc could contribute to each of these steps, although our findings that viral rna synthesis is inhibited by ampk suggests that rna replication is a key target. in addition to rvfv, we found that three additional viruses including the togavirus sinv, the flavivirus kunv, and the rhabdovirus vsv are restricted by ampk and lkb (figure ) . importantly, this group includes members of the three major families of arboviruses that contribute to human disease. members of the togavirus family including semliki forest virus and rubella virus have been described to induce characteristic modified endosomal and lysosomal structures termed cytopathic vacuoles that support the viral replication complex [ , , , ] . furthermore, a number of flaviviruses have been shown to have important lipid dependencies. kunv, a strain of west nile virus, has been described as forming two distinct membrane structures that include double membrane spherical vesicles that are the sites of viral replication, as well as arrays of convoluted membranes that are the sites of viral polyprotein processing [ ] [ ] [ ] [ ] [ ] [ ] . moreover, both fatty acid synthesis and oxidation have been shown to be essential for another flavivirus, dengue virus (denv). infection is characterized by virally-induced increases in cellular fatty acid synthesis and a redistribution of the enzyme fatty acid synthase to sites of denv replication [ ] . free fatty acids are also derived through autophagosomal processing of triglycerides, and exogenous addition of the fatty acid oleate was able to rescue denv infection when autophagy is inhibited [ ] . furthermore, induction of er-derived lipid droplet formation is necessary for denv particle formation [ ] . therefore denv and perhaps many other viruses require complex and unique interactions with cellular lipid metabolism through both synthesis and degradation pathways. in addition, hepatitis c virus (hcv), a distantly related flavivirus, induces formation of a membranous web derived from intracellular vesicles, whose formation requires fatty acid synthesis for replication [ , ] . interestingly, ampk has been implicated to play a role in hcv infections. ampk-activating drugs inhibited the replication of hcv replicons concomitant with a decrease in cellular lipid levels, while knock down of the upstream activator lkb led to increased replication, [ ] , consistent with our findings with rvfv, kunv, sinv, and vsv. importantly, kunv could be partially rescued from ampk-mediated restriction by the addition of the fatty acid palmitate. thus, ampk may restrict multiple families of viruses through this mechanism. since all positive strand rna viruses are thought to induce membrane modifications for viral rna replication, and include a large number of medically significant groups (e.g., picornaviruses, and coronaviruses) [ , , , ] , it will be important to determine the full scope of viruses restricted by ampk as well as the mechanism of restriction. since many disparate viruses are restricted by ampk, it is interesting to speculate how ampk could be activated in response to these viral infections. we have found that both live virus and uv-inactivated replication incompetent rvfv is capable of activating ampk via lkb . this suggests that the energy sensing pathway is responsible for this activation yet we were unable to detect global changes in cellular energy levels during the period in infection when ampk becomes phosphorylated. thus, we hypothesize that rvfv infection induces a localized drop in cellular energy to activate ampk. since this is independent of viral replication and can restrict a large panel of disparate viruses that have the commonality of entering cells via endocytic routes and fusing within these compartments, we postulate that a local energy drop may occur during these steps. since endocytosis is a highly energetic process usurped by many viruses, it is possible that increased levels could themselves could provide the trigger for this rapidly inducible antiviral response. we have previously reported that receptor-mediated endocytosis, employed by many viruses including kunv, sinv and vsv for entry is intact in ampk deficient cells [ ] . therefore at least some routes of endocytic entry used by viruses are unaffected by ampk, and may provide a trigger for activation rather than a point of restriction. this would allow broad activation of ampk by many types of viruses internalized by such routes and provide a rapid response to restrict virus infection by inhibiting fatty acid synthesis. since ampk activators are currently in the clinic to treat metabolic disorders such as type ii diabetes [ ] , and restrict rvfv and kunv replication in cell culture, they may prove to be useful antiviral therapeutics. several ampk activating drugs have been shown to reduce morbidity and mortality during lethal influenza infection in mice [ ] . in addition, treatment of ampkactivating drugs inhibited infection of hcmv and hiv in cells, and the addition of ampk-activating drugs such as metformin to current hcv treatment regimens had promising, albeit modest, effects on reducing patient viral loads [ , [ ] [ ] [ ] [ ] . infections with hcmv, hiv, and hcv have also been shown to inhibit ampk activity [ , , , ] . ampk may have multiple effects on these infections since different downstream mechanisms have been implicated [ , , , , ] ; however, this suggests the possibility that some viruses have developed mechanisms of immune evasion that target ampk. taken together, ampk plays a broad role in cellular innate immunity through potent inhibition of fatty acid synthesis, which is broadly utilized by viruses, suggesting that ampk and perhaps other modulators of lipid biosynthesis are potential targets for broad pan-antiviral therapeutics. mefs, bhk and u os cells were maintained at uc in dmem supplemented with % fbs (sigma), mg/ml penicillin/streptomycin, mm l-glutamine, and mm hepes. lkb / mefs [ ] were complemented with migr (vector) or flag-lkb -migr (lkb cdna) retrovirus and sorted on gfp+ cells by facs as previously described [ ] . rift valley fever virus mp- was grown in vero-e cells supplemented with % fbs [ ] . rvfv was uv-inactivated in a stratalinker. kunv (gift from m. diamond) was grown in bhk cells. vsv-gfp [ ] was grown in bhk cells as described [ ] . sinv-gfp virus [ ] was grown in c cells [ ] . all viruses were tittered by plaque assay in bhk cells. antibodies were obtained from the following sources: anti-rvfv id (gift from c. schmaljohn usamriid), anti-kunv ns (gift from r. doms), anti-tubulin (sigma), and anti-p-ampk, t-ampk, p-acc, t-acc, p-eef , t-eef (cell signaling technology). fluorescently labeled secondary antibodies and bodipy-tr were obtained from invitrogen. hrp-conjugated antibodies were obtained from amersham. a was obtained from santa cruz. other chemicals were obtained from sigma. viruses were plaqued on mefs as indicated. confluent monolayers were treated with serial dilutions of virus for two hours, after which the viral inoculums were removed, and cells were overlayed with . % agarose in mem, and incubated at uc for hours. cells were fixed in % formaldehyde, and stained with crystal violet. plaque number was determined manually, and plaque diameter was measured using metaxpress software and used to calculate areas. for all infections, washes and media changes were performed in the control untreated wells, as well as those infected with virus. viral immunofluorescence experiments were performed in well plates as previously described [ ] . briefly, cells were grown overnight in wells plates, media was removed and fresh media was added. when appropriate, drug was added at the indicated concentration in ml pbs, and cells were incubated at uc for hour before addition of virus. cells were infected with the indicated moi of virus in complete media and spinoculated for hour at rpm, and incubated at uc. cells were fixed and processed for immunofluorescence as previously described hours post infection for rvfv, sinv, and vsv, and hours post infection for kunv unless otherwise indicated [ ] . briefly, cells were fixed in % formaldehyde/pbs, washed twice in pbs/ . % tritonx- (pbst), and blocked in % bsa/pbst. primary antibodies were diluted in block, added to cells, and incubated overnight at uc. rvfv was stained with anti-rvfv id ; kunv was stained with anti-kunv ns . vsv and sinv expressed gfp, and did not require antibody staining. cells were washed three times in pbst, and incubated in secondary antibody with hoescht (sigma) counterstain for one hour at room temperature. plates were imaged at using an automated microscope (imagexpress micro), capturing four images per well per wavelength, and quantification was performed using metaxpress image analysis software. significance was determined using a student's t-test. for immunofluorescence assays, a minimum of three wells per condition was imaged, with four images taken per well. to control for variability in baseline level of infection, a student's t-test was performed on both the raw percent infection data in each individual experiment, and across a minimum of three replicate experiments where the untreated control had been normalized. significance was determined if p, . in all tests. mefs were infected with rvfv moi in well dishes and incubated at uc. two hours post infection, inoculums was removed, and fresh medium was added. at indicated time point, medium was removed from infected cells and tittered on bhk cells by plaque assay. mefs were grown overnight in a well dish. medium was replaced with ml of fresh complete medium and cells were chilled to uc for minutes. rvfv (moi ) was added on ice, and cells were incubated at uc for hour to allow virus binding. cells were washed in pbs, then treated with either pbs or . % trypsin to remove bound virus as previously described [ ] . cells were pelleted, then washed again, and lysed in trizol to extract total rna. samples were then prepared for quantitative rt-pcr. cdna was prepared from total rna using m-mlv reverse transcriptase (invitrogen) random primers, and transcripts were amplified by quantitative pcr. ddct was calculated for rvfv s segment using gapdh as a cellular loading control. time of addition experiments were performed as previously described [ ] . u os cells were grown overnight, and the media was replaced. cells were infected with rvfv (moi ), spun at rpm for hour, and subsequently incubated at uc. mm dg, mm a , or mm ammonium chloride were added either hour prior to infection ( ) , with infection ( ), or , or hours after infection. hours post infection cells were fixed in % formaldehyde in pbs and processed for immunofluorescence. significance was determined using a student's t test. mefs were infected with rvfv moi in well dishes (, % infection) and incubated at uc for indicated time point. for protein analysis, cells were washed briefly in cold pbs and lysed in np lysis buffer supplemented with protease (boehringer) and phosphatase (sigma) inhibitor cocktails. samples were separated by sds-page and blotted as described [ ] . hrp-conjugated secondary antibodies and western lightening chemiluminescence reagent were used for visualization. to analyze downstream effectors of ampk, mefs were treated with mm dg, mm oligomycin, or um a for hours, lysed and blotted as above. for rna analysis, cells were lysed in trizol buffer, and rna was purified as previously described [ ] . to detect viral mrna, total rna from infected cells was separated on a % agarose/ formaldehyde gel and blotted with the indicated probes as previously described [ ] . samples were quantified and normalized against controls using imagequant software. cellular lipids were stained as previously described [ , ] . mefs were grown to confluence overnight, and then treated with pbs vehicle or mm a for hours. cells were fixed in % formaldehyde for minutes and washed three times in pbs. staining was performed with mg/ml bodipy-tr and counterstained with hoescht in mm glycine in pbs overnight. cells were washed three times in pbs and imaged using the imagexpress micro automated microscope. integrated intensity of bodipy signal per cell area was calculated using metaxpress image analysis software. significance was determined using a student's t test. exogenous palmitate addition was performed as previously described [ ] . delipidated fetal calf serum and albuminbound palmitate were prepared as described [ ] and obtained as a kind gift from robert rawson. u os cells were set up on day in well plates and grown over night in normal growth medium. on day medium was removed and cells were washed briefly in pbs. cells were treated with low glucose dmem supplemented with % delipidated fetal calf serum with or without mm albumin-bound palmitate, and incubated overnight. on day cells were treated with mm a or pbs vehicle for hour, and infected with rvfv for hours. cells were fixed, processed for immunofluorescence, and imaged at using the automated microscope imagexpress micro, as described above. quantification was performed using metaxpress image analysis software. significance was determined using a student's t-test. figure s quantification of immunoblots using image j software. a-d. phosphorylation of ampk and downstream effectors upon rvfv infection. wt mefs were infected with rvfv (moi ) for or hours. lysates were collected, assayed by immunoblot and quantified for phospho-ampk (a), phospho-acc (b), phospho-acc (c), and phospho-eef (d) normalizing to the tubulin loading control. data are displayed as the average density relative to untreated at hours from triplicate experiments. e-h. phosphorylation of ampk and downstream effectors in wt and ampka /ampka / mefs. cells were treated with ampk activators dg ( mm), oligomycin (om, mm), and a ( mm) for hours. lysates were collected, assayed by immunoblot, and quantified as above for phospho-ampk (e), phospho-acc (f), phospho-acc (g), and phospho-eef (h) normalized to the tubulin loading control. data are displayed as the average density relative to untreated at hours from triplicate experiments. (tif) figure s uv-inactivated rvfv is replication incompetent. u os cells were infected with live (moi ) and uv-inactivated virus (equivalent volume to moi ) for hours, and processed for immunofluorescence. (rvfv-n, green; nuclei, blue) (tif) figure s ampk is not activated by rvfv in lkb null mefs.lkb / ;lkb and lkb / ;vec mefs were infected with rvfv (moi ) for hours. lysates were collected and assayed by immunoblot for phospho-ampk. total ampk and tubulin were assayed. representative blot of duplicate experiments is shown. (tif) figure s a: mtorc is not required for ampk-mediated restriction of rvfv. wt and ampka /ampka / mefs were pretreated with nm rapamycin or pbs for hour and infected with rvfv (moi ) for hours and processed for immunofluorescence. a representative of duplicate experiments is shown. b. autophagy does not restrict rvfv. rvfv was plaqued in mefs expressing a control hairpin rna or a hairpin against atg . c. atg mrna expression by qrt-pcr in mefs expressing a control hairpin rna or a hairpin against atg normalized to gapdh. (tif) figure s palmitate treatment does not inhibit ampk activation or signaling. u os cells were treated with palmitate overnight, then treated with dg ( mm) and a ( mm) for hours. lysates were collected and assayed by immunoblot for phospho-ampk, and phospho-acc. total ampk, acc and tubulin were assayed. representative blot of duplicate experiments is shown. (tif) text s the supporting information contains the methods used in figures s , s , s , s , s , s , s , s , s , s , s , s . (pdf) rift valley fever virus interepidemic rift valley fever virus seropositivity, northeastern kenya epidemic rift valley fever in saudi arabia: a clinical study of severe illness in humans hemorrhagic fever viruses as biological weapons: medical and public health management rift valley fever epidemic in saudi arabia: epidemiological, clinical, and laboratory characteristics modification of intracellular membrane structures for virus replication cellular origin and ultrastructure of membranes induced during poliovirus infection expression of hepatitis c virus proteins induces distinct membrane alterations including a candidate viral replication complex identification of the hepatitis c virus rna replication complex in huh- cells harboring subgenomic replicons biogenesis of the semliki forest virus rna replication complex rubella virus replication complexes are virus-modified lysosomes alphavirus rna replicase is located on the cytoplasmic surface of endosomes and lysosomes flock house virus rna replicates on outer mitochondrial membranes in drosophila cells polymorphism and structural maturation of bunyamwera virus in golgi and post-golgi compartments the unique architecture of bunyamwera virus factories around the golgi complex cytoplasmic viral replication complexes more than one door -budding of enveloped viruses through cellular membranes regulation of fatty acid synthesis and oxidation by the amp-activated protein kinase amp-activated protein kinase signaling in metabolic regulation ampk: an emerging drug target for diabetes and the metabolic syndrome a critical role of snf a/dampkalpha (drosophila amp-activated protein kinase alpha) in muscle on longevity and stress resistance in drosophila melanogaster lifespan extension induced by ampk and calcineurin is mediated by crtc- and creb metformin selectively targets cancer stem cells, and acts together with chemotherapy to block tumor growth and prolong remission metformin is an amp kinase-dependent growth inhibitor for breast cancer cells the antidiabetic drug metformin: a pharmaceutical ampk activator to overcome breast cancer resistance to her inhibitors while decreasing risk of cardiomyopathy amp-activated/snf protein kinases: conserved guardians of cellular energy cbs domains form energy-sensing modules whose binding of adenosine ligands is disrupted by disease mutations dissecting the role of -amp for allosteric stimulation, activation, and deactivation of amp-activated protein kinase investigating the mechanism for amp activation of the amp-activated protein kinase cascade structure of mammalian ampk and its regulation by adp lkb -dependent signaling pathways the tumor suppressor lkb kinase directly activates amp-activated kinase and regulates apoptosis in response to energy stress calmodulindependent protein kinase kinase-beta is an alternative upstream kinase for amp-activated protein kinase the ca +/calmodulin-dependent protein kinase kinases are amp-activated protein kinase kinases mammalian tak activates snf protein kinase in yeast and phosphorylates amp-activated protein kinase in vitro identification by amino acid sequencing of three major regulatory phosphorylation sites on rat acetyl-coa carboxylase location and function of three sites phosphorylated on rat acetyl-coa carboxylase by the amp-activated protein kinase malonyl-coa, a key signaling molecule in mammalian cells the fatty acid chain elongation system of mammalian endoplasmic reticulum a kinome rnai screen identified ampk as promoting poxvirus entry through the control of actin dynamics mutagen-directed attenuation of rift valley fever virus as a method for vaccine development physiological role of amp-activated protein kinase (ampk): insights from knockout mouse models knockout of the alpha but not alpha -amp-activated protein kinase isoform abolishes -aminoimidazole- -carboxamide- -beta- -ribofuranosidebut not contraction-induced glucose uptake in skeletal muscle -amp-activated protein kinase (ampk) is induced by low-oxygen and glucose deprivation conditions found in solid-tumor microenvironments characterization of the amp-activated protein kinase kinase from rat liver and identification of threonine as the major site at which it phosphorylates amp-activated protein kinase identification and characterization of a small molecule ampk activator that treats key components of type diabetes and the metabolic syndrome use of cells expressing gamma subunit variants to identify diverse mechanisms of ampk activation mechanism of action of a- , a valuable tool for activation of ampactivated protein kinase -amp activates the amp-activated protein kinase cascade, and ca +/ calmodulin activates the calmodulin-dependent protein kinase i cascade, via three independent mechanisms sto- , a specific inhibitor of the ca( +)/calmodulin-dependent protein kinase kinase rift valley fever virus infection of human cells and insect hosts is promoted by protein kinase c epsilon a novel system for identification of inhibitors of rift valley fever virus replication entry of bunyaviruses into mammalian cells innate immune recognition of viral infection interferon-inducible antiviral effectors viruses and the fuel sensor: the emerging link between ampk and virus replication regulation of peptide-chain elongation in mammalian cells lkb and amp-activated protein kinase control of mtor signalling and growth activation of amp-activated protein kinase leads to the phosphorylation of elongation factor and an inhibition of protein synthesis stimulation of the amp-activated protein kinase leads to activation of eukaryotic elongation factor kinase and to its phosphorylation at a novel site, serine autophagy in immunity and inflammation structure and function of eukaryotic fatty acid synthases role of amp-activated protein kinase in mechanism of metformin action prolonged aicar-induced amp-kinase activation promotes energy dissipation in white adipocytes: novel mechanisms integrating hsl and atgl virus factories: associations of cell organelles for viral replication and morphogenesis regulation of stearoyl-coa desaturases and role in metabolism lipid metabolism and hcv infection the intracellular dynamic of protein palmitoylation copi activity coupled with fatty acid biosynthesis is required for viral replication dengue virus nonstructural protein redistributes fatty acid synthase to sites of viral replication and increases cellular fatty acid synthesis synthesis of semliki forest virus rna requires continuous lipid synthesis role of cellular lipids in positive-sense rna virus replication complex assembly and function molecular biology of rift valley fever virus intracellular distribution of rubella virus nonstructural protein p characterization of rubella virus replication complexes using antibodies to double-stranded rna ultrastructure of kunjin virus-infected cells: colocalization of ns and ns with double-stranded rna, and of ns b with ns , in virus-induced membrane structures replication and gene function in kunjin virus kunjin rna replication and applications of kunjin replicons molecular and ultrastructural analysis of heavy membrane fractions associated with the replication of kunjin virus rna wrapping things up about virus rna replication kunjin virus: an australian variant of west nile? dengue virus-induced autophagy regulates lipid metabolism dengue virus capsid protein usurps lipid droplets for viral particle formation enhanced hepatitis c virus genome replication and lipid accumulation mediated by inhibition of amp-activated protein kinase seeking membranes: positive-strand rna virus replication complexes viral rna replication in association with cellular membranes the antidiabetic drug metformin activates the amp-activated protein kinase cascade via an adenine nucleotide-independent mechanism peroxisome proliferatoractivated receptor and amp-activated protein kinase agonists protect against lethal influenza virus challenge in mice ampkmediated inhibition of mtor kinase is circumvented during immediate-early times of human cytomegalovirus infection treatment of insulin resistance with metformin in naive genotype chronic hepatitis c patients receiving peginterferon alfa- a plus ribavirin pioglitazone as adjuvant therapy in chronic hepatitis c: sequential rather than concomitant administration with pegylated interferon and ribavirin? sirt regulates tat-induced hiv- transactivation through activating amp-activated protein kinase bryostatin modulates latent hiv- infection via pkc and ampk signaling but inhibits acute infection in a receptor independent manner loss of the lkb tumour suppressor provokes intestinal polyposis but resistance to transformation autophagy is an essential component of drosophila immunity against vesicular stomatitis virus a vesicular stomatitis virus recombinant expressing granulocyte-macrophage colony-stimulating factor induces enhanced t-cell responses and is highly attenuated for replication in animals natural resistance-associated macrophage protein is a cellular receptor for sindbis virus in both insect and mammalian hosts heterogeneous nuclear ribonuclear protein k interacts with sindbis virus nonstructural proteins and viral subgenomic mrna rnai screening for host factors involved in vaccinia virus infection using drosophila cells genomewide rnai screen reveals a specific sensitivity of ires-containing rna viruses to host translation inhibition ars regulates both mirna-and sirna-dependent silencing and suppresses rna virus infection in drosophila unsaturated fatty acids down-regulate srebp isoforms a and c by two mechanisms in hek- cells we would like to thank members of cherry lab for helpful discussions; sheri hanna, maggie nakamoto, and patrick rose for growing viruses; robert rawson for fatty acid reagents; craig thompson, tullia lindsten, and heesun cheong for autophagy-deficient mefs; bernard moss and stuart isaacs for vaccinia reagents; russell jones, morrie birnbaum, and russell miller for ampk reagents, technical help and suggestions. key: cord- -c lykari authors: irigoyen, nerea; firth, andrew e.; jones, joshua d.; chung, betty y.-w.; siddell, stuart g.; brierley, ian title: high-resolution analysis of coronavirus gene expression by rna sequencing and ribosome profiling date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: c lykari members of the family coronaviridae have the largest genomes of all rna viruses, typically in the region of kilobases. several coronaviruses, such as severe acute respiratory syndrome-related coronavirus (sars-cov) and middle east respiratory syndrome-related coronavirus (mers-cov), are of medical importance, with high mortality rates and, in the case of sars-cov, significant pandemic potential. other coronaviruses, such as porcine epidemic diarrhea virus and avian coronavirus, are important livestock pathogens. ribosome profiling is a technique which exploits the capacity of the translating ribosome to protect around nucleotides of mrna from ribonuclease digestion. ribosome-protected mrna fragments are purified, subjected to deep sequencing and mapped back to the transcriptome to give a global “snap-shot” of translation. parallel rna sequencing allows normalization by transcript abundance. here we apply ribosome profiling to cells infected with murine coronavirus, mouse hepatitis virus, strain a (mhv-a ), a model coronavirus in the same genus as sars-cov and mers-cov. the data obtained allowed us to study the kinetics of virus transcription and translation with exquisite precision. we studied the timecourse of positive and negative-sense genomic and subgenomic viral rna production and the relative translation efficiencies of the different virus orfs. virus mrnas were not found to be translated more efficiently than host mrnas; rather, virus translation dominates host translation at later time points due to high levels of virus transcripts. triplet phasing of the profiling data allowed precise determination of translated reading frames and revealed several translated short open reading frames upstream of, or embedded within, known virus protein-coding regions. ribosome pause sites were identified in the virus replicase polyprotein pp a orf and investigated experimentally. contrary to expectations, ribosomes were not found to pause at the ribosomal frameshift site. to our knowledge this is the first application of ribosome profiling to an rna virus. members of the family coronaviridae have the largest genomes of all rna viruses, typically in the region of kilobases (kb). several coronaviruses, including sars-cov and mers-cov, are of medical importance, with high mortality rates and, in the case of sars-cov, significant pandemic potential. other coronaviruses, such as porcine epidemic diarrhea virus and avian coronavirus, are important livestock pathogens. coronavirus infections are frequent in bats and other mammals [ ] and interactions between humans and non-human animal populations presents a constant risk of new zoonotic outbreaks [ ] . recent findings also indicate an evolutionary origin of the established human coronavirus species, human coronavirus e in hipposiderid bats [ ] . the family coronaviridae is divided into the subfamilies coronavirinae and torovirinae. torovirinae includes the genera bafinivirus and torovirus, infecting fish and mammals respectively, while coronavirinae includes the genera alphacoronavirus, betacoronavirus, gammacoronavirus and deltacoronavirus, commonly infecting mammals and birds. sars-cov and mers-cov are members of the genus betacoronavirus. therefore, a useful model for these two viruses, especially with regard to their structure and replication, is murine coronavirus, a betacoronavirus that is commonly referred to as mouse hepatitis virus (mhv). like all coronaviruses, mhv has a monopartite, positive-sense, single-stranded rna genome (grna) (fig a) . the two thirds of the genome contains two long open reading frames (orfs), orf a and orf b, which encode the replicative proteins. these orfs are expressed as two polyproteins pp a and pp ab, where pp ab is a "transframe" fusion of the orf a and orf b products, produced via − programmed ribosomal frameshifting (− prf) [ , ] . polyproteins pp a and pp ab are proteolytically cleaved by virus-encoded proteases, plp and plp (in nsp ) and cl (nsp ) to produce the non-structural proteins nsp to nsp . the third of the genome contains orfs that encode structural proteins and accessory proteins. these orfs are translated from a series of subgenomic mrnas (mrnas to ) produced during virus infection. each subgenomic mrna is identical to a -coterminal region of the virus genome with the exception of a nucleotide (nt) leader sequence at the end that is identical to the end of the grna. these leader sequences are added (as a reverse ribosome profiling is an emerging methodology that facilitates global mapping of the positions of translating ribosomes on the transcriptome, defining at the codon level the extent to which individual mrnas species are engaged in protein synthesis [ ] [ ] [ ] . the technique exploits the knowledge that translating ribosomes can protect from rnase digestion a defined fragment of mrna of around - nt in length [ ] . in ribosome profiling, often referred to as riboseq, cells are lysed under conditions optimised to minimise further ribosome movement (addition of translation inhibitors, rapid freezing), the lysate is treated with ribonuclease (often rnase ) to degrade regions of mrnas that are not physically protected, and the ribosomes harvested on sucrose gradients or through a sucrose cushion. the ribosome pellet is deproteinized, the ribosome-protected fragments (rpfs) harvested by elution from a polyacrylamide gel, ligated to adapters, subjected to rt-pcr, deep sequenced and mapped back to the genome. this analysis reveals the location and abundance of ribosomes on mrnas with up to single-nucleotide precision. the corresponding transcriptome is also determined from the same lysate: total rna is harvested, fragmented, cloned and sequenced to generate a parallel rna sequencing (rnaseq) library. ribosome profiling has been applied to a variety of cellular organisms to address a range of questions in translational control and global gene expression [ , [ ] [ ] [ ] [ ] [ ] [ ] . also, it has been employed in the study of the replication of large dna viruses; namely, human cytomegalovirus [ ] [ ] , kaposi's sarcoma-associated herpesvirus [ ] , herpes simplex virus [ ] , vaccinia virus [ ] , and bacteriophage lambda [ ] , providing insights into the temporal regulation of gene expression in these viruses and identifying numerous previously unrecognized translated orfs, including novel protein-coding orfs and short regulatory uorfs. in this paper, we describe the first analysis of rna virus replication and gene expression by ribosome profiling (and parallel rnaseq), using mhv as a model system. the data obtained allowed us to determine the time course of virus positive and negative-sense rna production, as well as the translation of each of the virus genes, the expression of short and/or previously unannotated orfs, and the efficiency of − prf. we also investigated early time points of infections at high multiplicity to visualise the translation of input genomes. the profiling data also revealed examples of prominent ribosomal pausing within the coding regions for nsp and nsp . nsp ribosomal pausing was confirmed in in vitro translation experiments. surprisingly, we found that ribosomes do not pause appreciably during − prf, arguing against a requirement for pausing in frameshifting. this study also provides insights into the challenges associated with the profiling of rna viruses and suggests strategies that may prove beneficial in future studies. to study the kinetics of virus rna and protein synthesis in a single cycle of virus replication, we performed two independent biological repeats (repeats and ) of an mhv infection time course in which murine clone cells ( cl- ) were infected with recombinant mhv-a at a multiplicity of infection (moi) of and cells harvested at , . , and h post-infection (p.i.), with mock-infected cells harvested at and h. for all time points, two dishes were prepared and, immediately prior to harvesting, cells were treated with cycloheximide (chx) alone, or harringtonine (har) then chx (as detailed in materials and methods), for analysis of elongating (chx) and initiating (har) ribosomes, respectively. subsequently, riboseq (chx), riboseq (har) and rnaseq (chx only) libraries were prepared for each time point, deep sequenced and reads mapped to host and virus sequences (see materials and methods). the composition of each library is summarised in s a table and s fig. virus gene expression at h p.i. as an example of the data provided by our experimental strategy, fig b shows the density of riboseq chx and rnaseq reads mapping to the virus genome at h p.i. in general, there is a to increasing gradient in total ribosome density, with the n orf being expressed at the highest level, the m, , s and orfs at intermediate levels, and orfs a and b at the lowest levels. as expected, very little ribosome density was observed within the defective genes he and . the step reduction in riboseq density between orf a and b reveals the proportion of ribosomes that terminate at the orf a stop codon instead of frameshifting into orf b. in contrast, rnaseq density is essentially constant across orfs a and b, and then steadily increases to , reflecting the cumulative density summed over the genomic rna and coterminal subgenomic transcripts. extra rnaseq density in the utr reflects the leader sequence that is present on all subgenomic transcripts as well as the genomic rna. riboseq density was also observed in the leader, although not corresponding to known coding regions (see below). negative-sense virus rna is present at much lower amounts than positive-sense rna, but follows roughly the same expression patterns, including high density in the (anti)-leader region, consistent with discontinuous transcription occurring during negative-sense rna synthesis [ ] . low levels of negative-sense riboseq reads were also observed but these had length distributions that did not match typical rpf length distributions (see below). thus, these are unlikely to derive from ribosomes loading onto negative-sense rnas (e.g. non-specifically onto uncapped, possibly degraded virus-derived rnas). instead, they may derive from low amounts of rna non-specifically co-sedimenting with ribosomes (see below). since the riboseq analysis represents the product of transcript abundance and translation efficiency, we also plotted the riboseq/rnaseq ratio along the genome (fig c) . this ratio was substantially lower in orf a and orf b than in the coding orfs (except the defective genes he and ), which may indicate that a substantial proportion of genomic rna is not being translated (e.g. sequestered in replication-transcription complexes [rtcs] or destined for packaging) or that genomic rna intrinsically has a relatively low translation efficiency. note, however, that this simple calculation ignores the fact that rnaseq density is present for all orfs on a transcript whereas riboseq density is only present for the translatable orfs (normally the proximal orf). this discrepancy is accounted for in the more detailed analysis of translation efficiencies below. fig shows enlarged views of the virus transcript utr and orfs with linear scales optimized separately for each region. this analysis shows that there is significant variability in the rnaseq read depth within a transcript, which we ascribe to biases such as fragmentation bias, pcr bias and ligation bias. similarly, variability in the riboseq data within a cds may be partly due to nuclease bias, pcr bias and ligation bias but also reflects real variations in ribosome progressivity. the depth of rnaseq reads in the utr is similar to that of the n orf, reflecting that the major contribution to leader sequence comes from mrna . peaks in the riboseq har data highlight the canonical translation initiation sites of the , s, , m and n orfs. in the same dataset, the orf a/ ab initiation peak is dwarfed by rpfs in the leader (presumably mostly coming from mrna ; see below). it should be noted that har arrests ribosomes at initiation, but not during elongation thus allowing elongating ribosomes to run-off. however, in these samples it is apparent that elongating ribosomes have not yet cleared the s orf. we considered it important to assess the quality of the datasets that were obtained by our experimental strategy. for rpfs derived from non-organellar ribosomes of eukaryotic organisms, mapping of the end positions to coding sequences (cdss) characteristically reflects the triplet periodicity (herein referred to as "phasing") of translational decoding [ ] . good phasing within datasets is beneficial in assigning orfs with confidence, particularly if such orfs are very short or overlap. the extent of phasing can vary between protocols and libraries due, presumably, to variation in the efficiency of rnase i (or other nuclease) trimming or other factors. s fig (repeat ) and s fig (repeat ) show, for each library, histograms of the codon positions to which the ends of host mrna reads map for different read lengths. the riboseq libraries show excellent phasing with the majority of rpf ends mapping to the first codon position. conversely, and as expected, the ends of rnaseq reads had a nearly uniform distribution between the three possible codon positions. the riboseq read length distributions were typically sharply peaked at around nt consistent with other analyses [ ] , while those of rnaseq were much broader, consistent with a length distribution set by the size of the gel slice excised during purification of fragmented rna in the rnaseq protocol (approximately - nt). s fig shows the distribution of host mrna rpf ends relative to initiation and termination codons, summed over all host mrnas in each of the riboseq libraries. for all samples, a discrete peak in rpf abundance was observed just upstream of the initiation site. as noted previously, the peak is probably largely a result of drug treatment-either har which specifically arrests initiating ribosomes, or chx which arrests elongating ribosomes but allows ribosomes to continue to accumulate at initiation sites [ ] . this peak corresponds to the ends of rpfs derived from initiating ribosomes with the aug codon in the ribosomal p-site, and allows calibration of the offset between the rpf end and rpf p-site position, which, for these libraries, is normally nt (e.g. s fig) . for many samples, a discrete peak was also observed nt upstream of the stop codon, corresponding to ribosomes pausing during termination (with the stop codon in the ribosomal a-site). the presence of this peak appears to be subject to minor variation in sample preparation as it was not consistent between repeats (cf. repeat and repeat , riboseq chx mock h in s fig) . in contrast to [ ] , we believe that the clear spike four codons downstream of the initiation peak is an artefact of ligation bias (and potentially also other biases): every read mapping to this position begins with -aug (thus compounding any ligation preferences), whereas reads that map to the initiation peak have different and ends in different mrnas (thus averaging out any ligation preferences). for -nt reads, a trough was also apparent four codons upstream of the termination peak (s fig); this corresponds to reads that all end in uag- , uaa- or uga- , and again is likely to be an artefact of ligation bias. peaks at the start and stop codons were also apparent for rnaseq data, corresponding to reads with ends aligning to the a of aug and the middle nucleotide of the stop codon, respectively (s fig, right) ; the latter is not visible in riboseq data due to low riboseq density in the utr. a peak nt upstream of the aug (more noticeable in repeat samples, s fig, left ) together with a very low level of phasing within the cds (s fig) likely represents a low level of contamination of rnaseq samples by riboseq samples, although the latter could potentially also be a result of codon usage bias, e.g. a preference for rny codons [ ] , compounded with ligation biases. averaged over all host mrnas, very few rpfs were observed in utrs while a larger but still low level of rpfs were observed in utrs (s fig and s fig) . the latter may largely derive from translation of uorfs in various locations and phases with respect to the main orf of each mrna [ ] . we also observed a remarkable perturbation in host cell translation at late time points (s fig, lower panels-riboseq chx, compare and h p.i. with and h mocks) that was not mirrored in rnaseq data (s fig) and could be a consequence of cell stress [ ] [ ] [ ] . this phenomenon and other host cell responses to virus infection will be discussed in future work. we also addressed the issue of possible contamination during sample preparation as we expected that rnaseq and riboseq analysis of virus-infected cells may present some specific challenges. for example, late in infection, virus rna can be produced at very high levels and extreme care is required to minimise cross contamination between late and early time-point libraries. indeed, a comparison of read length distributions of host-derived rna and virusderived rna revealed contamination of this type in some of our libraries, despite great care in processing experimental samples (s fig and s fig) . for example, in the first biological repeat (s fig) , the virus and host length distributions in the h p.i. riboseq chx sample were almost identical. however, for the and . h p.i. riboseq chx samples, virus and host length distributions were dissimilar to each other but instead the virus length distribution resembled the riboseq chx h p.i. length distribution, suggesting contamination of virus rpfs from the later time-point sample into the earlier time points. the absolute amount of contamination was very low and would have little effect on host mrna analyses; however, relative to the amount of virus rna at and . h p.i., it was significant. contamination was also apparent for the and . h p.i. riboseq har samples and the h p.i. rnaseq sample. similarly, the mock-infected controls each contained~ - virus reads (cf.~ - million at late time points of infection) (s a table) . in the second biological repeat, the mock samples were evidently less contaminated, containing from only to virus reads each (s a table) . nevertheless, traces of contamination were still apparent in the and . h p.i. riboseq chx and h p. i. riboseq har samples (s fig). a different type of contamination was observed for the h p. i. riboseq chx sample in repeat . here, the host read length distribution was broad compared to the virus read length distribution, and the host mrna phasing was poor (s fig and s fig, respectively) . this suggests that this sample is contaminated with rnaseq material from a sample containing little or no virus rna, thus affecting the host mrna length distribution but not the virus rna length distribution. in subsequent discussions of the mhv profiling data, any samples suffering from contamination have been excluded, or subjected to appropriate caveats. another potential source of "contamination" in our experimental strategy is the problem of non-ribosomal ribonucleoprotein (rnp) complexes. for example, certain virus proteins have rna binding properties and can associate with viral and, potentially, cellular rna. these rnp complexes may co-sediment with ribosomes and lead to contamination of riboseq libraries. such contamination may be revealed by unusually high read density in host mrna utrs (which normally have very low rpf occupancy) and differences in read length distributions [ ] . s fig and s fig show length distributions for all libraries for reads mapping within to codons upstream (green; cds) or downstream (orange; utr) of cds termination codons. in all riboseq libraries, the utr read density was extremely low compared to the cds read density (left plot of each pair). (it should be noted however that, as har enriches for initiating ribosomes, the above analysis is not well-suited to har samples.) for comparison, the rnaseq library utr read density was typically~ % of the cds read density (that it is not % likely reflects the presence of transcripts with utrs that are shorter than the annotated utrs). since the analysis is based on mapping to ncbi refseq mrnas, a low level of utr occupancy derives from genuine rpfs derived from coding exons in one splice form that have alternative mappings to the utr in another splice form. further, low levels of post-termination unrecycled s ribosomes may enter the utr [ ] [ ] [ ] . thus, for mock infections, the utr riboseq read length distributions largely matched those of the cdss (s fig and s fig, and h mock chx), albeit with some differences (e.g. a high-end tail) arising from unknown sources of contamination potentially including host protein:mrna rnps. while such contamination is expected to be present throughout the mrna, it is more apparent in the utr due to the much lower density of bona fide rpfs in this region. for infected samples, the host mrna utr density for chx samples was similar in magnitude ( . - . %) to that of the mocks ( . - . %), except for the h p.i. time points where the utr density was . - . % of the cds density (s fig and s fig) . consistent with the probable rnaseq contamination discussed above, the length profile of the h p.i. chx repeat sample was broad for both the cds and utr regions. on the other hand, the length profile of the h p.i. chx repeat sample was not qualitatively different from that of the mocks, suggesting that the increase in utr occupancy might not simply be explained by virus-induced rnps, but rather, or as well, by an increase in bona fide rpfs in the utrs. a mechanism for the latter could be overloading of the host cell ribosome recycling factors (abce and any cofactors), allowing an increase in post-termination unrecycled s ribosomes entering the utr [ ] . if a proportion of late time-point contamination results from virus proteins interacting with mrna to form rnps, it may be significantly higher for virus rna than for host mrna, as virus proteins are likely to interact selectively with virus rna; for example, through specific binding signals or via compartmentalization within the cell. excess contamination in the virus rpf fraction may be gauged by comparing length distributions of reads mapping to virus positive-sense rna with length distributions of reads mapping to host mrna cdss. reassuringly, in all cases, the virus positive-sense riboseq reads showed a similar or even tighter length distribution at late time points than the host riboseq reads (s fig and s fig; h.p.i and h p.i., chx and har). in contrast, as mentioned above, the small quantity of negative-sense virus reads in the riboseq samples had very different length distributions (s fig and s fig) indicating that they are unlikely to be true rpfs; such reads comprised < % of virus reads for all riboseq samples, and < . % for the two h p.i. chx repeats. fig a shows a time course of the total amount of virus rna expressed as a fraction of total virus rna plus host mrna, for both riboseq chx and rnaseq samples. samples with contamination (see above) could only be used to give upper bounds (grey symbols). total virus translation as a fraction of total cellular translation increased to , -fold from to h p.i., while virus positive-sense rna increased to -fold over the same time period. in repeat , virus translation and rna appeared to have reached a maximum by h p.i., while infection progressed a little slower in repeat . from h p.i. to . h p.i., the positive-sense rna fraction remained roughly constant (presumably reflecting the input rna) while the negative-sense rna fraction grew from essentially negligible amounts to~ . % of total virus rna and host mrna (fig a) . at late time points, virus negative-sense rna ceased to increase, whilst positive-sense virus rna showed significant increases (fig b) . at the later time points, virus translation had reached~ - % of total cell translation and positive-sense virus rna had reached~ - % of total virus rna plus host mrna. at the same time, negative-sense virus rna represented~ . % of total virus rna and host mrna ( fig b) . these findings are consistent with previous analysis of virus rna synthesis in mhv-a -infected cells [ ] . virus infection and the kinetics of viral protein expression over the time course were confirmed by western blot with antisera to the n, s and nsp proteins ( fig c) . we also calculated the levels of transcription and translation for each virus orf throughout the time course ( fig a) . note, again, that the data only provide upper bounds for the raw rnaseq densities represent the cumulative sum of densities for all mrnas that cover a given genome region. subtraction of the density for the immediately upstream inter-trs region gives an estimate of the rnaseq density for a specific mrna, herein referred to as the "decumulated" density. rnaseq densities for mrna are omitted as it is not expressed at a sufficiently high level relative to mrna to apply the "decumulation" procedure. lower right: estimated mean negative-sense rnaseq densities for each of the negative-sense subgenomic rnas , , , , and (anti)-grna. lower left: mean riboseq densities for each of orfs a, b, , s, , e, m and n. the density for n includes any rpfs deriving from the overlapping i orf. riboseq densities for the defective orfs he and are omitted. circles and solid lines correspond to repeat ; crosses and dotted lines correspond to repeat . due to low levels of reads and contamination (see text), values for h p.i. and . contaminated samples (as indicated in fig ) . the particularly contaminated repeat riboseq h p.i. data are omitted from fig a, while the upper bounds provided by the cleaner repeat are included as they are likely to be more accurate. to calculate translation efficiencies, it is necessary to determine the amount of each virus transcript but, in the case of coronaviruses, raw rnaseq densities represent the cumulative sum of genomic rna and all subgenomic transcripts. for example, for the n orf, rnaseq density includes contributions from mrnas to and grna. thus, to calculate the amount of mrna , we subtracted the positive-sense rnaseq density in the region of mrna upstream of the mrna trs from the density in the mrna region. we then followed a similar procedure for all other mrnas. the same analysis was also applied to the negative-sense virus rnas and these "decumulated" values are plotted in the right-hand panels of fig a. due to the low production of mrna relative to mrna , the amount of mrna could not be estimated in this way. we also omitted the h p.i. time point due to the low levels of virus reads (s a table) . translation efficiencies were calculated by dividing the raw riboseq densities for each orf by the decumulated rnaseq densities for the corresponding mrna. note also that initiation and termination peaks were excluded from the riboseq density calculations (see methods). the orfs , s, , e, m and n are all translated at comparable efficiencies ( fig b) . the translation efficiency of e was at the lower end, presumably due to it not being the proximal orf on its transcript (mrna ) [ ] . the translation efficiency of n was also at the lower end. the translation efficiency of orf a/ ab was, in comparison to the orfs, very low. as mentioned above, this could be due to a proportion of grna being present in an untranslatable pool, perhaps as rtcs or rnps destined for packaging, but may also indicate a real restraint on orf a/ ab translatablity (see below). the grna translation efficiency calculated in this way was low even at . h p.i. (repeat , orf a translational efficiency~ . ). on the assumption that grna will not be directed to a packaging pathway at early time points, this suggests that incoming and early synthesis grna is largely involved in rna synthesis, or is, indeed, inherently poorly translated. it should be noted that technically these calculations do not measure translational efficiency absolutely, as ribosome occupancy may also be affected by translational speed (though, when averaging over orfs, this effect is thought to be generally quite slight; [ ] ). further, as new transcripts enter the translation pool, there may not have been time to establish steady state ribosome loading on any particular transcript, while, at late time points, translational efficiencies may be below their optimal values due to saturation of the host cell protein synthesis machinery. transcript abundances can be calculated from the decumulated rnaseq densities (as above) or, independently, from the relative abundances of rnaseq reads spanning each leader/body junction. such "chimeric" reads (where the part maps to the leader sequence, and the part maps just downstream of a body trs) were not included in the initial mapping to the virus genome ( fig b) , but were identified subsequently (see materials and methods). fig com pares mrna abundances estimated using these two methods. the "trs method" has the advantage that it avoids the potential inaccuracies introduced by decumulation but may be more subject to fragmentation, ligation and pcr biases due to the relatively short window in which to calculate a mean rnaseq density. nonetheless there is a good correlation between the two estimates (r = . ). in mhv, the consensus for canonical trss is ucuaaac with minor exceptions being ucuauac for mrna and uccaaac for mrna [ ] [ ] [ ] [ ] . a variable number of tandem copies (two in mhv-a ) of ucuaa are present at the leader junction site, while an imperfect copy of ucuaa precedes the canonical ucuaaac at several body junction sites (s table) . heterogeneity in the number of copies of the pentanucleotide has previously been observed to occur in mrna for mhv-a , and both mrna and mrna for mhv-jhm, and this is presumably due to heterogeneity in the site of re-annealing following a polymerase jump [ ] . consistent with this, we also observed significant usage of a junction site nt upstream of the canonical site for mrna ( - % of mrna transcripts) (s table) . we also observed this phenomenon for mrna ( . - . % of mrna transcripts). the greater usage for mrna is likely due to it having an imperfect pentanucleotide uccaa at the canonical junction site but a perfect pentanucleotide ucuaa nt upstream; in contrast, other mrnas have a better pentanucleotide match at the canonical site than at the site nt upstream (s table) . for mrnas showing such heterogeneity, the summed values were used for fig . for mrna , where the upstream pentanucleotide is ccuaa instead of ucuaa, we observed that the first nucleotide could be templated either by the body sequence (i.e. 'c';~ %) or by the leader sequence (i.e. 'u';~ %) (s table, nt sequences) . we also observed many non-canonical leader/body chimeric sequences (s table) , though even the most frequent were present at % the level of leader/body chimeric reads for mrna (the least abundant canonical mrna). the coronavirus polymerase is known to engage in promiscuous jumping [ ] [ ] [ ] and there is no reason to suppose that the additional transcript species generated this way are functionally relevant. two of the most abundant (genomic coordinates and in s table) involved apparent backward jumping by the polymerase (although inter-template jumping is another possibility). the sequences at non-canonical junctions often partly resembled canonical trss (e.g. ucuaaaa at nt , ucucaac at nt , ccuacuu at nt , uccaagc at nt and uguaaua at nt ; canonical trs nucleotides in upper case). in cases where the nucleotides at + to + in the genome sequence differed from uc, the rnaseq read generally contained nucleotides templated by the genome sequence rather than the uc in the leader sequence (e.g. cc instead of uc for the nt junction), although there were exceptions (e.g. uc instead of aa for the nt junction) (s table) . this latter site, aauaagc, aligns with a trs previously identified for an he mrna in the jhm strain of mhv [ ] . the sequence in mhv-jhm is aauaaac, differing from the mhv-a sequence by a g to a substitution. an he mrna has not been observed for mhv-a and this is likely due to the greater deviation from the consensus trs, ucuaaac, in this strain [ , ] . although we observe some usage of this site in our sequencing, the levels are extremely low-just . - . % those of mrna (the least abundant canonical mrna). fig b. the latter are calculated on a per gene (rather than per transcript) basis, using rnaseq and riboseq reads contained entirely within annotated cds regions (i.e. excluding and utrs and also rpfs accumulating at or near to initiation or termination sites), and, like the virus values, are expressed relative to the mean levels for the cell (due to normalization by library size). the analysis shows that the virus translation efficiencies fall within the general range of those of host genes (except for orf a/ b which have particularly low translation efficiencies; see above) indicating that virus transcripts are not preferentially translated during virus infection. instead, massive production of virus proteins (in particular the n protein) is achieved through high levels of transcription. to study virus rna synthesis and translation during the earliest stages of infection, we did high moi (~ ) infections to maximize the number of virus reads in the libraries. the composition of the high moi libraries is summarized in s b table and s a fig. fig a shows the distributions of riboseq and rnaseq reads on the virus genome at h p.i. (where h p.i., is the time at which the inoculating virus is removed). a to decreasing gradient in rpf density is visible within orf a in the riboseq chx density profile, while very few rpfs were found within orf b, indicating that, at h p.i., ribosomes have only had time to translate part of the -codon orf a. this does not indicate the translate rate per se, as time is also required for uncoating, recruitment of ribosomes, translation of a uorf on the grna (see below), and potential delays with initiation and reinitiation (see also below). in the riboseq har data, a clear trough in rpf density is visible after the orf a initiation peak, followed by higher density further downstream in orf a. the trough reflects run-off of elongating ribosomes in the three minutes between addition of har (which inhibits new initiation events) and chx (which freezes the ribosomes). taking the width of the trough as~ codons, this gives an elongation rate of . amino acids s − , similar to that determined previously in mouse embryonic stem cells ( . amino acids s − ) [ ] . despite the very high moi, virus rna levels were low except, unexpectedly, in the n region where the mean rnaseq density was~ times that in the orf a region. to test whether this might be due to contamination from late time-point samples, we compared the length distribution of reads in the n region with the length distribution of reads mapping to host mrnas for the same sample ( fig b, right panel; red and green lines, respectively). the two distributions were very similar. in contrast, the length distributions of virus-derived reads from the and h p.i. rnaseq time points (from repeat which was co-processed with the high moi libraries) were different in shape (fig b, right panel; grey lines). while it is impossible to definitively rule out contamination in this way, the analysis suggests that the rnaseq density in the n region at h p.i. is not a result of contamination. since, for mrna , negative-sense rna is present at > . % of positive-sense rna at . , and h p.i. (fig ) , the absence of appreciable levels of negative-sense reads mapping to the n region in the high moi h p.i. sample ( negative-sense compared with , positive-sense reads; . %) also argues strongly against the positive-sense reads being inter-library contaminants. the near-complete absence of negative-sense reads also argues against this phenomenon being a result of early synthesis. moreover, the absence of equivalent rnaseq density in the leader region (cf. fig ) argues against the density in the n region deriving from bona fide mrna transcripts. closer inspection revealed a number of a relatively abundant chimeric reads suggesting a mosaic structure of rearranged n-orf sequences, reminiscent of defective interfering (di) rnas [ , ] . however, since coronavirus di rnas are expected to include parts of the end of the genome and a packaging signal, and only arise after multiple high-moi passages, we believe the n orf transcripts we have identified must represent a novel class of packaged transcripts. an alternative, albeit unlikely, explanation is that the excess density may be a result of selective degradation (either natural or artifactual) of~ % of the input grna. relative to rna levels, very few rpfs mapped to the n orf region and we were unable to ascertain whether or not they resulted from contamination from other samples as, in contrast to rpfs from orf a, their length distribution only partly matched the length distribution of host rpfs (fig b, left panel, red line). using these rpf counts, the n orf translation efficiency (normalized to total virus rna and host mrna) was calculated to be only . , compared to values in the range . to . at the . , and h p.i. timepoints, indicating that the early timepoint n orf rna revealed by rnaseq is not, or only barely, translated. the − prf signal that facilitates expression of mhv pp ab comprises two elements, a heptanucleotide slippery sequence (u_uua_aac), identical in all known coronaviruses, and an rna pseudoknot structure located a few nucleotides downstream [ , , ] (fig a) . during translation of the grna, elongating ribosomes either terminate at the orf a stop codon, yielding pp a, or frameshift on the slippery sequence to translate orf b, yielding pp ab. frameshifting likely provides a fixed ratio of translation products for assembly into a macromolecular complex [ , ] . studies of frameshifting using reporter constructs expressed in transfected cells or through in vitro translation of synthetic mrnas have indicated that the efficiency of the process in coronaviruses is generally in the region of - % [ , , [ ] [ ] [ ] however, the actual efficiency in the context of virus infection has not been previously determined. simplistically, one can calculate this value by dividing the riboseq density in orf b by the density in orf a. however, in principle, riboseq density represents the quotient of expression level and translational speed so the above calculation assumes that, on average, translation speed is the same in orfs a and b and that translation is steady state. such a calculation is, therefore, invalid immediately after infection (as ribosomes begin to translate orf a of the input grna but have not yet reached orf b; fig ) and may also be compromised if newly synthesised grna entering the translation pool represents a significant fraction of the grna undergoing translation. thus, we measured the frameshifting efficiency at h p.i., calculating values of % for repeat , and % for repeat ( fig b) . the former value ( %) is similar to previous in vitro measurements of the mhv frameshifting efficiency ( %) [ ] . as the infection appeared to be more advanced in repeat (fig ) , it is possible that the higher level measured ( %) is a consequence of depletion of the host cell protein synthesis resources, e.g. exhaustion of initiation factors (including free ribosomes) could decrease the density of ribosomes in orf a as elongating ribosomes run off, and a partial exhaustion of elongation factors could slow the establishment of a new steady state. we also measured the frameshifting efficiency by means of transfected reporter constructs. we began by cloning a -bp fragment including the mhv frameshift signal (fig a) into a dual luciferase frameshift reporter plasmid (pdluc; [ , ] ) between the renilla (rluc) and firefly (fluc) luciferase genes. in this plasmid, frameshifting permits expression of fluc as a fusion with rluc (analogous to the expression of mhv pp ab), while failure to frameshift results in expression of rluc alone. frameshifting efficiencies were calculated from the ratio of fluc activity to rluc activity, normalized by a control construct in which an extra c residue was inserted immediately downstream of the slippery sequence to place rluc and fluc in the same reading frame. the well-studied coronavirus frameshifting signal from avian coronavirus, infectious bronchitis virus (ibv) served as a positive control, alongside a lower efficiency control (the gag/pol − prf signal of hiv isolate hxb ) [ , ] . the mhv frameshifting efficiency was found to be % in cl- and % in bhk- cells, and similar in both instances to that of ibv ( fig c) . these data suggest that frameshifting in coronaviruses is not specifically modulated by virus infection, with the difference seen in the more advanced infection of repeat likely due to the non-specific effects mentioned above. the relevance of ribosomal pausing to the mechanism of − prf has long been a subject of debate [ ] [ ] [ ] . frameshift signal-associated pauses have been documented in a number of in vitro assays [ ] [ ] [ ] [ ] [ ] [ ] , but there is, as yet, little evidence for a causal relationship, and pausing has not been examined in infected cells. we therefore looked to see whether there was an accumulation of rpfs at the mhv frameshift site. in the initial riboseq time courses we failed to see significant pausing at the frameshift site. however, reasoning that the frameshift-stimulatory pseudoknot beginning nt of the slippery heptanucleotide u_uua_aac would be partly inside the mrna entrance channel at the onset of frameshifting, and might, due to its compact structure be somewhat resistant to rnase digestion, we considered the possibility that frameshift-associated pauses may generate longer rpfs, which would be excluded from the profiling analysis as a result of gel size selection ( - nt). thus we prepared new libraries from the h p.i. repeat riboseq chx samples (see s c table for composition) using a larger gel slice (nominally - nt). however, even in these samples we failed to see noticeable pausing at the frameshift site (s fig) . although we failed to identify significant pausing at the frameshift site, there were other sites at which rpfs accumulated to a much higher level than at neighbouring sites. we frequently observed such accumulations at initiation sites (possibly an artifact of chx treatment; [ ] ) (fig and fig ) , but also at internal sites within orfs. besides ribosome pausing, fluctuations in rpf density may occur as a result of nuclease, ligation, and pcr biases. the latter two occur also for rnaseq, whilst in rnaseq nuclease bias is replaced by fragmentation bias. following [ ] , we compared the distributions of variability in riboseq and rnaseq densities within orf a, which revealed that riboseq densities were more variable than rnaseq densities ( fig a) , with the extra variability presumed to be a result of fluctuations in ribosome progressivity. we focused on two of the highest riboseq density peaks in the orf a region (blue arrows in fig b) . rpfs at the second of the two pause sites, located in the nsp region, have ends that map almost exclusively to nt which, unusually, corresponds to the second codon position (fig c, right) . the end positions of these rpfs were, as is normal, more variable, with the most abundant ends mapping to nt - for repeat and nt - for repeat , giving read lengths of - and - nt which are within the typical range for the respective samples ( s fig and s fig) . for these samples, riboseq chx h p.i. repeats and , % and %, respectively, of host mrna rpfs have ends mapping to codon position , with only % and % mapping to codon position . the reason for the deviation at the pause site is unknown but may be a result of "tension" within the mrna or perturbation of the ribosome conformation [ ] . due to the unusual codon position of the end, it was not possible to definitively predict the p-site position of ribosomes at this pause site, but it is more likely to be at nt to (aaa codon) than at nt to (cag codon) as the lengths of the most abundant reads ( - and - nt in repeats and , respectively) are more consistent with reads being nt shorter than normal at the end, rather than nt longer. the nascent peptide sequence (i.e. peptide sequence within the ribosome exit tunnel that could potentially affect pausing) here is. . .ikhkhlyltmyimpvlctlfytnylvvykq (p-site amino acid underlined). an additional smaller peak was apparent nt upstream (in repeat ) and potentially corresponds to a following ribosome stacking behind a proportion of paused ribosomes. rpfs at the first of the two pause sites, located in the nsp region, have ends that map to nt in repeat but nt in repeat (fig c, left) . however, this nt difference is made up in the length of the reads (top three read lengths , and nt in repeat , but , and nt in repeat ) so that the ends of the rpfs map to similar positions in both repeats. incidentally, this difference in end position between the two repeats makes it highly unlikely that the peak is an artefact of ligation bias. in general the nuclease trimming seems to be less stringent in repeat than in repeat (host mrna rpf lengths peak at - nt in repeat but at nt in repeat ; s fig and s fig). the pause site read length in repeat is unusually long, indicating that the extra~ nt at the end are, for some reason, partially protected, resisting trimming in repeat but not in the more stringently trimmed repeat . the nature of this protection (scrunching of extra mrna into the mrna exit channel, formation of an rnase-resistant rna structure -adjacent to the ribosome, conformational changes in the ribosome, or an additional ribosome/mrna-associated protein factor) and whether and how it is linked to pausing remain undetermined. the nascent peptide sequence is. . .ekcqvtsvagtkalslqlaknlcrdvkfvt (p-site amino acid underlined). the nascent peptides at both pause sites lack the e-or p-site prolines or a-site gaa codons that are commonly associated with pausing in ribosome profiling meta-analyses [ ] , though many diverse nascent peptides are also known to perturb ribosome progressivity [ , ] . as an alternative possibility, we analysed the rna downstream of the pause sites for evidence of stable rna structures that could induce pausing, but nothing was apparent. an alternative explanation is that these pauses are induced by trans-acting factors, e.g. rna binding proteins, or chaperones of the nascent peptide. coronaviruses induce substantial membrane rearrangements in the infected cell, including formation of a reticulovesicular network composed of two types of membrane modifications, double-membrane vesicles (dmvs) and convoluted membranes (cm). the reticulovesicular network is contiguous with the endoplasmic reticulum (er) and is the site of virus rna synthesis [ , ] . nsp , nsp and nsp are integral membrane proteins whose topology has been determined in vitro for sars-cov and mhv [ ] [ ] [ ] [ ] , and, in the case of sars-cov, have been shown to be necessary and sufficient for double-membrane vesicle formation [ ] . nsp is the largest protein encoded in mhv orf a and contains multiple domains, including two small ubiquitin-like domains (ubl and ubl ), two papain-like cysteine proteinase domains (plp and plp ), a poly-adp-ribose-binding activity (adrp domain), the newly determined domain preceding ubl and plp (dpup; [ ] ), the nucleic-acid binding domain (nab), the betacoronavirus marker (g m), a transmembrane domain (tm) and domain y (fig a) . the apparent ribosome pause occurs within the sequence dvkfvtnac (p-site at pause underlined; fig a) which is located between the adrp and the dpup domains. we investigated the nsp pause in vitro in rabbit reticulocyte lysate (rrl) translations using edeine assays [ ] . initially, a cdna fragment comprising the first , residues of nsp (nsp à ; excluding the nab, g m, tm and y domains) was cloned into pcdna . and synthetic mrnas translated in rrl for min prior to addition of the translation initiation inhibitor edeine. incubation was continued, samples withdrawn at the indicated times post-edeine addition and translation products separated on a % sds-page gel (fig b) . to accurately mark the position of the predicted pause product, a control mrna in which a uaa stop codon had been introduced at the pausing a-site was also translated. as seen in fig b (marked by a red asterisk) a distinct translational pause was observed during translation of nsp à , migrating at the same position as the "pause control" and accumulating and then samples were withdrawn at the indicated times after edeine addition, and translation products separated on a % sds-page gel and detected by autoradiography. mw indicates c-labelled molecular weight standards and h o as a negative control. the predicted position of the pause product was determined from the "pause control" lane (see text). (c) time course of translation of pps /nsp -derived mrna in rrl as above. pps contains, under the control of the sp promoter, a copy of the influenza virus pb gene into which has been inserted cdna encoding the nsp pause region (red) plus upstream residues. (d) ribosomal pausing assays of pps -nsp mutant mrnas in rrl ( min at °c). in each case, positively charged or aromatic amino acids were changed to alanine. in mutant , lys-phe at the pausing site was changed to ala-ala, and subsequent mutants were prepared sequentially from this clone, thus mutant contains six substitutions (see text). (e) ribosomal pausing assay of pps /nsp mut mrna in rrl as described above. in all panels, the pause product is indicated by a red asterisk. diminishing as translation proceeded. to more closely define the stalling sequence, a region encoding amino acids of nsp including the putative pausing peptide was cloned into the influenza pb reporter gene in transcription vector pps (fig c) [ ] and edeine assays performed as above. once again, a clear ribosomal pause was evident (fig c, red asterisk) . the nsp sequence within pps includes five upstream positively charged amino acids (four lys and one arg) and one aromatic residue (phe) that could potentially contribute to pausing [ ] . these residues were mutated to alanine sequentially and incrementally (pps -nsp mutants), such that in mut , lys-phe adjacent to the pausing site was changed to ala-ala, mut had these changes plus arg to ala, and so on, as shown in fig d. edeine assays were performed and a single time point ( min) from each mutant analysed by sds-page. as seen in fig d, pausing was obviated in mut , mut and mut , indicating that the residue substituted by alanine mut is likely to be a major contributor to the ribosomal pause. a complete timecourse of pps -nsp mut confirmed the lack of pausing (fig e) . fig a) , is present on all mrnas so reads mapping to this region may derive from any mrna, although most are expected to derive from the highly abundant mrna . the plot excludes "chimeric" reads (i.e. reads that span a trs transcriptional discontinuity), so the rnaseq density drops close to the trs site and the same is also expected to happen for riboseq. probing of initiation sites through harringtonine treatment revealed unexpectedly that a substantial number of reads accumulate at or near the end of the leader, despite an absence of aug codons. these -proximal reads have a tight length distribution characteristic of true rpfs (fig b; left panel) so are likely to be bona fide rpfs rather than some form of contamination. the portion of the leader contains a number of potential near-cognate non-aug initiation codons, but most of the harringtonine reads do not obviously map to these. for example, the most abundant rpf position corresponds to a gcg codon (genome coordinates [ ] [ ] [ ] ; initiation at this point would generate a amino acid peptide, but it should be noted that gcg is not a recognised non-aug initiation codon. elongation profiling with cycloheximide revealed a similar pattern of reads in the part of the leader but also a larger peak on a uug codon close to the end of the leader sequence ( fig a) . uug is a known, albeit quite inefficient non-aug initiation codon [ , ] and, in this case, it is also in a poor initiation context (cucuuguag; in mammals contexts with an a at − , or a g at − and a g at + , may be regarded as "strong"; [ ] ), so only a very small proportion of ribosomes would be expected to initiate here. consistent with this, the har peak is very small compared to that seen at the n initiation codon ( . %; fig c) (though similar in magnitude to initiation peaks at the uorf and orf a on the genomic rna; fig a) . interestingly, the difference between the uug peak and the n initiation peak was much less for the chx samples ( %; fig c) . the reasons for this are unknown, but may be related to the uug codon being immediately followed by a termination codon, with the peak potentially being derived from both initiation and termination pauses (uug in p-site, uag in a-site). we note also that, on mrna , the uug codon is nt upstream of the n initiation codon, so that initiation at n might lead to stacking of ribosomes on the uug codon, potentially increasing initiation on this ostensibly very weak start codon. downstream of the leader trs but upstream of orf a, is a single, short aug-initiated uorf that is present in many coronaviruses and believed to play a role as a regulator of genomic [ ] . upstream orfs are present in~ % of mammalian mrnas and have been shown generally to cause repression of translation of the downstream (main) orf [ , ] . we observed rpfs mapping specifically and in-frame to the uorf, confirming that it is translated. indeed, at h p.i. it appeared to be translated as efficiently as orf a (fig a) despite its poor initiation context (uccaugc; cf. auaaugg for orf a) suggesting that it inhibits ribosomal access to orf a. this effect appeared less pronounced at early time points, suggesting a potential role for temporal regulation of replication protein synthesis (fig a, bottom panel) . interestingly, we observed the greatest density of rpfs on the second codon (proline) rather than the first codon (methionine) of the uorf, both for har and chx-treated samples. prolines are often associated with ribosome pausing due to their restrained geometry in the decoding centre and/or ribosome exit tunnel [ , , ] . to see if n-terminal met-pro was associated with ribosomal pausing on other mrnas, we compared mean ribosome profiles for host mrnas with cdss beginning with aug-ccn with mean ribosome profiles for generic host mrnas and found that, particularly under conditions of virus infection, ribosomes tend to pause more at the second codon in the former ( fig b) , although the ratio of ribosome occupancy between the aug and ccn averaged over host mrnas was less extreme than is the case for the virus uorf. it should be noted that, although presence of the uorf is conserved in of ncbi betacoronavirus refseqs, ccn occurs as the second codon in only six of these. har) and rnaseq reads that map near to the ends of the he, and orfs. again, "chimeric" leader/body reads spanning transcriptional discontinuities at the trs sites are excluded from these plots. in the laboratory-adapted strain mhv-a , the he orf is disrupted by a premature termination codon (red diamond, fig a) [ ] , and, furthermore, the trs upstream of he in mhv-a is defective (open green box, fig a) [ ] , leading to only very low levels of he mrnas (see above). although ribosomes were observed to initiate at the authentic he aug codon, upstream of the premature termination codon (fig a, har) , very little riboseq density was observed downstream of the premature termination codon. translation of the annotated he orf (i.e. the long fragment; grey, fig a and fig a) was negligible, consistent with the presence of numerous aug codons in other reading frames downstream of the "authentic" he start codon, which would be expected to inhibit ribosomal access to the fragment of he. the low level of initiation noted at the "authentic" he start codon is likely explained by the very low levels of he mrna production inferred from the observation of a few rnaseq reads crossing the he leader/body transcriptional discontinuity (see above and s table) , since leaky scanning on mrna is unlikely to allow access to he due to the large number of intervening aug codons. similarly, in mhv-a the natural orf coding sequence is split by a frameshift mutation into a short orf a (pale yellow, fig b) and a longer orf b (grey, fig a and fig b) [ ] . again, we observed ribosomes initiating at the orf a aug codon (fig b, har , the blue peak is in the orf a frame), but very little riboseq density in the annotated orf b. ribosome access via leaky scanning to orf b would be inhibited not only by the orf a aug but also by an additional out-of-frame aug codon (fig b) . upstream of orf a, but downstream of the mrna trs junction, a low level of initiation appeared to occur on an auu codon (riboseq, har, orange peak). ribosomes initiating here would translate a -codon orf resulting in the peptide mysiliatwprkrqs (assuming the initiating codon auu is decoded as met). a similar orf is present in other strains of mhv. upstream of orf , we identified an alternative initiation site at a cug codon (fig c, har , blue peak) which may have some bearing on the mechanism of expression of the e orf, which lies downstream of orf on the bicistronic mrna (fig c) . the cug codon in question is in the same reading frame as the upstream orf and initiation here would result in translation of the last codons of orf with peptide sequence mvvhillrhcpgi (assuming the initiating codon cug is decoded as met). the cug is downstream of the mrna trs and appears to be utilized only on this mrna as the riboseq density on the upstream part of the defective orf (see above) is negligible. the level of initiation at the cug was comparable to that at the orf aug ( fig c) and translation of this short orf might be utilized to shunt a proportion of ribosomes past the orf aug codon. we also observed utilization of an aug codon just downstream of the orf aug codon (fig c, har , orange peak, sixcodon orf, peptide sequence mdlace). access to this aug is likely facilitated by the poor initiation context of the orf aug (cauauga). after translating a very short orf (e.g. < codons), the small subunit of the ribosome can remain associated with the message, resume scanning, and reinitiate translation at a downstream aug codon [ ] . after translation of a short orf, the s subunit of the ribosome is not immediately competent to reinitiate, but becomes competent after scanning for some distance. thus, after translating the short cug-initiated orf, it is possible that the post-termination s subunits can scan past the five aug codons present within the first nt of orf (green +s, fig c) , before becoming initiation competent and able to reinitiate translation at the next available aug codon, which is the initiation codon for the e orf some nt (fig c) (see also [ ] ). the presence of an upstream cug-initiated short orf is preserved in other strains of mhv, though most (other than mhv-a ) also have a separate aug-initiated (albeit in a weak initiation context) short orf that could be used to shunt even more ribosomes past the orf initiation codon. these viruses also preserve a conserved absence of aug codons (in any reading frame) throughout orf except for the -most nt (where there are from one to five aug codons, depending on species and strain) and the very end where the e orf aug is situated [ ] . in contrast, related viruses such as betacoronavirus (including bovine coronavirus and equine coronavirus) have aug codons spaced throughout orf , but produce a separate mrna for e protein expression so that bicistronic expression from the same mrna as orf is not required [ , ] . it should be noted, however, that expression of e (but not protein ) can occur from artificial reporters in which an additional orf is added upstream of orf , and therefore appears to involve internal ribosome entry [ , ] . it is possible that multiple strategies are used to enhance e expression. alternatively, presence of the cug-initiated uorf could simply be to downregulate production of protein . a long internal orf (i) is present within the n orf of mhv and many other coronaviruses, encoding a largely hydrophobic polypeptide that is thought to confer a minor growth advantage to the virus [ , ] . as shown in fig a, however, har profiling did not reveal an initiation spike for the i protein of mhv-a , suggesting that it might not be expressed. however, western blotting of infected-cell lysates using anti-n and anti-i sera revealed unambiguous expression of n and i from h p.i. (fig b) . to further confirm expression of the i protein, the n coding sequence was cloned into pcdna. and the mrna translated in rrl ( fig c) and immunoprecipitated (fig d) with anti-n and anti-i sera, and, as a negative control, anti-s serum. as shown, both n protein ( kda) and i protein ( kda) were expressed from the synthetic n mrna, with i produced at a level of about % of n. although we were unable to obtain strong evidence for the expression of i from the profiling data, a comparison of the phasing of rpfs (a) in the region where the i orf overlaps the n orf, and (b) in the region of the n orf downstream of the i termination codon, revealed in the former a slight excess of rpfs with ends mapping to the second position of n-frame codons (blue in fig e; upper panels) . the excess is consistent with - % of ribosomes translating the + (i.e. i) reading frame in this region. it is possible that leader sequence present in mrna (e.g. the uug-initiated uorf), but absent from the pcdna. -transcribed mrna, promote access to the i orf. to ensure that the phasing difference was not due to a single rpf peak (as individual peaks can sometimes map to a non-standard phase; cf. fig c) , mean phasing was also determined in a -codon sliding window and, consistent with the previous result, the proportion of rpfs mapping to the second position of n-frame codons (blue in fig e; lower panels) was found to decrease abruptly around the i orf stop codon. a caveat to note is that, in mhv-a , an upstream aug (bold) is present in the i frame followed by a stop codon (asterisk) prior to the "designated" i aug codon (underlined;. . .mpvaeapl à talvmessrrp; both augs are in a strong context). in some related virus sequences, the stop codon is replaced with a sense codon such that i is probably initiated from the upstream aug. thus, mhv-a may be somewhat defective with regards to i expression. respectively. (a) of the he orf. a defective trs for a very low abundance he mrna is annotated with an open green box. in mhv-a , the he orf is disrupted with a premature termination codon (red diamond). out-of-frame aug codons that would inhibit ribosomal access via leaky scanning to the next he-frame aug codon downstream of the premature termination codon are indicated in green. (b) of orf . in mhv-a , orf is split by a frameshift mutation into orf b (grey) and a very short orf a (pale yellow). an upstream auu-initiated short orf and a short out-of-frame aug-initiated orf are shown in orange. (c) of orf . a cug codon in the same frame as the upstream orf , and a short out-of-frame aug-initiated orf are indicated. we have used ribosome profiling to investigate virus gene expression kinetics, relative translational efficiencies, ribosomal frameshifting, ribosome pausing, and uorf translation in cells infected with mhv, a representative of the betacoronavirus genus of the coronavirus family of rna viruses. these studies provide the highest resolution data on coronavirus translation to date. using parallel rnaseq data, we examined the kinetics of virus replication and transcription, the relative abundances of different transcripts, and the degree of promiscuous polymerase jumping. we explored a number of data quality issues that can arise when applying ribosome profiling to the study of rna viruses that replicate to high titres in cell culture and describe ways to bioinformatically assess and quantify potential contamination. despite identifying low levels of different types of contamination, we were able to use impartial tests to avoid drawing incorrect conclusions from our data. viruses present particular challenges in profiling experiments. one of these is library contamination, which in this study may have been derived from two sources. the first was lowlevel contamination of one sample by another, a problem that is compounded by the high levels of virus rna synthesised in infected cells at late time points. we took precautions to avoid this source of contamination, including the use of designated work spaces, buffers and equipment, and avoiding parallel processing of early and late time points where possible. potentially, contamination may also have been introduced through the multiplex adaptor sequences. in the relatively small number of published studies on virus ribosome profiling, data from mockinfected samples and tests for contamination are often not reported, so the level of contamination suffered by others is uncertain. a second potential source of contamination could derive from rnps comprising virus or host mrna complexed with virus or stress-induced host rna binding proteins. such rnps might co-sediment with ribosomes during the sucrose cushion centrifugation step and contaminate riboseq libraries. although we were mindful of the possibility of such contamination, we found little evidence for it occurring as a result of mhv infection. an increased utr riboseq (chx) density was not apparent until h p.i. (when the plateau of virus production has been reached and virtually all cells are involved in extensive syncytium formation) and, even then, the read length distributions were similar to those of mock-infected cells; suggesting that the increased utr occupancy was as much due to bona fide rpfs as contaminating rnps. the former could be due to depletion of ribosome recycling factors resulting in increased amounts of unrecycled post-termination ribosomes accessing the utr [ ] . the high level of phasing in our riboseq data (s fig) allowed us to carefully assess contamination issues, and our observations reinforce the essentiality of basic data quality checks (e.g. s -s figs) in profiling studies. despite these challenges, the profiling and rna-seq analysis of mhv infection still showed itself to be a powerful tool to investigate specific aspects of mhv replication at high resolution. the kinetics of virus transcription in mhv-infected cells as observed through rnaseq were consistent with previous studies [ , [ ] [ ] [ ] . up to . h p.i., there was little amplification of positive-sense rna, whilst negative-sense rna levels rose from undetectable to about . % of total virus rna and host mrna. subsequently, positive-sense rna levels increased rapidly -with the accumulation of negative-sense rna plateauing at about h p.i.-such that, at late time points, the former comprised - % of total virus rna plus host mrna, while the latter comprised only~ . %. despite differences in abundance, the patterns of expression of positive and negative-sense rnas were similar, including high densities in the leader region, consistent with discontinuous transcription occurring during negative-strand synthesis [ ] . the measurement of decumulated rnaseq densities and the analysis of specific rnaseq leader/body chimeric reads at trss determined the relative abundance of mrnas at h p.i. to be mrna > mrna > mrna /mrna /mrna > mrna /mrna . an earlier study of mhv-a transcription using [ p] pulse labelling in the presence of actinomycin d provided a similar but slightly different order (mrna > mrna > mrna > mrna /mrna / mrna > mrna ) [ ] although it should be noted that, while mrnas and are nearly always the most abundant subgenomic transcripts, the relative abundances of the other transcripts can vary greatly between different isolates, strains and mutants of mhv [ , ] . the translation of virus proteins was detectable at a very early stage of infection. indeed, using a high moi infection, we were able to visualize input grna translation at h p.i., a stage when the majority of ribosomes had not yet reached the pp b orf. using riboseq (har) data at this time point, we were able to estimate a translation rate of . amino acids s − , consistent with previous estimates for mammalian systems [ ] . during the course of infection, we found that virus mrnas - were translated with generally similar efficiencies and, importantly, were not preferentially translated relative to host mrnas. rather, the synthesis of large quantities of virus proteins, especially n, is achieved through high levels of transcription (note that, due to library normalization, the quotient of riboseq and rnaseq does not inform on global virus-induced host shut-off, which is likely to be occurring at late time points of infection [ ] ). the virus genomic rna, however, appears to be poorly translated, as judged by the quotient of riboseq and rnaseq. during infection, much of the grna pool may, of course, be unavailable for translation. at earlier time points, it is, perhaps, sequestered in replication-transcription complexes; whereas at later time points, it may also be involved in packaging complexes. at . h p.i., when grna is unlikely to be a substrate for packaging, its translational efficiency was still low, but at this point in the replication cycle, the formation of replicationtranscription complexes would preclude the massive amplification of viral rna that takes place between and h p.i. [ ] . it may also be the case that the pp a and pp b orfs on the grna are inherently poorly translatable, e.g. due to translation of the uorf (see below) inhibiting ribosomal access to orf a. we also observed significant amounts of rnaseq reads mapping to the n orf region at h p.i., a time point at which negative-sense rnaseq reads were essentially absent. this suggests that the n orf rna is not newly synthesised. further, the absence of similar amounts of rnaseq density in the leader region, together with a very low translation efficiency, suggest that the n orf rna does not correspond to bona fide mrna . there has been considerable debate regarding the presence of subgenomic rnas in coronavirus particles [ , ] but recent analyses [ ] suggest that there is a very selective incorporation of mhv grna into virus particles and, although immunopurified virus particles may contain detectable amounts of mrna , it is minimal. the n orf rna observed in our study may represent a part of a defective viral genome with some structural similarity to di-like rnas. an alternative possibility, namely that the rnaseq density corresponding to the n orf may arise by selective degradation of the genomic rna, is not without precedent in other virus infections [ ] . however, it seems very unlikely to occur to~ % of the input grna prior to replication complex formation. further studies are needed to determine the source of this rna and whether or not it has any biological relevance. our data indicate that in mhv-infected cells, in addition to the "standard" coding sequences, ribosomes access and translate a number of short orfs. in general, translation of upstream short orfs (uorfs) is thought to regulate translation of downstream protein-coding orfs, with the peptide product of the uorf only rarely being functional in itself [ ] . the aug-initiated uorf of the grna has been characterised previously and may play a role in attenuation of translation of orfs a and b, with a beneficial but non-essential role in coronavirus replication in cell culture [ , ] . we found that translation of this uorf occurred at a level similar to that of orf a, reflecting its upstream position but poorer initiation context. interestingly, ribosomes on this uorf paused predominantly at the second codon (proline), probably as a consequence of the restrained geometry of this amino acid in the decoding centre [ ] . other translated uorfs included a uug-initiated -codon orf in the leader sequence, an auu-initiated -codon orf upstream of orf a, and a cug-initiated -codon orf upstream of orf . the function, if any, of the first two is unknown, but we speculate that the latter uorf may play a role in expression of the e protein, which is encoded downstream of orf on mrna . e is a small, hydrophobic viroporin that plays multiple roles during infection, including a role in virion morphogenesis [ ] . as the second cistron on mrna , it is not clear how the e aug is accessed for translation initiation. previous evidence indicates that e can be expressed via internal ribosome entry [ ] , although the experiments that led to this conclusion did not test for the production of alternative transcripts that might allow e expression in the system used. we now hypothesize, however, that e could also be expressed via a form of leaky scanning, where, after translating the short uorf on mrna , the small subunit of the ribosome remains associated with the mrna, resumes scanning, and re-initiates at the aug of the e orf. intervening augs within the nt of orf could be bypassed, as the scanning s subunit may not have had time to reacquire the relevant initiation factors [ ] . we were also able to confirm expression of the previously characterized internal (i) orf embedded within the n gene [ ] through western blotting, while analysis of profiling data (taking advantage of the phasing quality to gauge translation levels in different frames) was consistent with translation of i at a level not more than % of n protein expression. the mechanism of i expression is uncertain, but leaky scanning of ribosomes that fail to initiate at the n aug is a possibility and the low level of i expression is consistent with such a mechanism. note that failure to detect i orf initiation (and weak detection of e orf initiation) may indicate a shortcoming of the ribosomal profiling technique in the detection of initiation codons accessed by non-standard mechanisms. coronavirus − prf signals have been useful models for studies of ribosomal frameshifting in vitro, both from the perspective of structure-function relationships of rna pseudoknots, and also because they stimulate efficient frameshifting [ ] . from the profiling analysis presented here, we now know that frameshifting in the context of mhv infection is also extremely efficient, with around half of the ribosomes that translate orf a continuing on to translate orf b. we find little evidence that − prf is modulated by mhv infection, with similar efficiencies observed both in infected cells and in transfected cells expressing a frameshift-reporter mrna. intriguingly, there is no evidence that ribosomes pause upon encountering the mhv frameshift-promoting pseudoknot. several published in vitro studies have shown that rna pseudoknots (and certain other rna structures) can pause ribosomes [ , [ ] [ ] [ ] and recent kinetic studies have revealed that the translocation step of protein synthesis is significantly slowed by frameshift-stimulatory rna structures [ ] [ ] [ ] . whilst the in vitro systems used to study pausing and frameshifting kinetics could be inappropriate, it may be that profiling is insufficiently sensitive to register what may, in vivo, be pseudoknot-induced ribosomal pauses of short duration. relevant to this, despite the burgeoning literature on ribosomal profiling, only relatively few studies have addressed whether riboseq pauses can be generally correlated with intra-mrna structure [ ] [ ] [ ] . until this is better understood, the significance of these observations remains to be determined. in this study, we did identify a number of strong ribosomal pauses, however, and confirmed the occurrence of pausing within nsp in an in vitro translation assay. the nsp pausing site is located in the linker region between two modular domains of the protein, i.e. adrp [ ] and the recently identified dpup [ ] , and we hypothesize that the pause may occur after synthesis of the first domain in order to allow it to fold properly before synthesis of the second domain. ribosomal pausing as a way to optimize protein folding has been reported increasingly in recent years [ ] [ ] [ ] . we show that replacing four residues (lys, arg, lys, phe) in the nascent peptide sequence (within aa upstream of the pausing p-site) is sufficient to largely abrogate pausing, indicating that the pause is nascent peptide mediated and depends, at least in part, on positively charged residues acting within the ribosome exit tunnel, consistent with other ribosome profiling data where positively charged residues have been linked to ribosome retardation [ ] . our analysis of mhv by ribosomal profiling is the first such investigation for an rna virus. together with rnaseq, the datasets provide a high-resolution examination of mhv replication and gene expression and provides a basis for the subsequent analyses of virus-host responses (manuscript in preparation). we anticipate that the information will also be valuable to researchers with an interest in translation and virology, not least due to the excellent phasing in the riboseq datasets and the good coverage of reads on virus and cellular mrnas. murine clone ( cl- ) [ ] and bhk- [c- ] (atcc ccl- ) cells were maintained in dulbecco's modification of eagle's medium supplemented with % (vol/vol) fetal calf serum (fcs). recombinant mhv strain a (mhv-a ) was derived as previously described [ ] . cl- cells ( ) were plated in cm dishes and, upon reaching - % confluence, were infected with mhv-a at a multiplicity of infection (moi) of pfu/cell (or pfu/ cell in the "high moi" experiment) in hank's balanced salt solution (hbss) containing μg/ ml deae-dextran and . % bovine serum albumin (bsa). after min at °c, the inoculum was removed and the cells were incubated in dmem containing % fcs, u/ml penicillin and μg/ml streptomycin at °c until harvest. at the appropriate time point, cells were treated with chx (sigma-aldrich; to μg/ml; min), or har (lkt laboratories; μg/ml, min) then chx (to μg/ml; min). cells were rinsed with ml of ice-cold pbs, the dishes were submerged in a reservoir of liquid nitrogen for s and then transferred to dry ice and μl of lysis buffer [ mm tris-hcl ph . , mm nacl, mm mgcl , mm dtt, % triton x- , μg/ml cycloheximide and u/ml turbo dnase (life technologies)] dripped onto the cells. the cells were scraped extensively to ensure lysis, collected and triturated with a -g needle ten times. lysates were clarified by centrifugation for min at , g at °c, the supernatants recovered and stored in liquid nitrogen. cell lysates were subjected to riboseq and rnaseq. the methodologies employed were based on the original protocols of ingolia and colleagues [ , ] , except ribosomal rna contamination was removed by treatment with duplex-specific nuclease (dsn) and library amplicons were constructed using a small rna cloning strategy [ ] adapted to illumina smallrna v to allow multiplexing. the methods used were as described [ ] , with minor modifications for the analysis of ribosomal pausing at the mhv − prf signal, namely a broader range of rpfs, migrating between and nt, were harvested prior to amplicon construction, and longer pcr amplicons of~ - bp were gel purified. amplicon libraries were deep sequenced using an illumina hiseq platform (repeat samples at the wellcome trust centre for human genetics-oxford genomics centre; repeat , moi , and long read samples at the beijing genomics institute). adaptor sequences were trimmed using the fastx-toolkit and reads shorter than nt were discarded. trimmed reads were mapped first to mus musculus rrna (genbank accession numbers nr_ , nr_ , nr_ , nr_ , nr_ and gu ), followed by the mhv genome (genbank accession number ay . ) and subsequently mus musculus mrna, ncrna and genomic dna databases. in order to select good-quality samples of host mrna-derived rpfs for analyzing rpf length, framing, and position-on-transcript distributions, the mouse mrna database comprised ncbi refseq mrnas. the non-coding rna and genomic dna databases comprised the ensembl mus_musculus.ncbim . . ncrna.fa and release- dna chromosome files, respectively. reads that map to the gdna, but none of the rna databases, are expected to derive from unannotated transcripts as the sequencing protocol is rna-specific. reads were mapped using bowtie version [ ] with parameters -v -best (i.e. maximum mismatches, report best match). the order of mapping was tested to check that virus-derived reads were not lost accidentally due to mis-mapping to host rna, or vice versa; a slight reduction (~ . %) in virus-derived reads was observed only on mapping to the entire host genome (gdna) and thus mapping to virus rna and host mrna was considered to be specific. for host mrna mapping, no specific consideration was given to the presence of multiple isoforms within the refseq database; reads that could be mapped to multiple transcripts were assigned at random to one transcript. except where specifically stated, virus reads that mapped discontinuously to the mhv genome (due to transcriptional discontinuities at trs sites) were excluded from the analyses. host mrna riboseq and rnaseq phasing distributions (s fig and s fig) were derived from reads mapping to the "interior" regions of annotated coding orfs; specifically, the end of the read had to map between the first nucleotide of the initiation codon and nt of the last nucleotide of the termination codon, thus, in general, excluding rpfs of initiating or terminating ribosomes. histograms of end positions of host mrna reads relative to initiation and termination codons (s fig, s fig, s fig) were derived from reads mapping to refseq mrnas with annotated cdss ! nt in length and annotated and utrs ! nt in length. all figures are based on total numbers of mapped reads, rather than weighted sums for highly expressed mrnas [ ] , because virus-induced shut-off of host cell translation at late time points reduces the efficacy of the latter approach for our data. read length distributions ( s fig and s fig) are based on total mapped reads (to positive-sense host mrna, or to positive or negative-sense mhv genome, as indicated) without restriction to annotated coding regions. to compare read densities between cdss and utrs (s fig and s fig) , we used reads whose end offset by + nt (i.e. estimated p-site positions for rpfs) mapped within the regions from nt to nt upstream of stop codons (cdss), or from nt to nt downstream of stop codons ( utrs). this analysis was restricted to mrnas with annotated coding orfs ! nt in length and annotated utrs ! nt in length. the presence of transcript isoforms with utrs shorter than the annotated (! nt) utrs leads to a modest underestimation of the actual utr density. for fig b, refseq mrnas with annotated cdss ! nucleotides in length and annotated utrs ! nt in length (with no restriction on annotated utr length) were used, as only the end of cdss was analysed, and the more relaxed thresholds increased the sample size [important for the more restricted set of cdss beginning with aug-ccn (met-pro); of ncbi refseq mrna accessions, have cdss beginning with aug-ccn]. transcripts with ! rpfs with ends mapping between − and + relative to the annotated initiation codon were used, and histograms of end positions for individual transcripts were down-weighted by the number of rpfs mapping to this region before summing over the different transcripts (i.e. a weighted sum of "highly expressed" mrnas, [ ] ). fig b is based on sums over and transcripts for generic cdss and cdss beginning with aug-ccn, respectively. plots showing reads mapped to the mhv genome (figs , , , , , a, and and s fig) show histograms of the positions to which the ends of reads map, with a + nt offset to indicate (for rpfs) the approximate p-site. (more precisely, the + nt offset means that rpfs whose end aligns to the first position of a codon are mapped to the first nucleotide of the p-site codon, and rpfs whose end aligns to the third position of a codon are mapped to the last nucleotide of the codon preceding the p-site codon.) in contrast, plots showing reads summed over large numbers of host mrnas (fig b and s fig, s fig, s fig) show histograms of the positions to which the ends of reads map, without the + nt offset. this is because the host mrna plots are used for calibration whereas the virus plots are used to illustrate specific features of virus gene expression. to normalize for different library sizes, while taking into account global shut-off of host gene expression in response to virus infection, counts expressed as reads per million mapped reads (rpm) or reads per kb per million mapped reads (rpkm) use the sum of total virus rna (positive and negative-sense) plus total host mrna (reads that map to ncbi mrna refseqs) as the denominator. the same library normalization factors were also used for fig , s fig and s fig. to calculate the expression of individual virus orfs (fig , riboseq) , we counted rpfs whose end mapped between the first nucleotide of the initiation codon and nt of the termination codon, thus excluding rpfs of ribosomes paused during initiation or termination (or nearby). the corresponding sequence length was used to calculate counts per kb. we used a similar procedure to calculate rnaseq densities for each inter-trs region (fig , rnaseq) , with the inter-trs regions (prior to the nt buffer) being to (mrna ), to (mrna ), to (mrna ), to (mrna ), to (mrna ), to (mrna ), and to (mrna ). frameshifting efficiencies (fig ) were calculated using reads whose end offset by + nt (i.e. estimated p-site positions for rpfs) mapped within the regions to (for orf a) and to (for orf b). these coordinates leave a nt buffer after the orf a initiation codon (nt ), before the frameshift site (nt ), after the orf a termination codon (nt ) and before the orf b termination codon (nt ), respectively. read counts were divided by region lengths to obtain read densities. phasing distributions in the n and i orfs (fig e) were calculated with respect to the n reading frame, using reads whose end offset by + nt (i.e. estimated p-site positions) mapped within the regions to (for the i/n overlap) and to (for n downstream of i). for comparison, the coordinates of the n and i orfs are to (n) and to (i). for the analysis of riboseq and rnaseq count variability within orf a (fig a) , counts were first smoothed with a -nt running mean filter and then the fold-change relative to mean was calculated using reads whose end mapped between nt (the start of orf a) and nt ( nt of the frameshift site). for the above analyses, virus reads with discontiguous mappings to the mhv genome (i.e. reads spanning sites of discontinuous transcription-generally at the trs sites) were excluded. to identify such reads we re-mapped raw trimmed reads to host rrna, virus genome, host mrna, ncrna and gdna databases, this time permitting zero mismatches. we then pooled the remaining unmapped reads with the reads that mapped to the virus genome and, for each library, searched this set of reads for the query sequence uuuaaaucuaa (ay . nt to ; -adjacent to the leader trs). reads were selected that had at least nt of the query sequence and classified according to whether nucleotides + to + after the query sequence were compatible with mrna , , , , , or , or were derived from non-canonical chimeric sequences. these criteria were motivated by previous data indicating that, in leader/ body chimeras, nucleotides up to and including uuuaaaucuaa are templated by the leader, nucleotides at + and + may be templated by leader or genome, and nucleotides at + and above are templated by the genome sequence [ ] . counts were normalized to reads per million mapped reads as described above. a possible source of error here is that different libraries have different rnaseq read length distributions (due to variation in the gel-slice boundaries); libraries with longer reads will have proportionally more reads found to span leader/body discontinuities due to the requirement of at least nt of the -nt query sequence for selection. for this reason, inter-library comparisons are avoided. to calculate host translational efficiencies, after removing reads mapping to rrna with bowtie as above, remaining reads were mapped to the mouse genome (ucsc, assembly mm ) using tophat (parameters: -no-novel-juncs -bowtie -prefilter-multihits -maxmultihits , with -transcriptome-index defined using the genes.gtf file from the ucsc mm annotation available from the tophat website) [ ] . reads entirely contained within annotated cdss were enumerated with htseq-count (parameters: -t cds -m intersection-strict -i gene_id -s yes) [ ] , reporting read counts per gene rather than per transcript. read counts were normalized for library size as above, and for cds length according to the sum of all coding exon fragment lengths for a given gene id in the genes.gtf file. this will tend to result in an overestimate of cds lengths since many transcripts (alternative splice forms and/or alternative transcription initiation sites) will lack some coding exons. while this is likely to have only a modest effect on riboseq/rnaseq translation efficiencies (fig , y-axis) it will tend to result in underestimates for rnaseq rpkm values (fig , x-axis) . the sequencing data have been deposited in the arrayexpress database (http://www.ebi.ac. uk/arrayexpress) under the accession number e-mtab- . the mhv frameshift signal, and the n, nsp and nsp protein coding sequences were amplified using specific oligonucleotides (s table) and cdna derived from cl- cells infected with mhv-a at an moi and harvested at h p.i. for assessing frameshifting efficiencies in transfected tissue culture cells, the dual-luciferase reporter vector pdluc was employed (kind gift from dr m. howard, university of utah; [ ] ). dna fragments of bp spanning the mhv frameshift signal and flanked by xhoi and bglii restriction sites were derived by pcr amplification and ligated into appropriately cleaved pdluc vector. an in-frame control (mimicking % frameshifting efficiency) was also constructed. pdluc-ibv and pdluc-hxb have been described elsewhere [ , ] . bamh -xhoi-digested pcr fragments were cloned into pcdna . (+) (life technologies) previously digested with bamh -xhoi. in pps plasmids, pcr reactions were carried out using the pcdna. nsp plasmid as a template and cloned into a digested xhoi/pvuii-pps plasmid. pps -nsp mutants were subjected to site-directed mutagenesis. for all pcdna. and pps constructs, a "pause control" was also generated in which a uaa stop codon was introduced to generate a protein whose size corresponded to that produced by the predicted ribosomal pause. all sequences were confirmed by dideoxy sequencing. frameshifting assays in tissue culture cl- and bhk- cells were seeded in dishes of a -well plate and grown for h until % confluence was reached. plasmids were transfected using a commercial liposome method (transit-lt , mirus). transfection mixtures [containing plasmid dna, serum-free medium (opti-mem; gibco-brl) and liposomes] were set up as recommended by the manufacturer and added dropwise to the tissue culture cell growth medium. cells were harvested h post transfection (h.p.t.) and reporter gene expression was determined using a dual-luciferase assay system kit (promega). frameshifting efficiencies were calculated by dividing the fluc/rluc ratios of the test samples by the fluc/rluc ratio of the in-frame controls. proteins were separated by %, % or % sds-page depending on the molecular weight of the protein of interest and transferred to nitrocellulose membranes. these were blocked for - min with % powdered milk (marvel) in pbst [ mm nacl, . mm kcl, mm na hpo , . mm kh po (ph . ), and . % tween ] and probed with mouse monoclonal antibodies raised against nsp (am pu-n, acris antibodies, inc, : in marvel-pbst), n ( : , ), s ( : ), gapdh (g , sigma-aldrich, : , ) or a polyclonal rabbit anti-i ( : , , a kind gift of prof. p. s. masters, wadsworth center, new york state department of health). membranes were incubated in the dark with an irdye-conjugated secondary antibody in pbst [irdye cw donkey anti-mouse igg (h+l), irdye cw donkey anti-rabbit igg (h+l) and irdye rd goat anti-mouse igm (μ chain specific)]. blots were scanned and bands quantified using an odyssey infrared imaging system (licor). pcdna. and pps plasmids were linearized with xhoi and avaii respectively and capped run-off transcripts generated using t rna polymerase and sp rna polymerase respectively as described previously [ ] . rnas were recovered by a single extraction with phenol-chloroform ( : vol/vol) followed by ethanol precipitation. remaining unincorporated nucleotides were removed by gel filtration through a nucaway spin column (ambion). the eluate was concentrated by ethanol precipitation, the mrna resuspended in water, checked for integrity by agarose gel electrophoresis and quantified by spectrophotometry. rnas were translated in nuclease-treated rabbit reticulocyte lysate (rrl) (promega) programmed with~ μg/ml template mrna. a typical reaction mixture had a volume of μl and was composed of % (vol/ vol) rrl, μm amino acids (lacking methionine), and . mbq [ s]-methionine. reaction mixtures were incubated for min at °c and stopped by the addition of an equal volume of mm edta, μg/ml rnase a followed by incubation at room temperature for min. in ribosomal pausing assays, conditions were the same except that the reaction mixture had a volume of μl and the translational inhibitor edeine was added min after the start of the reaction in order to obtain synchronous initiation (final concentration, μm). aliquots of . μl were withdrawn from the translation reaction mixture at specified intervals and mixed with an equal volume of edta/rnase a mixture, as above. in immunoprecipitations, μl of rrl was mixed with either mouse anti-n, anti-s or rabbit anti-i for min at °c prior to binding to protein a-sepharose cl- b (pharmacia biotech ab, uppsala, sweden) and subsequent washing. samples were prepared for sds-page by addition of volumes of x laemmli's sample buffer and boiling for min. proteins were resolved on % or % sds-page gels. c-labelled molecular weight standards (mw) were from amersham international (united kingdom). dried gels were exposed to a carestream kodak biomax mr film (sigma-aldrich) and scanned. supporting information s table. genomic sequences flanking the leader and body junction sites. trss (ucuaaac or similar) are indicated in bold. nucleotides consistent with tandem copies of the pentanucleotide ucuaa are indicated in red (copy at the canonical junction site) and blue (copy nt upstream of the canonical junction site). note also the high similarity between the sequences at the leader (mrna ) and mrna junction sites: when polymerase jumping for mrna occurs nt upstream of the canonical site, nt of sequence are required to distinguish grna reads from mrna reads. (docx) s table. frequencies of canonical and non-canonical leader/body chimeric reads. chimeric reads utilizing the leader trs were identified by searching for all reads containing the sequence uuuaaaucuaa (ay . nt to ), and classified according to the identity of the following nucleotides at positions + to + . these nucleotides are listed in column . the genomic coordinate of the first nucleotide of the is given in column . nucleotides at positions + to + in the rnaseq read are listed in column . the corresponding two nucleotides from the genome are listed in column . also, the nucleotides preceding these in the genome are listed in column . the numbers of junction/body chimeric reads containing each sequence are given in column (repeat ) and column (repeat ). only sequences with three or more occurrences in repeat and ten or more occurrences in repeat are shown. data are shown for the h p.i. rnaseq libraries. . reads were counted in windows from to codons upstream (cds; green) or downstream ( utr; orange) of annotated termination codons, and summed over all host mrnas. the left panel in each pair shows the absolute read counts, allowing comparison of the cds and utr read densities; the density ratio ( utr / cds) is indicated in purple in each panel. for all riboseq samples, utr occupancy is very low compared to cds occupancy, whereas, for rnaseq, utr occupancy is typically around % of cds occupancy (the rnaseq value is less than unity due to differences in the transcript isoforms present in the sample compared to the refseq mrna database). the right panel in each pair shows the distributions normalized to have equal total sums so that the shapes of the cds and utr distributions can be compared. for rnaseq, the two distributions have essentially identical shapes. for riboseq, differences in the two distributions provide an indicator of the level of non-rpf contamination present in the sample. bats as reservoirs of severe emerging infectious diseases middle east respiratory syndrome coronavirus: another zoonotic betacoronavirus causing sars-like disease evidence for an ancestral association of human coronavirus e with bats an efficient ribosomal frame-shifting signal in the polymerase-encoding region of the coronavirus ibv the primary structure and expression of the second open reading frame of the polymerase gene of the coronavirus mhv-a ; a highly conserved polymerase is expressed by an efficient ribosomal frameshifting mechanism a contemporary view of coronavirus transcription genome-wide analysis in vivo of translation with nucleotide resolution using ribosome profiling ribosome profiling of mouse embryonic stem cells reveals the complexity and dynamics of mammalian proteomes ribosome profiling: new views of translation, from single codons to genome scale ribosome pausing and stacking during translation of a eukaryotic mrna ribosomal footprints on a transcriptome landscape molecular biology. translation goes global ribosome profiling: a hi-def monitor for protein synthesis at the genome-wide scale ribosome profiling reveals the what, when, where and how of protein synthesis the awesome power of ribosome profiling the use of duplex-specific nuclease in ribosome profiling and a user-friendly software package for ribo-seq data analysis decoding human cytomegalovirus the transcription and translation landscapes during human cytomegalovirus infection reveal novel host-pathogen interactions kshv . : a comprehensive annotation of the kaposi's sarcoma-associated herpesvirus genome using next-generation sequencing reveals novel genomic and functional features widespread disruption of host transcription termination in hsv- infection deciphering poxvirus gene expression by rna sequencing and ribosome profiling high-resolution view of bacteriophage lambda gene expression by ribosome profiling a new model for coronavirus transcription ribosome profiling reveals sequence-independent post-initiation pausing as a signature of translation on the prevalence of certain codons genome-wide ribosome profiling reveals complex translational regulation in response to oxidative stress widespread regulation of translation by elongation pausing in heat shock cotranslational response to proteotoxic stress by elongation pausing of ribosomes ribosome profiling reveals pervasive translation outside of annotated protein-coding genes dom rescues ribosomes in untranslated regions rli /abce recycles terminating ribosomes and controls translation reinitiation in utrs in vivo modified ribosome profiling reveals high abundance of ribosome protected mrna fragments derived from ' untranslated regions coronavirus minus-strand rna synthesis and effect of cycloheximide on coronavirus rna synthesis characterization of an internal ribosome entry site within mrna of murine hepatitis virus coding sequence of coronavirus mhv-jhm mrna three intergenic regions of coronavirus mouse hepatitis virus strain a genome rna contain a common nucleotide sequence that is homologous to the ' end of the viral mrna leader sequence the '-end sequence of the murine coronavirus genome: implications for multiple fusion sites in leader-primed transcription identification of a new transcriptional initiation site and the corresponding functional gene b in the murine coronavirus rna genome discontinuous transcription generates heterogeneity at the leader fusion sites of coronavirus mrnas coronavirus mrna synthesis: identification of novel transcription initiation signals which are differentially regulated by different leader sequences mutagenic analysis of the coronavirus intergenic consensus sequence heterogeneity of gene expression of the hemagglutinin-esterase (he) protein of murine coronaviruses the production of recombinant infectious di-particles of a murine coronavirus in the absence of helper virus defective-interfering particles of murine coronavirus: mechanism of synthesis of defective viral rnas characterization of an efficient coronavirus ribosomal frameshifting signal: requirement for an rna pseudoknot a threestemmed mrna pseudoknot in the sars coronavirus frameshift signal a second, non-canonical rna-dependent rna polymerase in sars coronavirus achieving a golden mean: mechanisms by which coronaviruses ensure synthesis of the correct stoichiometric ratios of viral proteins an 'elaborated' pseudoknot is required for high frequency frameshifting during translation of hcv e polymerase mrna the q-base of asparaginyl-trna is dispensable for efficient − ribosomal frameshifting in eukaryotes programmed ribosomal frameshifting in decoding the sars-cov genome a dual-luciferase reporter system for studying recoding signals processive selenocysteine incorporation during synthesis of eukaryotic selenoproteins structure-function analysis of the ribosomal frameshifting signal of two human immunodeficiency virus type isolates with increased resistance to viral protease inhibitors spacer-length dependence of programmed − or − ribosomal frameshifting on a u a heptamer supports a role for messenger rna (mrna) tension in frameshifting translational frameshifting: implications for the mechanism of translational frame maintenance ribosomal pausing at a frameshifter rna pseudoknot is sensitive to reading phase but shows little correlation with frameshift efficiency pseudoknot-dependent − ribosomal frameshifting: structures, mechanisms and models. in recoding: expansion of decoding rules enriches gene expression ribosomal movement impeded at a pseudoknot required for frameshifting ribosomal pausing during translation of an rna pseudoknot kinetics of ribosomal pausing during programmed − translational frameshifting programmed − frameshifting by kinetic partitioning during impeded translocation dynamic pathways of − translational frameshifting a frameshifting stimulatory stem loop destabilizes the hybrid state and impedes ribosomal translocation the anti-shine-dalgarno sequence drives translational pausing and codon choice in bacteria rrna:mrna pairing alters the length and the symmetry of mrna-protected fragments in ribosome profiling experiments regulatory nascent peptides in the ribosomal tunnel arrest peptides: cis-acting modulators of translation sars-coronavirus replication is supported by a reticulovesicular network of modified endoplasmic reticulum new insights on the role of paired membrane structures in coronavirus replication rna replication of mouse hepatitis virus takes place at double-membrane vesicles mutation in murine coronavirus replication protein nsp alters assembly of double membrane vesicles topology and membrane anchoring of the coronavirus replication complex: not all hydrophobic domains of nsp and nsp are membrane spanning detection of non-structural protein in murine coronavirus-infected cells and analysis of the transmembrane topology by using bioinformatics and molecular approaches severe acute respiratory syndrome coronavirus non-structural proteins , , and induce double-membrane vesicles x-ray structural and functional studies of the three tandemly linked domains of non-structural protein (nsp ) from murine hepatitis virus reveal conserved functions positively charged residues are the major determinants of ribosomal velocity translation initiation at non-aug triplets in mammalian cells point mutations define a sequence flanking the aug initiator codon that modulates translation by eukaryotic ribosomes reselection of a genomic upstream open reading frame in mouse hepatitis coronavirus '-untranslated-region mutants upstream open reading frames cause widespread reduction of protein expression and are polymorphic among humans a perspective on mammalian upstream open reading frame function slow peptide bond formation by proline and other n-alkylamino acids in translation accounting for biases in riboprofiling data indicates a major role for proline in stalling translation sequence of mouse hepatitis virus a mrna : indications for rna recombination between coronaviruses and influenza c virus the ns gene of mouse hepatitis virus (mhv), strain a contains two orfs and thus differs from ns of the jhm and s strains termination and post-termination events in eukaryotic translation coronavirus mhv-jhm mrna has a sequence arrangement which potentially allows translation of a second, downstream open reading frame leader-mrna junction sequences are unique for each subgenomic mrna species in the bovine coronavirus and remain so throughout persistent infection genomic characterization of equine coronavirus internal ribosome entry in the coding region of murine hepatitis virus mrna the nucleocapsid protein gene of bovine coronavirus is bicistronic the internal open reading frame within the nucleocapsid gene of mouse hepatitis virus encodes a structural protein that is not essential for viral replication coronavirus transcription: subgenomic mouse hepatitis virus replicative intermediates function in rna synthesis coronavirus transcription: a perspective the rna structures engaged in replication and transcription of the a strain of mouse hepatitis virus the virus-specific intracellular rna species of two murine coronaviruses: mhv-a and mhv-jhm coronavirus non-structural protein : common and distinct functions in the regulation of host and viral gene expression presence of subgenomic mrnas in virions of coronavirus ibv transmissible gastroenteritis coronavirus packaging signal is located at the end of the virus genome functional analysis of the murine coronavirus genomic rna packaging signal functional non-coding rnas derived from the flavivirus ' untranslated region mouse hepatitis virus stem-loop functions as a spacer element required to drive subgenomic rna synthesis coronavirus envelope (e) protein remains at the site of assembly determinants of translation elongation speed and ribosomal profiling biases in mouse embryonic stem cells causal signals between codon bias, mrna structure, and the efficiency of translation and elongation secondary structure across the bacterial transcriptome reveals versatile roles in mrna regulation and function proteomics analysis unravels the functional repertoire of coronavirus nonstructural protein protein folding within the cell is influenced by controlled rates of polypeptide elongation transient ribosomal attenuation coordinates protein synthesis and co-translational folding monitoring cotranslational protein folding in mammalian cells at codon resolution enhanced growth of a murine coronavirus in transformed mouse cells recombinant mouse hepatitis virus strain a from cloned, full-length cdna replicates to high titers in vitro and is fully pathogenic in vivo the ribosome profiling strategy for monitoring translation in vivo by deep sequencing of ribosome-protected mrna fragments mammalian micrornas predominantly act to decrease target mrna levels ultrafast and memory-efficient alignment of short dna sequences to the human genome tophat: discovering splice junctions with rna-seq htseq-a python framework to work with high-throughput sequencing data mutational analysis of the "slippery-sequence" component of a coronavirus ribosomal frameshifting signal we thank paul masters for providing anti-i sera. key: cord- -imn g authors: ciminski, kevin; pfaff, florian; beer, martin; schwemmle, martin title: bats reveal the true power of influenza a virus adaptability date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: imn g nan influenza a viruses (iavs) circulate among a wide variety of different hosts. they can cross species barriers and establish new virus lineages in avian and mammalian species. as a consequence, iavs are exposed to recurrent selective pressures, leading to a range of virus variants that are able to cope with the new host environment [ ] . bats were not considered to be part of this iav habitat until recently, when two phylogenetically distinct iav lineages, designated h n and h n , were identified in the new world bats sturnira lilium and artibeus spp, respectively [ ] [ ] [ ] . likewise, old world bats can harbor influenza viruses, as exemplified by the genetically distinct h n virus found in egyptian fruit bats (rousettus aegyptiacus) [ ] . the discovery of influenza viruses in new and old world bats did severely challenge our previous conception of iav host range and phylogeny. it, moreover, provided unexpected insights into the remarkable adaptive potential of these viruses and their evolutionary origin. the genetic divergence of iavs circulating in new and old world bat species is much broader than initially anticipated. bat-derived iavs have a higher overall genetic diversity than conventional non-bat iavs [ , ] . internal gene segments that are usually highly conserved among iavs cluster as one narrow clade on a phylogenetic branch when non-bat iavs are analyzed, whereas the corresponding gene segments of new and old world bat iavs form distant outgroups [ , ] (fig a and s fig) . new and old world bat iavs did most likely split into two genetic branches as a result of either geographic separation or multiple early spillover events of ancient viruses. phylogenetic backdating of the internal gene segments suggests that the precursor of the new world bat iavs separated from all other lineages more than years ago, although some uncertainties commonly associated with molecular clock analyses exist (fig b) . interestingly, although the old world bat h n subtype arose from a common ancestor dated around ce (fig b) , its h hemagglutinin (ha) sequence is phylogenetically similar to younger sequences isolated from avian hosts (fig c) . this disparity is best explained by the occurrence of more recent reassortment events between old world bat ancestral viruses and avian strains. furthermore, our phylogenetic analyses reveal that the newly discovered bat iavs share a common ancestor with non-bat iavs (fig a and b) . it is therefore conceivable that all present-day iavs originate from bats. it is equally possible, however, that an even older iav precursor was circulating in avian species and was later introduced into mammals-including bats. what is the biological role of the bat iav surface glycoproteins? iavs initiate infection via the viral ha surface glycoprotein that binds to sialic acid receptors on host cell glycoproteins (fig a) . until recently, it was thought that sialic acids serve as the universal receptors for all influenza virus strains, thereby facilitating cross-species transmission. indeed, the ha of the newly discovered old world bat h n virus binds to α , -sialic in contrast, the internal gene segments of the bat-derived iavs form two outgroups that are located at a more basal position. notably, old world (red) and new world bat iavs (purple) are widely separated. the parallel lines indicate uncertainties between the phylogenetic trees that make up the presented phylogenetic network. (b) a timecalibrated phylogeny was calculated for pb as a representative iav internal gene segment. the timeline (presented in ce) shows that the new world bat iav segments branched off more than years ago (purple node) and that the last common ancestor of old world bat iavs (red node) and conventional iavs (blue node) is around years old. (c) surprisingly, a comparable time-calibrated phylogeny of the old world bat h ha (red node) along with conventional h , h , and h has (blue nodes) points to a much more recent common ancestor for the old world bat-derived ha. see supporting information for a detailed description of the performed phylogenetic analysis (s technical appendix) and a full-sized version of the phylogenetic network (s fig) acid moieties [ ] . surprisingly, however, h and h of the new world bat influenza strains were found to lack this property and cannot use sialic acid receptors for infection [ , , ] . instead, both new world bat-derived ha subtypes utilize major histocompatibility complex class ii (mhc-ii) molecules for cell entry [ , ] . importantly, mhc-ii proteins of multiple species, including chicken, pigs, mice, and humans, can serve as receptors, indicating that receptor usage by bat iavs does not provide a tight species barrier but is compatible with a broad host range ( fig b) [ ] . mhc-ii molecules are normally found on immune cells of the lymphoid tissue, such as b cells, macrophages, and dendritic cells [ ] , but they can also be expressed on epithelial cells [ ] . although mhc-ii molecules are indispensable for h -and h -mediated cell entry [ , ] , it is presently not clear whether they serve as bona fide binding receptors or else as necessary entry cofactors. physical interactions between h /h and mhc-ii molecules need to be demonstrated, and the binding interface(s) need to be defined. conventional iavs and the bat h n virus possess the surface glycoprotein neuraminidase (na), which removes sialic acid residues from infected cells to facilitate the release of newly formed viral particles (fig a) . the new world bat-derived iavs also encode an na protein which, however, has no detectable sialidase activity [ , , , ] . its real function has remained enigmatic until recently. preliminary data now suggest that bat n down-regulates the surface expression of mhc-ii molecules by an as yet unknown mechanism [ ] (fig b) . if confirmed, these data demonstrate that the surface glycoproteins of new world bat iavs have both receptor binding and destroying activities and hence serve the same function as the ha and na proteins of conventional iavs. a well-known characteristic of iavs is their capacity to rapidly evolve and adapt to new environments. their adaptability is based mainly on ( ) gene reassortment (i.e., the ability to exchange gene segments between strains) and ( ) genetic drift that generates a vast number of viral quasispecies due to the infidelity of the viral polymerase [ ] . reassortment between two different yet compatible viruses may generate antigenically novel strains with pandemic potential [ ] . favorable mutations acquired by genetic drift allow evasion from host immune responses and promote successful adaptation to new hosts. for instance, specific amino acid substitutions in ha (e d/g d and q l/g s) enable avian iavs to bind and infect human cells [ ] . interestingly, as the h and h proteins of new world bat iavs are homologs of the ha glycoproteins of conventional iavs, they must have undergone drastic changes to accommodate mhc-ii molecules as entry factors. an exchange of key amino acids in the putative receptor binding site prevents any interaction with sialic acid residues [ , , ] . the underlying selection pressure that resulted in the switch of receptor usage is not known and remains a matter of speculation. a plausible scenario would be that the precursor of the presently known iavs harbored a ha protein with a dual affinity for sialic acids and for an unknown-possibly mhc-ii-related-cellular surface protein, allowing infection of both birds and bats. possibly because of ecological separation of this ancestral influenza virus in new world bats, subsequent evolution in the new host led to increased affinity for mhc-ii. simultaneously, the evolutionary process fostered additional species-specific adaptations. these comprise for example the co-selection of genome packaging signals in conjunction with a specific set (code) of amino acids in the viral nucleoprotein (np) [ ] [ ] [ ] . both the segmentspecific packaging signals and a compatible np amino acid code are compulsory to orchestrate the incorporation of the viral genome into progeny virions [ ] . therefore, because of nonmatching packaging sequences and a different np amino acid code, new world bat and conventional non-bat iavs are unable to reassort their genomes [ ] -an additional unique feature of new world bat iavs, making them a truly divergent entity of iavs. conventional sialic acid-binding ha and na proteins are well studied and known to evolve rapidly in face of a rigorous host immune response [ ] . the surface glycoproteins of the new world bat iavs are still poorly understood and their adaptive potential is presently unknown. first studies show that serial passages of the h n subtype in non-bat epithelial cells resulted in the emergence of efficiently replicating mutant viruses. surprisingly, the selected mutants harbored a truncated nonfunctional n protein yet were able to replicate in mice, ferrets, and bats [ ] (fig c) . sequence analysis showed that these virus variants had acquired at least two mutations in the h head domain that appeared to compensate for the loss of a functional n . perhaps these acquired mutations reduced the binding affinity of h to mhc-ii and supported n -independet replication. virus release may have been further facilitated by the fact that new world bat iav particles bud at the apical membrane of epithelial cells [ ] , where mhc-ii molecules are scarce [ ] . remarkably, h n and h n are the first iav subtypes that utilize a proteinaceous entry receptor. the amazing ability of h to rapidly overcome the absence of a functional n suggests that the structure of h (and possibly h ) may provide a broader scope of evolutionary flexibility than that of conventional sialic acid-dependent iavs. it is therefore tempting to speculate that bat iav ha proteins, and in particular h , might have the potential to adapt to novel and so far unknown entry receptors different from mhc-ii (fig d) . up to now, more than different viruses have been isolated from or detected in bats. some of these bat-borne viruses, such as rabies, ebola, severe acute respiratory syndrome (sars), and middle east respiratory syndrome (mers) virus, have caused human and animal diseases, highlighting their zoonotic and epizootic threat [ ] . the risk for zoonotic transmission and the associated pandemic potential that emanates from an influenza virus considerably depends on its degree of human preadaptation and the ability to overcome host restrictions. whereas avian iavs carry gene segments with species-specific determinants that allow efficient replication in avian but not necessarily human cells [ ] , the new and old world bat iavs appear to possess a human-compatible gene signature due to their mammalian origin. indeed, upon serial passaging of chimeric new world bat iavs in eggs and avian cells, these viruses readily acquired mutations in several genes. in sharp contrast, no such mutations occurred in primary human airway epithelial cell cultures [ ] , indicating that the internal viral proteins are well suited for the replication in human cells. moreover, new world bat iavs might well have the potential to infect humans, because their has can utilize the human mhc-ii homolog human leukocyte antigen-dr isotype (hla-dr) for cell entry [ , ] . nevertheless, h n viruses did not induce any pathogenicity in and were not transmissible among ferrets [ ] , which are considered to be the best animal model for human influenza [ ] . aside from this, h n is also not able to overcome the intracellular restriction imposed by major host defense factors such as the human type i and iii interferon-induced antiviral factor mx (in humans, mxa), because of the lack of mxa escape mutations in np [ ] . as to bat h n , the usage of α , instead of α , -sialic acid residues, together with the lack of obvious mxa resistance mutations in np [ ] , suggests that the old world bat virus possesses a low degree of human preadaptation. thus, based on the currently available data, there is a clear, albeit low, risk for zoonotic spillover of the different bat iavs. in view of their human-adapted internal gene segments and the ability to rapidly acquire new mutations, future transmissions to humans cannot be excluded, and proper surveillance of bat iav distribution is indicated. the presented phylogenetic network is based on the segments pb , pb , pa, np, m, and ns from representative iavs (highlighted in green) and six influenza b viruses (highlighted in yellow). a more detailed close-up zoom of iavs is presented in the main text (fig a) , and methods are described in detail in s technical appendix. iav, influenza a virus; np, nucleoprotein. (tiff) s technical appendix. technical information and methods used for the phylogenetic analysis. (docx) evolutionary processes in influenza viruses: divergence, rapid evolution, and stasis a distinct lineage of influenza a virus from bats new world bats harbor diverse influenza a viruses bat influenza a (hl nl ) virus in fruit bats isolation and characterization of a distinct influenza a virus from egyptian bats bat-derived influenza hemagglutinin h does not bind canonical avian or human receptors and most likely uses a unique entry mechanism hemagglutinin homologue from h n bat influenza virus exhibits divergent receptor-binding and ph-dependent fusion activities entry of the bat influenza h n virus into mammalian cells is enabled by the mhc class ii hla-dr receptor mhc class ii proteins mediate cross-species entry of bat influenza viruses receptor for bat influenza virus uncovers potential risk to humans the ins and outs of mhc class ii-mediated antigen processing and presentation epithelial mhc class ii expression and its role in antigen presentation in the gastrointestinal and respiratory tracts structural and functional characterization of neuraminidase-like molecule n derived from bat influenza a virus crystal structures of two subtype n neuraminidase-like proteins from bat influenza a viruses reveal a diverged putative active site bat influenza viruses transmit among bats but are poorly adapted to non-bat species getting the flu: key facts about influenza virus evolution origins and evolutionary genomics of the swine-origin h n influenza a epidemic host and viral determinants of influenza a virus species specificity a conserved influenza a virus nucleoprotein code controls specific viral genome packaging an infectious batderived chimeric influenza virus harbouring the entry machinery of an influenza a virus the structure of the influenza a virus genome packaging of the influenza virus genome is governed by a plastic network of rna-and nucleoprotein-mediated interactions highly polarized hla class ii antigen processing and presentation by human intestinal epithelial cells synthetically derived bat influenza a-like viruses reveal a cell type-but not species-specific tropism bats and viruses: current research and future trends the ferret: an animal model to study influenza virus human mxa is a potent interspecies barrier for the novel bat-derived influenza a-like virus h n application of phylogenetic networks in evolutionary studies we thank otto haller for critically reading the manuscript and for constructive comments. key: cord- - vl hks authors: epstein, jonathan h.; quan, phenix-lan; briese, thomas; street, craig; jabado, omar; conlan, sean; ali khan, shahneaz; verdugo, dawn; hossain, m. jahangir; hutchison, stephen k.; egholm, michael; luby, stephen p.; daszak, peter; lipkin, w. ian title: identification of gbv-d, a novel gb-like flavivirus from old world frugivorous bats (pteropus giganteus) in bangladesh date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: vl hks bats are reservoirs for a wide range of zoonotic agents including lyssa-, henipah-, sars-like corona-, marburg-, ebola-, and astroviruses. in an effort to survey for the presence of other infectious agents, known and unknown, we screened sera from pteropus giganteus bats from faridpur, bangladesh, using high-throughput pyrosequencing. sequence analyses indicated the presence of a previously undescribed virus that has approximately % identity at the amino acid level to gb virus a and c (gbv-a and -c). viral nucleic acid was present in of sera ( %) from a single colony of free-ranging bats. infection was not associated with evidence of hepatitis or hepatic dysfunction. phylogenetic analysis indicates that this first gbv-like flavivirus reported in bats constitutes a distinct species within the flaviviridae family and is ancestral to the gbv-a and -c virus clades. bats (order chiroptera), after rodents, comprise the most diverse group of mammals with more than , species. they are present on six continents, often have substantial habitat overlap with humans [ ] and harbor several zoonotic viruses causing significant human morbidity and mortality, including ebola-and marburgvirus, nipah virus (niv), and sars-like coronaviruses [ ] [ ] [ ] [ ] . proximity of bats to human populations may facilitate the zoonotic transmission of viruses either through direct contact, via amplifying domestic animal hosts, or through food-borne routes [ ] [ ] [ ] . the current study was set up as part of a viral discovery effort to target key wildlife reservoirs in emerging disease hotspots. bangladesh is a 'hotspot' for emerging zoonotic diseases [ ] , with a relatively high diversity of wildlife that likely harbors new zoonotic pathogens, one of the densest human populations on the planet, and a high level of connectivity between people, domestic animals and wildlife. in bangladesh and india, frugivorous pteropus giganteus bats have been identified as a reservoir for niv [ , ] , which has been recognized as the cause of several outbreaks of encephalitis [ ] [ ] [ ] . pteropus giganteus bats are common throughout the indian subcontinent, living in close association with humans and feeding on cultivated fruit [ ] . niv transmission from bats to humans has been linked with the harvest and consumption of raw date palm sap, which becomes contaminated with bat feces, urine or saliva overnight when bats such as p. giganteus come to feed from the collecting pots [ , ] . date palm sap or other foods eaten by both bats and people, may also serve as a vehicle for transmission of other bat-borne agents. several zoonotic flaviviruses, including japanese encephalitis virus, west nile virus, and kyasanur forest virus have been identified in bats; however, to date, gb viruses have not [ ] . gb viruses a and c (gbv-a and -c) represent two recently identified species that are currently unassigned members of the family flaviviridae [ ] . gbv-a viruses have been described in new world primates and are not known to infect humans [ ] [ ] [ ] , while gbv-c (also known as hepatitis g virus (hgv)) have frequently been isolated from humans in many regions of the world, including india and bangladesh [ ] [ ] [ ] [ ] [ ] , and from wild chimpanzees (pan troglodytes) in africa [ , ] . here we describe discovery of a virus in the serum of healthy bats in bangladesh, tentatively named gb virus d (gbv-d), that is distantly related to gbv-a and -c and represents a new member of the family flaviviridae. every effort was made to minimize bat stress and avoid injury during capture, restraint, and sampling procedures. this study was conducted following wildlife trust institutional guidelines under iacuc approval g issued by tufts new england medical center, boston, massachusetts. as part of a longitudinal surveillance study of nipah virus in bats, free-ranging p. giganteus bats were caught from a colony of approximately individuals in the faridpur district of bangladesh in december ( figure ). each bat was anesthetized using isoflurane gas; morphometric measurements (weight, forearm length, head length, and body condition) were taken and bats were aged [ ] . each bat was marked for future identification using an rfid microchip (avid corp, www.avidid. com) implanted subcutaneously between the scapulae. three ml of blood were collected and placed into serum separator tubes (vacutainer; becton dickinson, franklin lakes, nj, usa). serum was allowed to separate overnight at uc then drawn off without centrifugation and immediately frozen using a liquid nitrogen dry shipper. to inactivate potentially infectious agents, serum samples were heat-treated at uc for min and then stored at uc. for rna extraction, ml of serum was added to ml tri-reagent ls (molecular research center, cincinnati, oh, usa). saliva was collected from the bat's throat using a sterile cotton swab. urine was collected either by catching urine in a . ml sterile cryovial while the bat was urinating, or by urethral swab. urine and saliva swabs were immediately placed into ml tri-reagent ls and frozen in liquid nitrogen. total rna from serum was extracted for uhts analysis to screen for the presence of microorganisms. five microliters of total rna from each bat were combined into pools: pregnant bats; non-pregnant female bats, and pools of adult male bats, respectively. reverse transcription (rt) was performed on dnase i-treated (dna-free, ambion inc., austin, tx, usa) rna pools to generate cdna using superscript ii rt (invitrogen, carlsbad, ca, usa) and random octamers linked to a defined arbitrary, -mer primer sequence tail (mwg, huntsville, al, usa) [ ] . after rnase h treatment cdna was amplified by the polymerase chain reaction (pcr), applying a : mixture of the defined -mer primer sequence and the random octamer-linked -mer primer sequence, respectively [ ] . products of . base pairs (bp) were selected by column purification (minelute, qiagen, hilden, germany) and ligated to specific linkers for sequencing on the genome sequencer flx ( life sciences, branford, ct, usa) without dna fragmentation [ , ] . sequences were analyzed using software applications implemented at the greeneportal website (http:// tako.cpmc.columbia.edu/tools/). multiple forward and reverse primers for rt-pcr (available upon request) were designed using the sequences obtained by uhts in order to fill gaps between fragments. amplifications were performed with bio-x-act (bioline, london, uk) according to manufacturer's protocols. products were size fractionated by electrophoresis and directly sequenced in both directions with abi prism big dye terminator . cycle sequencing kits (perkin-elmer applied biosystems, foster city, ca, usa) at a commercial facility (genewiz, south plainfield, nj, usa). additional methods applied to obtain the genome sequence included touch-down pcr [ ] , -step walking pcr [ ] , and -and -race (invitrogen). a real time taqman pcr assay was developed to screen bat samples for gbv-d. reactions were performed in a ml volume by using commercial taqman universal master mix (applied biosystems, foster city, ca, usa). primers and probe were designed to target a nt region in the ns a gene region: fadiforward, -gcagctgcgtgtgcca; fadi-reverse, -acacc-catgatgttaccacgac; fadi-probe, -fam-aggacccgg-tcgctccagca-t-bqx (tib molbiol, adelphia, nj, usa). cycling conditions were: uc for min, and uc for min, followed by cycles at uc for sec and uc for min. thermal cycling was performed in an abi real-time pcr system (applied biosystems). a liver function panel was conducted at the international center for diarrheal disease research (dhaka, bangaldesh) using non heat-treated bat sera (automated chemistry analyzer au , olympus corporation, tokyo, japan). the following parameters were analyzed: total protein, albumin, globulin, albumin:globulin ratio, total cholesterol, total bilirubin , alkaline phosphatase, alanine transferase, aspartate aminotransferase, gamma glutamyltransferase , and lactate dehydrogenase. sequence alignments were generated with clustalw software [ ] and phylogenetic relationships deduced using geneious software [ ] . statistical significance was assessed by bootstrap re-sampling of pseudoreplicate data sets. sequence relations were determined from p-distance matrices calculated with pairwise deletion for missing data and homogeneous patterns among lineages based on clustalw alignments as implemented in mega software [ ] . sliding window similarity analysis was performed using simplot [ ] . potential signalase cleavage sites, glycosylation sites, and phosphorylation sites were analyzed using the respective prediction servers available at the center for biological sequence analysis (http://www.cbs.dtu.dk/services/). bats are important reservoirs for emerging zoonotic viruses with significant impact on human health including lyssaviruses, filoviruses, henipaviruses and coronaviruses. opportunities for transmission to humans are particularly prominent in countries like bangladesh, where people live in close association with bats. whereas previous studies of bats have employed assays that test for known pathogens, we present the first application of an unbiased molecular approach to pathogen discovery in this reservoir for emerging zoonotic disease. unbiased pyrosequencing of serum from pteropus giganteus bats enabled identification of a novel flavivirus related to hepatitis c and gb viruses. viral nucleic acid was present in of ( %) sera, and in the saliva of one animal. sequence identification of two strains of the virus, tentatively named gbv-d, suggests p. giganteus as a natural reservoir. detection of viral nucleic acid in saliva provides a plausible route for zoonotic transmission. phylogenetic analysis indicates that gbv-d is ancestral to gbv-a and -c, and separate from the recently classified genus hepacivirus. our findings provide new insight into the range of known hosts for gb-like viruses and demonstrate the power of unbiased sequencing to characterize the diversity of potentially zoonotic pathogens carried by bats and other reservoirs. total rna from the serum of healthy bats captured at a roost in the faridpur district of bangladesh was extracted for uhts analysis. extracts of individual bats were combined into pools consisting of pregnant adult bats, non-pregnant adult female bats, or adult male bats. each pool yielded between , and , assembled contigs or singlton reads (representing , - , reads ranging in size from - nt). two reads of and nucleotides (nt) derived from the pregnant bat pool had distant homology to gbv-a sequences at the deduced amino acid (aa) level in the e and ns a gene regions respectively (blastx); no homology was detected by searches at the nt level (blastn; local copy of the executables with standard settings except that the reward for a nucleotide match was set to instead of ). no viral sequences were detected in other pools at the nt or aa levels. screening of the individual rna preparations from the pregnant bat pool using primers derived from the uhts reads confirmed the presence of the gbv-like sequence in the serum of bat . a quantitative real time pcr assay indicated a load of approximately rna copies in bat- serum extract, and identified an additional positive bat sera from the original samples ( / ; %), indicating serum loads ranging from to , rna copies per assay. these positive samples came from male bats that were not included in the initial uhts pools. extracts of saliva from the five positive bats indicated a load of approximately rna copies in bat ; no signal was obtained with urine extracts from the five positive bats. near full-length genome sequence was generated from bat- and a second positive serum (bat ), applying primers crossing mature structural proteins in gb viruses, as well as other flaviviruses, are the product of cleavage by host signal peptidase [ ] . in gbv-d the first potential signal sequence cleavage site is present after a stretch of , largely basic aa ( kda, pi = ), followed by sequence homologous to e (pfam , http:// pfam.sanger.ac.uk/) ( figure ). the single glycosylation site n it present in that sequence is located in a position comparable to gbv-c, -a, -b and hcv glycosylation sites. identification of the downstream e termini is less apparent as the next aa contain multiple potential signal sequences and potential glycosylation sites that indicate no homology to hepaciviral e /ns (pfam ), until the sequence aligns with n-terminal ns motifs (pfam ) (figure , figure ). however, despite similarity to pfam no signal sequence compatible with cleavage at a /a was found; cleavage may occur at g /r, which combined with potential signalase cleavage at a /f may indicate the existence of a heavily glycosylated potential kda product instead of the p transmembrane protein identified in hcv [ ] [ ] [ ] or the kda variant described in gbv-b [ , ] . conserved c-terminal motifs of the autocatalytic ns /ns endoprotease domain are compatible with ns /ns cleavage at s /a and comparable to other gbv and hcv [ ] . figure indicates potential cleavage sites for ns (peptidase s , pfam ; dead box helicase, pfam ; helicase c, pfam ), ns a (pfam ), ns b (pfam ), ns a (domain- a zinc finger, pfam ; domain- b, pfam ), and ns b (pfam ). conserved aa motifs were recognized in ns proteins. rnadependent rna polymerase (rdrp) motifs in rdrp block iii that are conserved with respect to other gbv and hepaciviruses were identified in ns b (figure ) [ ] [ ] [ ] [ ] . potential phosphorylation sites are present at multiple serine ( ), threonine ( ) and tyrosine ( ) residues in ns a, compatible with its possible function as a phosphorylation-regulated mediator of viral replication [ ] . however, significant conservation of primary sequence is not obvious for phosphorylation sites, proline-rich, or interferon-sensitivity determining region motifs [ ] [ ] [ ] . the c-terminal portion of ns has homology to conserved ntpase/helicase motifs [ ] ; the nterminal portion includes conserved active triad residues h , d , s of serine protease [ ] , the viral protease responsible for cleavage of mature non-structural proteins [ ] . likewise, the active triad h , e , c of the cis-acting protease activity in the cterminal portion of ns is conserved with respect to other gbv and hcv [ ] . the only other discernable motif identified was a wellconserved n c/d c motif at the n-terminus of e (figure ) [ ] . phylogenetic analysis of gbv-d was performed in comparison to selected representatives of gbv-a, gbv-b, gbv-c and hcv. analysis of ns b aa sequence ( figure a ) confirmed a closer relationship of gbv-d to gbv-a and -c than to gbv-b or hcv as also indicated by pairwise sequence comparisons ( table ) . the same relationships were also apparent when ns , or the complete polyprotein sequence were analyzed ( figure b and c, respectively). all three trees show gbv-d consistently at the root of the gbv-a/-c viruses, indicating an independent phylogenetic clade compatible with a separate species distinct from the recently created genus hepacivirus [ ] . a liver serum chemistry panel was conducted on sera from bats, the five gbv-d infected and non-infected animals. standard assays to detect hepatitis and/or impaired liver function were performed [ ] . levels of total protein, alanine transferase, aspartate aminotransferase and total cholesterol were within published ranges reported for p. giganteus, except for bat (infected) and bat (uninfected), which had modest elevation in aspartate aminotransferase. reference values for albumin, globulin, albumin:globulin ratio, total bilirubin, alkaline phosphatase, gamma glutamyltransferase and lactate dehydrogenase are not available for p. giganteus, however, values were comparable to those reported for other pteropus species [ ] . mean values did not significantly differ between infected and uninfected bats ( table ). molecular analyses of sera from pteropus giganteus bats from faridpur, bangladesh led to the identification of a , nt sequence consistent in genomic organization with known gbv and other species within the family flaviviridae [ ] . whereas previous studies of bats have employed assays that test for known pathogens, ours is the the respective sequences are indicated. entebbe bat virus was used as an outgroup; distance in substitutions per site is indicated by scale bars; percent bootstrap support for values greater than % is indicated at respective nodes. doi: . /journal.ppat. .g table . liver function values from pteropus giganteus bats. no indication of hepatitis or impaired liver function was observed; no significant differences between mean values for infected (bold) or non-infected bats were apparent. the first report of an unbiased molecular approach to pathogen discovery in this important reservoir of emerging infectious diseases. the modest yield of novel microbial sequences may reflect the choice of sample (e.g., serum vs feces, tissue or another specimen), competition between host and microbial template during unbiased amplification, or both. efforts to address template competition are under way that include subtraction of host nucleic acids or the use of semi-random primers that do not amplify host sequences. such efforts will likely enhance the sensitivity and throughput of unbiased sequencing technologies for pathogen discovery. the discovery of this chiropteran flavivirus broadens both the taxonomical and geographical distribution of gb-like viruses. three types of gb viruses have been described: gbv-a, -b and -c [ , , , , , ] . gbv-b, which has never been found in humans and was only reported in captive tamarins after serial passage of the original human gb serum [ ] , is most closely related to hcv and was recently classified together with hcv into a new genus, hepacivirus, within the family flaviviridae [ ] . gbv-a and -c remain unclassified members of the family. gbv-a have been isolated from several new world monkeys. different genotypes appear to be associated with specific monkey species of the genera saguinus, callithrix (callitrichidae family) and aotus (aotidae family), without any clinical signs associated with infection [ , , ] . gbv-c have been isolated from humans with non-a-e hepatitis; however, its pathogenicity is unknown and the virus is widespread in the human population [ , [ ] [ ] [ ] . population studies showed that gb viruses are enzoonotic and species-specific within both old and new world nonhuman primates as well as humans, and have likely co-evolved with their hosts over long periods of time [ ] . previously, the only gbv found in the old world was gbv-c from chimpanzees (in africa) and humans. although gbv-c were found in humans, gb viruses have not been previously reported in primates or other animals on the indian subcontinent. gbv-c and -a are remarkable for a truncated or missing capsid (c) protein [ , ] . due to exhaustion of our samples we were unable to complete assessment of the -terminal sequence; nonetheless, race experiments suggest that gbv-d likely codes for a short basic peptide, instead of a full-length c protein. the first methionine (m ) predicts a peptide of aa (pi = ); however, the more favorable kozak context [ ] of m indicates a aa peptide. after signalase cleavage from the polyprotein precursor, this peptide may be functional, possibly influencing maturation of, or directly binding to, the e and/or e glycoproteins. phylogenetic analyses of ns b, ns and complete polyprotein sequence place gbv-d at the root of the gbv-a and -c clades and are consistent with a model wherein gbv-d is ancestral to gbv-a and -c clades. mixed relationships indicative of recombination events [ ] were not evident (figure , figure ). both pteropid bats and chimpanzees are restricted to the old world. while the range of chimpanzees (africa) and p. giganteus (the indian subcontinent) do not overlap, it is possible that other primate species in bangladesh or india, such as macaques, or other fruit bats in africa such as eidelon spp., whose range overlaps that of chimpanzees, may carry related viruses. while gbv-a is only known from primates of the new world, an african origin has been suggested for gbv-c based on a -aa indel sequence in ns a [ ] . although the ns a sequence of gbv-d, similar to that of gbv-a, appears elongated in the indel region, compatible with their respective earlier phylogenetic branching compared to gbv-c, little sequence conservation is observed in that region. the bats in this study, like primates infected with their associated gbv [ ] , all appeared to be healthy. the lack of chemical evidence of hepatic inflammation or dysfunction suggests that this virus may not target hepatic cells in bats. this is consistent with the behavior of gbv-a in its natural primate hosts [ ] . in contrast, elevated alanine transferase levels and mild hepatitis are observed in experimental infections of macaques with gbv-c isolates from humans [ ] . five percent of the bats we studied were infected with one of at least two different strains of gbv-d, which suggests widespread viral circulation within this species. the observation that bats are asymptomatically infected with diverse strains that constitute a distinct phylogenetic clade is compatible with a co-evolutionary relationship between gbv and their hosts [ , ] , and supports the hypothesis that p. giganteus bats may be a natural reservoir for gbv-d. in one case we were able to detect gbv-d nucleic acid in saliva. this suggests a potential route for viral transmission via fighting or grooming behavior, or via food shared by bats. pteropus giganteus is a frugivorous bat species that carries niv, a zoonotic paramyxovirus [ , ] . this species lives in close association with humans in bangladesh and bats have been observed drinking from (and urinating into) date palm sap collecting pots [ ] . human consumption of contaminated palm juice is proposed to be a major route of niv transmission [ ] . although it is unclear whether infectious virus was present in bat saliva, the observation that saliva can contain gbv-d nucleic acids provides a biologically plausible mechanism for transmission from infected bats to other hosts. while it is currently unknown whether gbv-d virus occurs in humans, up to % of non-a-e hepatitis cases remain unexplained [ ] . bats: important reservoir hosts of emerging viruses marburg virus infection detected in a common african bat nipah virus: impact, origins, and causes of emergence bats are natural reservoirs of sars-like coronaviruses fruit bats as reservoirs of ebola virus nipah virus: a recently emergent deadly paramyxovirus infection of humans and horses by a newly described morbillivirus global trends in emerging infectious diseases henipavirus infection in fruit bats (pteropus giganteus) nipah virus encephalitis reemergence nipah virusassociated encephalitis outbreak person-to-person transmission of nipah virus in a bangladeshi community recurrent zoonotic transmission of nipah virus into humans foodborne transmission of nipah virus family flaviviridae genomic analysis of two gb virus a variants isolated from captive monkeys the sequence and genomic organization of a gb virus a variant isolated from captive tamarins sequence and genomic organization of gbv-c: a novel member of the flaviviridae associated with human non-a-e hepatitis molecular cloning and disease association of hepatitis g virus: a transfusiontransmissible agent gbv-c in the aetiology of fulminant hepatitis infection with hepatitis g-virus and viral hepatitis in india genotype of gb virus c hepatitis g virus by molecular evolutionary analysis detection in chimpanzees of a novel flavivirus related to gb virus-c hepatitis g virus isolation of a gb virus-related genome from a chimpanzee panmicrobial oligonucleotide array for diagnosis of infectious diseases detection of respiratory viruses and subtype identification of influenza a viruses by greenechipresp oligonucleotide microarray a metagenomic survey of microbes in honey bee colony collapse disorder genome sequencing in microfabricated high-density picolitre reactors touchdown pcr for increased specificity and sensitivity in pcr amplification characterization of bacterial operons consisting of two tubulins and a kinesin-like gene by the novel two-step gene walking method multiple sequence alignment with the clustal series of programs geneious v mega : molecular evolutionary genetics analysis (mega) software version . full-length human immunodeficiency virus type genomes from subtype cinfected seroconverters in india, with evidence of intersubtype recombination flavivirus genome organization, expression, and replication processing in the hepatitis-c virus e -n region -identification of p and distinct e -specific products with different c-termini hepatitis-c virus glycoprotein e products with different c-termini signal peptide cleavage and internal targeting signals direct the hepatitis c virus p protein to distinct intracellular membranes characterization of gb virus b polyprotein processing reveals the existence of a novel -kda protein with partial homology to hepatitis c virus p protein functional analyses of gb virus b p protein: development of a recombinant gb virus b hepatitis virus with a p protein hepatitis g virus encodes protease activities which can effect processing of the virus putative nonstructural proteins the modeled structure of the rna dependent rna polymerase of gbv-c virus suggests a role for motif e in flaviviridae rna polymerases the phylogeny of rna-dependent rna polymerases of positive-strand rna viruses identification of conserved motifs among the rna-dependent polymerase encoding elements rift-valley fever virus l-segment -correction of the sequence and possible functional role of newly identified regions conserved in rna-dependent polymerases phosphorylation of hepatitis c virus ns a nonstructural protein: a new paradigm for phosphorylation-dependent viral rna replication src homology domain of hepatitis c virus ns a protein interacts with bin and is important for apoptosis and infectivity how hepatitis c virus counteracts the interferon response: the jury is still out on ns a ns a, a nonstructural protein of hepatitis c virus, binds growth factor receptor-bound protein adaptor protein in a src homology domain/ligand-dependent manner and perturbs mitogenic signaling rna translocation and unwinding mechanism of hcvns helicase and its coordination by atp gb virus b and hepatitis c virus ns serine proteases share substrate specificity the ns / a proteinase of the hepatitis c virus: unravelling structure and function of an unusual enzyme and a prime target for antiviral therapy molecular and serologic analysis in the transmission of the gb hepatitis agents diagnosis and monitoring of hepatic injury. i. performance characteristics of laboratory tests amer assoc clinical chemistry comparative rectal bacterial flora of four species of flying fox (pteropus sp.) five new or recently discovered (gbv-a) virus species are indigenous to new world monkeys and may constitute a separate genus of the flaviviridae studies on transmission of human viral hepatitis to marmoset monkeys. . transmission of disease serial passages and description of liver lesions acute non-a-e hepatitis in the united states and the role of hepatitis g virus infection gb virus type c/hepatitis g virus clinical impact of gb virus c viremia on patients with hiv type infection in the era of highly active antiretroviral therapy phylogenetic analysis of gb viruses a and c: evidence for cospeciation between virus isolates and their primate hosts initiation of translation in prokaryotes and eukaryotes homologous recombination in gb virus c/ hepatitis g virus african origin of gb virus c hepatitis g virus species-specific variants of gb virus a in captive monkeys serological and histological findings in infection and transmission of gbv-c/hgv to macaques transmission of human infection with nipah virus we thank the forestry department of bangladesh for permission to conduct this research; md. sheikh gofur and md. pitu biswas for help in sampling bats; a. bennett, a. tashmukhamedova, and r. tokarz for technical support, and k. olival for critical comments on the manuscript. conceived and designed the experiments: jhe tb pd wil. performed the experiments: jhe plq dv sh. analyzed the data: jhe plq tb cs oj dv skh me pd. contributed reagents/materials/analysis tools: jhe tb cs sc sak mjh skh me spl wil. wrote the paper: jhe plq tb wil. coordinated field and lab activities and logistics for work in bangladesh; contributed to paper-writing: mjh. coordinated permissions, field activities and logistics for work in bangladesh; contributed to paperwriting: spl. key: cord- -qu zin o authors: wu, nannan; nguyen, xuan-nhi; wang, li; appourchaux, romain; zhang, chengfei; panthu, baptiste; gruffat, henri; journo, chloé; alais, sandrine; qin, juliang; zhang, na; tartour, kevin; catez, frédéric; mahieux, renaud; ohlmann, theophile; liu, mingyao; du, bing; cimarelli, andrea title: the interferon stimulated gene protein (isg ) is an innate defense antiviral factor that discriminates self versus non-self translation date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: qu zin o isg is a broad spectrum antiviral protein thought to directly degrade viral rna. however, this mechanism of inhibition remains controversial. using the vesicular stomatitis virus (vsv) as a model rna virus, we show here that isg interferes with viral replication by decreasing protein synthesis in the absence of rna degradation. importantly, we demonstrate that isg exerts a translational control over a large panel of non-self rna substrates including those originating from transfected dna, while sparing endogenous transcripts. this activity correlates with the protein’s ability to localize in cytoplasmic processing bodies. finally, these functions are conserved in the isg murine ortholog, whose genetic ablation results in mice with increased susceptibility to viral infection. overall, our results posit isg as an important defense factor able to discriminate the self/non-self origins of the rna through translation modulation. isg was identified in by the mechti laboratory as a type-i interferon-induced protein associated to promyelocytic leukemia (pml) nuclear bodies and then to nucleoli and cajal bodies [ ] [ ] [ ] . the protein belongs to the dnaq-like (or dedd, for asp-glu-asp-asp) '- ' exonuclease superfamily that includes several enzymes with dna or rna specificities. members of this superfamily share three conserved exonuclease motifs (exo i, ii and iii) that surround the protein's active site and contain the four dedd residues important for metal ions chelation [ , ] . isg exhibits rna, but no apparent dna exonuclease activities [ , ] and this property has been associated with inhibition of a broad range of rna viruses such as flaviviridae (yellow fever, west nile, dengue, hepatitis c and bovine viral diarrhea viruses), picornaviridae (hepatitis a virus), togaviridae (sindbis, chikungunya and venezuelan equine encephalitis viruses), rhabdoviridae (vesicular stomatitis virus, vsv), orthomyxoviridae (influenza virus), retroviridae (human immunodeficiency type virus), hepadnaviridae (hepatitis b virus) and more recently several bunyaviridae [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , although certain rna viruses do resist isg as human coronavirus and phleboviruses [ , ] . direct viral rna degradation by isg has been proposed as the main mechanism of viral inhibition based on two main evidences: its potent exonuclease functions in vitro and the loss of antiviral effects observed upon the mutation of dedd residues in the protein's catalytic site, which constitute at present the only isg mutants described in the literature. however, a number of studies have observed viral inhibition by isg in the absence of viral rna degradation [ , ] , raising the possibility that alternative mechanisms may be at play. in this study, we determine that isg acts by modulating mrna translation and we provide evidence that this mechanism broadly targets mrna transcripts originated from exogenous genetic material independently of its viral origins (that we globally refer to here as of non-self origins), while sparing mrnas of endogenous chromosomal origins (self), or selfmimicking ones. using vsv as a model for highly replicative rna viruses, we show here that isg decreases translation from a broad spectrum of viral as well as non-viral mrnas, as long as they are ectopically expressed. while this mechanism of discrimination relies on an intact catalytic core, it takes place in the absence of target mrna degradation and correlates with the ability of isg to localize in cytoplasmic processing bodies (p bodies). the properties of human isg are also conserved in its murine orthologue and through the crispr/cas -mediated generation of isg knockout mice, we provide clear evidence that isg plays an important role in the modulation of virus replication in vivo. overall, our study reveals a novel role for isg as a translational modulator capable of discriminating self from non-self mrnas, offering novel perspectives on strategies of viral control and an appropriate animal model in which to test them. to apprehend the role/s of isg during viral infection, dox-inducible jurkat, thp- and hela cell lines stably expressing isg were generated by retroviral-mediated gene transduction followed by the selection of the pool of transduced cells. isg was then expressed at levels comparable to those observed in primary monocyte-derived macrophages and dendritic cells stimulated with ifnα, not only to keep within physiologically relevant boundaries, but also because we noted that higher expression levels of isg were cytotoxic in established cell lines (s a and s b fig). as such, all subsequent experiments were carried out at non-cytotoxic levels of isg expression. under these conditions, wt isg inhibited vsv spread in the different cell lines tested, in contrast to a catalytically inactive isg mutant (fig a, m , that corresponds to the already described . however, when hela cells were examined closely, we noted only a moderate reduction in the levels of viral-coded gfp rna in isg -expressing cells in contrast to the drastic decrease in the amount of accumulated viral proteins by wb (fig b, in this recombinant virus, gfp behaves as an additional transcriptional viral unit inserted between the m and the g genes, [ ] ). this was not due to loss of the rna exonuclease abilities of isg , as these were readily measurable when isg was immunoprecipitated from cell lysates and incubated with different exogenously-provided substrates (single or double stranded rnas, as well as dna:rna hybrids, although not dnas as expected [ ] , s a and s b fig) . this lack of specificity was not present in cells however, given that the ectopic expression of isg did not lead to the indiscriminate degradation of cellular rnas (ribosomal as well as small nucleolar rnas previously shown to be associated to isg [ ] ), nor of an hepatitis c virus (hcv)-derived luciferase reporter mrna produced in vitro and then transfected into cells (s c and s d fig) , suggesting that in cells the rnase activity of isg is tightly controlled. given that the protein accumulation defect was also observed after mg -mediated inhibition of proteasome (s fig), we decided to determine whether the effects of isg related to translation inhibition. to this end, hela cells were metabolically labelled hours post vsv infection for one hour with s methionine and cysteine, prior to cell lysis and phosphor imager analysis (fig c and d ). under these experimental conditions, wt isg imposed a strong translational inhibition to the expression of viral-coded proteins (n and gfp) in contrast to the catalytically inactive m -isg mutant. cellular translation rates remained overall unperturbed, indicating that under these experimental conditions isg was capable of discriminating viral from cellular mrnas. overall, these findings indicate that isg may exert a novel antiviral inhibitory mechanism through the selective translational control of viral mrnas. the antiviral activity of isg strictly correlates with its ability to act as a general translational modulator of non-self genetic material to determine whether this mechanism of control could target more broadly non-self genetic material, we used transient dna transfection which indeed represents a massive introduction of exogenous genetic material into the intracellular milieu. transfection of isg -expressing hela cells with dnas coding for a gfp reporter yielded a mutually exclusive pattern of expression upon confocal microscopy analysis, so that cells expressing isg were rarely gfppositive and vice versa, mirroring the results obtained during ongoing vsv infection (fig a, for qualitative results). this pattern could be observed with wt isg , but not with the catalytically inactive m mutant and interestingly, loss of gfp protein accumulation was observed in the absence of effects on several endogenous proteins tested, as assessed by wb and in the absence of detectable changes in gfp dna and mrna levels (fig b) . similar results were obtained with pcr amplicons placed at different location on the gfp mrna and with reverse transcription reactions started with random hexamers or at the polya tail with oligo-dt primers, excluding the possibility of partial end degradation of the gfp mrna (s a fig) . at the protein level, similar defects in gfp accumulation were observed in the presence of isg both by wb using limiting dilutions of samples, as well as by facs by adding defects in both the accumulation of gfp-positive cells and in their mean fluorescent intensity (s b and s c fig). lastly, the inhibitory effect of isg was dose-dependent (s d fig) and was . b) sixteen hours post infection, a fraction of hela cells expressing or not isg were lysed and analyzed by rt-qpcr for the viral coded gfp rna and by wb. c) as in b, but cells were incubated for one hour with s met/cys prior to cell lysis. samples were then directly loaded onto an sds-page gel and analyzed by phosphor imager. d) viral translation was quantified on the viral-coded proteins n and gfp, while cellular translation was quantified on the indicated portion of the gel (normalized to each no dox. condition). nd, not determined. the graphs present means and sem of two to four individual experiments, while the panels present typical results obtained. � ; p� . between the indicated condition and control, according to a student t test. observed for a large spectrum of ectopically expressed proteins independently from their dna backbone, transfection method used or origins (viral or cellular genes, s e fig) . given the lack of described mutants in isg outside its dedd residues, a series of mutations were engineered in isg outside the exo domains, in sequences of potential interest (phosphorylation, sorting and nuclear import, through the expasy and the cbs protein sequence analysis prediction tools) to better apprehend the relationship between antiviral functions and translation control abilities. not all generated mutants could be detected upon transient transfection and wb analysis, likely due to a compromised protein stability. however, five novel isg mutants could be analyzed functionally (fig c) . although not pursued in depth in this study, these mutations could potentially affect phosphorylation on serine (m and m - ), or trafficking via mutations in dileucine domains (m and m ), or in nuclear localization signals (m ). three isg mutants lost their ability to inhibit vsv spread in the antiviral properties of isg strictly correlate with its ability to act as a general modulator of translation of non self genetic elements. a) hela cell lines stably expressing isg were analyzed by confocal microscopy twenty-four hours after vsv-gfp challenge at an moi of . , or after transfection with dna coding for gfp, as indicated. b) the same cells used in the bottom panels in a were also analyzed by wb, rt-qpcr (rna), qpcr (dna), as indicated. the pictures and the panels present typical patterns of expression, while the graph presents data obtained from four experiments. c) schematic representation of the isg mutants used here. the gray boxes indicate schematically the position of the three exo domains of isg . the color code is used throughout the figure and refers to the antiviral activities of the indicated mutant: purple = lost; yellow = preserved. d) dox-inducible isg stable jurkat t cells expressing the different mutants were obtained and infected with vsv, as specified above at an moi of . . the graph presents typical replication curves obtained out of three independent experiments. e) hek t cells were ectopically transfected with dnas coding for isg mutants and trex , used here as an additional reporter of isg activity on ectopic gene expression. cells were then metabolically labelled for one hour with s met/cys prior to cell lysis. a fraction of the soluble lysate was loaded to appreciate global translational effects (input), while the rest was used for immunoprecipitations with anti-flag antibodies specific for tagged isg and trex proteins. samples were then loaded onto an sds-page gel for phosphor imager analysis. the graph presents means and sem of three to five independent experiments. � ; p� . between the indicated mutant and control, according to a student t test. f) correlation between antiviral and translation inhibitory properties of individual isg mutants. the ability of these mutants to inhibit translation from ectopically expressed genes was assessed either directly by s metabolic labeling, or by wb (fig e and s b fig, using trex or gfp as examples of exogenous genes, respectively). under these conditions, the m and m mutants behaved as wt and strongly reduced the rate of protein synthesis from the exogenous reporter genes, while in contrast the m , m and m - mutants behaved as the catalytically inactive m -isg and did not significantly affect protein translation, highlighting a near perfect correlation between antiviral abilities and translation inhibition of non-self material ( fig f) . with the exception of the catalytically inactive m , all mutants exhibited robust rnase activities in vitro (s c and s d fig) . interestingly, two of the three mutations that altered the properties of isg were spatially close and exposed at the surface of the crystal structure of isg [ ] , potentially defining a region that may be involved either directly or indirectly in the regulation of the protein's functions (s e fig). self mimicry allows the escape of target genes' mrnas from the effects of isg vsv infection and ectopic dna or rna transfection do not bear much in common apart from the fact that both represent artificial injections into the cell of non-self genetic material from without. to determine whether isg could be an ifn-induced mechanism of defense acting at the translational level, we determined the effects of isg on exogenous expression cassettes that mimicked cellular host genes. to this end, retroviral-mediated gene transduction was used to stably integrate into the host genome the identical cmv-gfp expression cassette used in fig a. after a three weeks selection, cells were transiently transfected with wt isg , lysed and analyzed by wb (fig a) . in this case, isg did not modify the amount of produced gfp, indicating that, when expressed from the cell's genome, the gfp-coding mrna is isg -resistant and suggesting that cellular mrnas mimicry (meant here as the ability to behave as, or similarly to, cellular genes) could constitute an escape mechanism from isg . some dna viruses as the epstein-barr virus (ebv, herpesviridae) can be stably maintained in an extrachromosomal latent form in the nucleus of target cells (referred to as episomes) using the cellular dna machinery for their duplication and segregation into dividing cells. although both transiently transfected dna and the ebv genome are extrachromosomal, only the latter presents a chromatin status identical to the host genome [ , ] . to determine whether this, rather than integration per se, could be a determining factor in the susceptibility to isg , hek t cells bearing stable ebv-gfp episomes were transiently transfected with dnas coding for wt isg , in addition to a control plasmid coding for hiv- gag (that served as a control for non-self genetic material within the same cells, fig a) . upon cell lysis and wb analysis, isg did not affect the amount of gfp expressed from the ebv genome, whereas it did affect the amount of gag. to further strengthen this point, the effect of isg on ebv infection was determined in hone- cells upon the reactivation of the viral lytic cycle with tpa/ba (fig b) [ ]. isg did not affect the production of several ebv proteins that typically initiate its lytic cycle indicating that ebv mrnas were, as cellular ones, largely resistant to the action of isg . also, isg did not modify the extent of ebv infection, when virion particles produced under these conditions were used to challenge raji cells (s fig) . overall, these results indicate that isg discriminates mrnas based on their self/non-self genesis and also underline the fact that certain viruses, as ebv, may have devised mechanisms to resist this inhibition due to their specific modes of replication. to determine whether isg could affect translation through modifications in either splicing or transport of mrnas from the nucleus to the cytoplasm, its activity on intron-or intronlessluciferase based constructs was assessed [ ] and the accumulation of target mrnas was determined in cytoplasmic and nuclear fractions (s a and s b fig) . isg exerted similar inhibitory effects on both intron and intronless constructs and it did not appreciably modify the distribution of target mrnas in cytoplasm and nucleus, overall suggesting that isg does not affect the nucleo-cytoplasmic transport of mrnas. lastly, isg could not be detected in crude ribosomal fractions, indicating a lack of stable association with ribosomes (s c fig). a series of constructs was used to determine whether isg affected translation initiation by comparing more specifically its effects on cap-dependent versus internal ribosomal entry site (ires)-translation (fig c, through the use of bicistronic vectors), as well as its effects on rnas with different ' untranslated regions ( ' utrs) that require or not eif factors to initiate translation (fig d) . gene mimicry of self through integration into the host genome, or ebv-mediated episomal maintenance, allows escape from isg translational targeting. a) hek t cells were first stably transduced with a retroviralbased vector bearing a cmv-gfp expression cassette and then transiently transfected with an isg -expressing dna vector. cells were analyzed forty-eight hours after by wb. similarly, hek t cells carrying a stable ebv-gfp episomal genome were transiently transfected with dnas coding for isg and hiv- gag, used here as a reporter of the activity of isg on ectopic expression. b) hone- cells that bear a stable latent episomal ebv genome were transfected with isg prior to stimulation with -o-tetradecanoylphorbol- -acetate (tpa) and butyric acid (ba) that reactivates the life cycle of ebv via a pkc-dependent mechanism. cells were lysed twenty-four hours later and analyzed by wb for the indicated ebv proteins. panels present typical results obtained. c) hek t cells were transfected with isg along with the indicated bicistronic dna vectors. the first cistron bears the 'utr of the cellular gene globin (glo). the second cistron is under the control of several ireses derived from the cellular gene myc (myc), polyomavirus (pv), or the encephalomyocarditis virus (emcv). mrna levels have been normalized to controls (no isg condition) and luciferase activities are reported after mrna normalization. d) translationcompetent mrna generated in vitro were transfected in isg expressing cells, prior to cell lysis one hour afterwards and analyses by rt-qpcr or luciferase assays. the 'utrs of these mrnas varied with respect to the requirement of specific eifs and were derived from the cellular gene myc (myc), the cricket paralysis virus (crpv) and vsv. the graphs present means and sem of (monocistronic) and (bicistronic) independent experiments. � ; p� . between control and isg , as indicated, according to a student t test. isg was capable of inhibiting both cap-and ires-dependent translation from bicistronic mrnas in the absence of mrna degradation and interestingly this occurred on ires sequences in which translation initiation required ribosomal scanning along the mrna or not (fig c, ireses obtained from c-myc, polyomavirus-pv, or the encephalomyocarditis virus-emcv, respectively). to further determine whether isg acted on translation initiation and more specifically through specific initiation factors (eifs), capped and polyadenylated mrnas coding luciferase under the control of several ' untranslated regions ( ' utrs) were generated in vitro and directly transfected in isg -expressing cells (fig d) . under these conditions, isg was able to inhibit translation of target rnas containing either the ' utr of c-myc, that requires the complete set of eifs, or of the cricket paralysis virus (crpv) that requires none in the absence of mrna degradation. we noted that in the case of direct mrna transfection the extent of isg inhibition was lower than in the case of dna transfections, but we believe this is mainly due to the shorter time frame elapsing between transfection and luciferase assay (one hour, as opposed to overnight). as expected, the catalytically inactive m -isg mutant lost its ability to inhibit translation from these reporter constructs (s fig) . overall, these analyses underline the fact that the translational interference of isg proceeds in the absence of overt mrna degradation in a manner that appears independent from the presence of specific initiation factors during ' cap loading. when expressed in two clonal mouse embryonic fibroblast (mef) cell lines, isg has been recently described to drive type i interferon responses resulting in the expression of different interferon-sensitive genes among which the interferon-induced protein with tetratricopeptide repeats (ifit ) [ ] . ifit recognizes viral uncapped rnas (often defined as non-self, although the definition used in our study includes more broadly all foreign genetic elements) and has been extensively associated to translation inhibition [ - ], this work raised the possibility that isg could inhibit translation indirectly via the induction of ifit . to address this possibility, we analyzed ifit expression in both ectopically transfected hek t cells, as well as in stable hek t and u cells, a myeloid cell line highly sensitive to ifn (fig a) . when hek t cells were ectopically transfected with increasing amounts of isg -coding dnas, ifit remained undetectable unless ifnα was provided ( . u/ml). similarly, isg induction by doxycycline in stable hek t and u cells did not induce a concomitant ifit production, indicating that isg does not induce ifit expression under the experimental conditions used here. to more generally appreciate whether isg induced an ifn program, we measured the levels of type-i ifns potentially secreted upon induction of isg in the supernatants of u cells that are more responsive to ifn stimulation than hek t. to this end, isg expression was induced via doxycycline in stable u cells and hours later supernatants were harvested and incubated with reporter hek t cells bearing an integrated expression cassette responsive to type i interferons ( - :luc). reporter cells were then lysed twenty-four hours after and luciferase measured. the levels of type-i ifn measured via this assay remained below the threshold of detection ( u/ml, in our hands), indicating the absence of major ifn up-regulation following isg induction (fig b) . lastly, we wished to determine whether ifit could exert similar activities than isg . to this end, hek t cells were transiently transfected with dna coding these proteins along with gfp as non self genetic material (fig c) . under these conditions, isg but not ifit inhibited the accumulation of gfp, while both inhibited vsv infection (s fig). ifit has been described to act in complex with other ifit proteins [ ] . given that its ectopic expression alone is sufficient to inhibit vsv replication, we believe that other ifits are expressed at a basal level under the conditions used here and that ifit may be a limiting component of the complex. on the whole, our results indicate that ifit is not involved in the mechanism of translation inhibition by isg given that: ) isg does not induce the expression of ifit , at least under the experimental conditions used in this study and ) that isg , but not ifit , is capable of inhibition of translation from non-self genetic material. isg is largely distributed throughout the entire cell [ , ] , although certain studies reported a more specific association with nuclear bodies (pml nuclear bodies, nucleoli and cajal bodies [ , ] ). to determine whether this was the case, we employed a commonly used technique to reveal protein association to nuclear bodies. cells were transiently transfected in duplicate with isg (transfection rates � %) and while one sample was immediately fixed, the other was previously treated with detergent prior to fixation to reveal proteins strongly associated to in parallel, ifit expression levels were analyzed by wb in hek t and u stable-isg cells stimulated for twenty-four hours with different concentrations of doxycycline, or with ifnα ( . u/ml). b) to measure the possible presence of ifn subtypes in the supernatant of cells expressing isg , u cells that express the most readily detectable levels of ifit were stimulated as indicated and the supernatant was then transferred to reporter cells expressing luciferase under the control of the ifn-inducible - promoter. luciferase production from reporter cells was then determined twenty-four hours later. the linearity and limit of detection of the assay ( u/ml in our hands) are routinely determined with external dilutions of ifn. c) hek t cells were transiently transfected with plasmids coding for either isg or ifit along with a gfp-coding plasmid followed by wb analysis. the graph present results obtained from independent experiments, while the wb panels display representative results obtained from independent experiments. https://doi.org/ . /journal.ppat. .g nuclear bodies. under these conditions, partial co-localization of isg was observed with splicing speckles (sc nuclear bodies, s a fig), but the signal was essentially lost upon detergent pre-extraction, suggesting that the association between isg and nbs may be very weak. instead, we noted a partial colocalization between isg and both the endogenous forms of the trinucleotide repeat containing a protein (tnrc a) and the dead-box helicase (ddx ), two well-established markers of p bodies [ , ] (fig a for hek t cells expressing isg and s b fig for the complete panels of control cells) both in unstimulated cells, as well as in cells treated for one hour with puromycin, drug that induces translational stress and increases both the proportion of cells exhibiting p bodies, as well as their number on a per cell basis. however, isg did not affect the proportion of cells presenting or not p bodies over controls, suggesting that isg does not induce p bodies by itself. to study the correlation between the antiviral phenotype of isg and p bodies, hek t cells expressing the different isg mutants were examined by confocal microscopy after ectopic expression of a destabilized version of tnrc a, that allows for an easier visualization of p bodies (fig b for representative pictures and graph for comprehensive quantification of the proportion of p bodies within cells that scored positive for isg ). under these conditions, wt isg was present in % of p bodies in double-positive cells, a proportion similar to the functional isg m and m mutants ( % and %, respectively). in contrast, the proportion of isg present in p bodies dropped substantially in all the non-functional mutants examined (m , m and m , with rates of %, % and %, respectively). thus, these results indicate isg co-localizes with p bodies. a) hek t isg -expressing cells were analyzed by confocal microscopy along with two markers of p bodies, tnrc a and ddx in the presence or absence of a one-hour incubation with μg/ml of puromycin that induces a translational stress known to increase the number of cells expressing p bodies as well as the number of p bodies on a per cell basis [ ] . control cells not expressing isg are shown only as a zoomed overlay, while the complete panel are presented in s b fig. the graph presents the proportion of cells exhibiting detectable p bodies in the presence or absence of puromycin and/or isg . b) the different isg mutants were similarly analyzed by confocal microscopy with the exception that an ha-tagged, destabilized form of tnrc a was also concomitantly expressed by transfection to increase the extent of p bodies accumulation. representative pictures and distributions (out of > cells per condition/mutant in two to three independent experiments) are shown. the graph presents the proportion of p bodies with or without isg in double-positive cells. that p bodies and/or p bodies localization may be critical for translation inhibition and antiviral properties of isg . murine and human isg are highly conserved and display more than % identity at the amino acidic level (s fig) and a recent report indicated that murine isg was also endowed with anti-viral properties [ ] . to determine whether the remaining functions of isg described here were also similarly conserved, murine isg (misg ) was ectopically expressed in hek t cells and compared to its human counterpart (hisg ). under these conditions, misg was able to inhibit vsv spread through the culture (fig a) , it impaired expression from transfected dna coding for a luciferase reporter (fig b) and was also able to cluster along with tnrc a bodies (fig c) . therefore, these results indicate that the key functions of isg described here are mainly conserved between these two animal species. to determine the importance of isg during viral control in vivo, isg knockout mice were generated by crispr mediated gene editing (fig a, left panel) and the functional ablation of isg was controlled upon in vitro stimulation of bone-marrow derived macrophages (bmdm, fig a, right panel) . isg -/-mice were viable and displayed the expected mendelian frequency. when animals were challenged intraperitoneally with vsv, the survival rates of isg -/-mice were severely compromised with respect to age-and sex-matched control groups (fig b) , indicating that isg plays an important controlling role during viral containment in vivo. the higher replicative capacity of vsv was evident in different organs and correlated with higher infiltration of inflammatory cells as observed upon hematoxylin and eosin staining (fig c and d , lung sections). as expected, peritoneal macrophages (pem) isolated from isg -/-mice exhibited an increased susceptibility to infection (fig e, in both percentage of infected cells as well as in their mfi), thus confirming a primary role for isg in intracellular innate defenses. overall, these results indicate that isg is an important contributor of viral replication control in vivo, corroborating similar results obtained in a previously published animal model [ ] . in this study, we determine that isg acts as an important antiviral factor in vitro, as well as in vivo, given that mice genetically deficient in isg exhibit higher mortality following viral challenge. the antiviral properties of isg are not linked to its ability to directly degrade viral rna, but rather to its capacity to interfere with the process of translation. this mechanism of translational regulation targets and is able to distinguish viral rnas as well as rna originated from ectopically transfected dna (that we globally refer to as non-self, for mrnas originated from non-self genetic elements) from cellular mrnas (or self) that are instead resistant to isg , under the experimental conditions used here. finally, we determine that resistance to isg can be achieved via integration into the host genome, or through ebv-like maintenance in stable episomes through what can be defined as self mimicry. isg is a potent and non-specific rna exonuclease in vitro and this property had long been hypothesized to underpin a broad mechanism of inhibition via the direct degradation of the rna genome of a large spectrum of rna viruses, albeit not all [ , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . however, a few studies failed to observe a decline in viral rna levels in the presence of isg [ , ] and evidence of direct binding of target rnas to isg in cells is lacking. the data presented in our study also argue against overt degradation of viral rna and point instead to an interference with the process of translation that not only targets viral rnas, but more generally distinguishes mrnas according to their self/non-self origins (according to the broader definition used in this study). despite the fact that the catalytically inactive mutant m loses both its exonuclease and translation inhibition activities, whether the former is required for translation inhibition remains unclear. indeed, mutations in the dedd residues may potentially exert multiple effects on the protein functions in addition to inhibiting its rnase activity and the in vitro assays used in the field (as well as in this study) do not seem to recapitulate the stringency of action of isg in cells. it is important to note that although our results do not support a mechanism based on the direct degradation of target mrnas by isg , they do not exclude that its exonuclease activity may be turned against a cellular rna component, itself involved in translation. rather, we favor this hypothesis in which isg may attack ribosomal rnas or small nucleolar rnas that are required for ribosomal rna methylation, possibly affecting the ribosomes' behavior. given that cellular rnas continue to be translated, this implies that isg may affect some, but not all ribosomes. we do not know whether this occurs here, however recent genome-wide studies have clearly highlighted that ribosomes are not only heterogeneous in composition, but also in function [ - ], so that it is possible that the activity of isg is directed only against a specific subset of ribosomes. the ability to discriminate rnas according to the global definition of self/non-self origins we use here during translation is an interesting concept and sting has also been recently shown to inhibit the translation of viral, as well as of ectopically expressed mrnas through a novel ifn-independent activity [ ]. notable differences exist however with the mechanism described here, as sting seems to act on the phase of translation initiation, thus sparing mrnas that require no initiation factors (such as crpv) [ ], while on the contrary isg targets also these mrnas, strongly pointing in this case to a mechanism of regulation at a step other than translation initiation. a previous study also suggested that isg could interfere with viral translation in the absence of target rna degradation [ ] . in this study however, this inhibition was linked to the ability of isg to induce ifn when expressed in mef cell lines and more specifically the interferon-protein with tetratricopeptide repeats protein (ifit , [ ] ), a known translational modulator that impairs uncapped rnas [ , , ]. however, we have failed to obtain evidence of ifn and of ifit upregulation in our experimental system and since ifit cannot target the translation of mrnas expressed from plasmid dnas its involvement in the isg -mediated inhibition we describe here is unlikely. furthermore, if isg expression resulted in the direct upregulation of an ifn program, isg would be expected to inhibit far more viruses than currently reported. it is therefore possible that this aspect of the biology of isg can be observed solely under very particular conditions. if translation initiation does not seem to be the main step affected by isg , our results highlight a tight correlation between the ability of the different isg mutants to inhibit translation and their localization in p bodies, which is in line with the key role of these cytoplasmic structures in translational silencing and which would suggest rna re-routing by isg . p bodies are involved in numerous aspects of rna metabolism and although initially described as sites of rna degradation [ ], their functions have been further complexified by studies indicating their role in rna storage as well as in rna translational repression in the absence of rna degradation [ ] [ ] [ ] [ ] [ ] . thus, the results we have obtained at this stage support the hypothesis that p bodies play an instrumental role in the action of isg . however, it remains possible that these structures form as a consequence of the translational block itself [ ] [ ] [ ] , so that additional work will be required to firmly establish the functional role of these structures during isg -mediated translation inhibition. our data indicate that the main determinant of susceptibility to isg is the self/non-self nature of the genetic element from which the mrnas originate and we hypothesize that the chromatin status of the transcribed elements may be a determining factor of this phenomenon. specific co-or post-transcriptional modifications themselves linked to the chromatin status (as for example, n -methyladenosine, m a, reviewed in [ , ] ) are known to modulate different steps of mrna biology and it is possible that a major difference between genetic elements embedded in the host chromatin and the others resides in the identity of marks deposited on the mrna. lack of such signals may expose the mrna to isg and to its translational regulation, providing an interesting level of regulation of foreign elements. an alternative and non-mutually exclusive hypothesis may be that the levels -and not the identity-of post-transcriptional modifications may be lower in elements that are either present in high number of copies or that are expressed at high levels, simply because the system is overrun, thus exposing the rna to the action of isg . this hypothesis is also of interest and may be viewed as a basic threshold control mechanism of transcription/translation that is automatically turned on when genetic elements that replicate (or transcribe/translate) rapidly at high levels, as is the case during viral infection or following ectopic dna transfection. this hypothesis may certainly explain the different isg -susceptibility of rna viruses with respect to cellular genes, because most rna viruses replicate to exponential levels in cells in a very brief period of time, but it may also explain the isg -susceptibility of ectopically transfected dnas/genes, as their chromatin status is distinct from the one of the host genome [ , , , ] and their expression levels are generally much higher when compared to integrated cellular genes. in this respect, integration into the host genome or latent episomal maintenance may constitute a self mimicry defense strategy used by certain viruses, as we have shown here for ebv, to escape isg surveillance. from a more general perspective, it will be of high interest to determine the extent of the translational control activity of isg on viruses such as retroviruses, retroelements, as well as more broadly on different dna viral families. ifn responses rely on a complex network of more than . isgs. along with its multiple effects on the cell physiology, a major antiviral block of type i ifn responses takes place during translation initiation, by protein kinase r (pkr)-mediated inactivation of the eukaryotic initiation factor alpha (eif α, [ , ] ). given that the effects of ifn on translation are not absolute and vary according to the cell type, the fact that isg targets a step downstream translation initiation is of high interest, because it suggests the existence of a second layer of translational control in which isg would target rnas that have escaped the eif α -block. by extension, this may suggest the existence of yet additional layer of translational control during ifn responses all of which concur in limiting viral replication. overall, our study reveals that isg is an important modulator of antiviral responses in vitro and in vivo and points to a novel function through which this protein is able to distinguish self from non-self mrnas at translation. n-terminal flag-tagged human isg mutants (gene id: ) were produced by overlapping pcr from an original pcdna /frt-flag-isg plasmid obtained from ju-tao guo (baruch s. blumberg institute, doylestown, pennsylvania, usa) and cloned into pcdna (invitrogen), or pretrox-tight-puro backbones by standard molecular biology techniques (clontech). the latter is a murine leukemia virus (mlv)-based genome suitable for retroviral gene transduction and it allows for stable selection of recipient cells in which expression of the gene of interest is under the control of doxocycline of a modified e. coli tet repressor coded by a second mlv-based vector (pretrox-tet-on, rtta-advanced, clontech). murine isg (gene id: ) was obtained following the same cloning scheme. the remaining vectors were obtained as follows: prrl-cmv-destabilized gfp (hiv- -based lentiviral vector genome bearing an expression cassette for destabilized gfp, described in [ ] and clontech); flag-trex (in pcdna , this study), hiv- gag (in [ ] , respectively), ha-ubiquitin (gift of pierre jalinot, ens-lyon), vsv-tagged samhd (in prrl-cmv, this study); ha-destabilized tnrc a was engineered by standard molecular biology techniques from the double-tag pfrt/to/ flag/ha-dest-tnrc a (gift from thomas tuschl, addgene, # , [ ] ). an n-terminal ha tagged ifit (gene id: ) was cloned by standard molecular biology techniques into pcdna . (invitrogen). the following bicistronic vectors were used to examine the role of different ireses and were described in [ ] and [ ]: bi-glo-fluc-cmyc -rluc; bi-glo-fluc-emcv-rluc and bi-glo-fluc-pv-rluc. the following constructs code for a renilla luciferase gene under the control of the beta-globin promoter and contain or lack an intron: pglobin-intron-rluc and pglobin-rluc (described in [ ] , gift from fabrice mure, ciri, lyon, france). the following vectors have been used for in vitro production of rna and they allow upon linearization the t -based synthesis of a polyadenylated mrna that is then ' capped: phcv-rluc, pcrpv-rluc, pcmyc-rluc. the pvsv-rluc was constructed by standard molecular biology techniques by placing the region - upstream of the n gene of vsv (ef ) in front of the renilla luciferase. upon synthesis, rnas are dnase treated, precipitated and used for cell transfection. antibodies directed against the following proteins were as follows: tubulin (t ), actin (a ), flag (f and f ), gfp (g ), ha (h ), vsv-g (c ), tnrc a (hap ) from sigma; msin a (ab ); donkey anti-rabbit igg (alexa fluor conjugate, a- ) and donkey anti-mouse igg (alexa fluor conjugate, a- ) from invitrogen; creb ( ) and akt ( ) from cell technology; ddx (bet - a) from ozyme; isg ( - -ap, in our hands this antibody is highly unreliable on human isg but is more efficient on murine isg ) from proteintech; sc (nb - ) from novus biologicals; anti bmrf (mab , merck millipore); anti bmlf , anti brlf and bzlf were described in [ , ] ; anti-ifit (pa - , life technologies). the anti-gag/p antibody (clone -h c) was obtained from the aids reagents program of the nih. human embryonic kidney t (hek t) and human cervix epithelial hela cells (hela) were maintained in complete dmem media supplemented with % fcs, while human peripheral blood t lymphocyte jurkat (jurkat), human peripheral blood monocytic thp- cells (thp- ) and u were maintained in complete rpmi medium with % fcs. cells were obtained from the atcc (crl- ; ccl- ; tib- ; tib- , crl- . respectively). hek t-ebv [ ] , hone [ ] and raji cells were obtained from the laboratory of oncogenic herpesviruses of henri gruffat (ciri, lyon) and were maintained in complete dmem or rpmi , respectively. ebv reactivation was induced in hone cells upon incubation with -o-tetradecanoylphorbol -acetate (tpa) and butyric acid (ba) provided at ng/ml and mm, respectively (sigma). when indicated the proteasome inhibitor mg was added at a final concentration of μg/ml (sigma). hl cells, derivative of ht cells that bear an integrated luciferase under the control of the interferon alpha-inducible protein promoter ( - :luc) were obtained from gilles uze (university of montpellier, montpellier, france [ ] ) and were maintained in complete dmem. primary human blood monocytes were obtained by negative depletion as described in [ ] (monocyte isolation kit ii, catalogue n˚ - - , miltenyi) prior to their differentiation in macrophages or dendritic cells upon incubation for to days in m-csf or gm-csf and il (at ng/ml, -a - , pcyt- and pcyt- , eurobio). when indicated, cells were incubated for further hours with . u/ml of human ifnα (catalogue n˚ - , tebu bio), or in the case of murine bmdm with . u/ml of murine ifnβ ( -m h, sino biological inc.). primary mouse bone marrow derived macrophages (bmdms) were prepared from isg -/and isg +/+ mice, as described before [ ] . briefly, bone marrow cells were isolated from mice femurs and tibias, washed with pbs and filtered through a μm cell strainer to remove cell clumps. the single-cell suspension was then cultured in rpmi medium containing % fbs and ng/ml of m-csf ( -ml, r&d systems) for days with daily media replenishment. differentiation was monitored by flow cytometry analysis of cd b+f / + cells (bd) and reached proportion higher than %. pools of stable cell lines allowing for doxycycline (dox)-inducible isg expression were generated upon mlv-based retroviral gene transduction based on the pretrox-tight system of clontech. briefly, the corresponding mlv vectors were produced by transient dna transfection of hek t cells with plasmids coding the structural mlv protein gag-prol-pol, the pantropic vesicular stomatitis virus g protein (vsvg), pretrox-tight-puro and pretrox--tet-on vectors that code for a miniviral genome bearing expression cassettes for a resistance gene (puromycin and neomycin, respectively) and either isg , or the teto transcriptional rtta activator (ratios : : : , respectively, for a total of μg of transfected dna for a -cm dish). virion particles released in the supernatant of transfected cells were filtered, titered by exogenous rt activity [ ] and used to challenge the indicated target cells. pools of transduced cells were obtained upon selection with puromycin and g (p and g -ro, sigma). isg expression was induced by incubation with doxycycline ( , clontech). for mrna transfection, hek t cells were electroporated in quadruplicates with ng of in vitro transcribed capped and polya mrna in a total volume of μl (settings of v, ms, pulses, neon electroporator, invitrogen). cells were then transferred into μl pure rpmi medium and cultured for hour prior to cell lysis and luciferase analysis (see below). mrnas were generated by an in-house in vitro transcription reaction and then capped. briefly, μg of linearized dna was incubated for hours at ˚c with u of t rna polymerase, u of rnasin (both from promega) and . mm for each ribonucleotide triphosphate, mm dtt, mm tris-hcl (ph . ), mm mgcl , mm spermidine, and mm nacl. to induce mrna cap addition, the rgtp concentration was reduced to . mm, and . mm of m gpppg cap analog (new england biolabs) was added. rnas were then precipitated and finally resuspended in rnase-free water. dna transfections were carried out generally by calcium/phosphate transfection, or lipofectamine for confocal microscopy (invitrogen). generally, for transient dna transfections a ratio of : between reporters and isg was used (from : to : in the dose curve presented in s fig) not surpassing an overall amount of μg of isg per well of well plate to avoid cytotoxic effects. cells were analyzed in triplicates and lysed between eighteen and twenty-four hours after dna, or hour after rna transfection. one fifth of the cell lysates was used for firefly or renilla luciferase assays (luciferase assay system or renilla luciferase assay system, promega; mithras lb multimode microplate reader, berthold), while the remaining fractions were used for rna extraction and quantitative real-time pcr assay. the luciferase activities are normalized for the amount of rna present in each sample. cells were starved in ml/well of met-/cys-free dmem medium ( , gibco) for hour and then medium was replaced with μl of the same medium containing μci s met/cys (amersham) for a pulse of hour. labeled cells were then lysed thoroughly with ripa buffer ( mm nacl, % np , . % deoxycholate, . % sds, mm triscl ph . ) in the presence of protease inhibitors (roche), by vigorous pipetting and cycles of freezethawing. lysates were centrifuged at . g for minutes to remove insoluble material and then either directly analyzed by sds-page gels or incubated with μl anti-m -flag beads for hours to overnight at ˚c. after extensive washing, material was separated by sds-page gel and upon gel fixation in methanol and drying, radioactivity was quantified by phosphor imager (typhoon system, ge healthcare). stable or transfected cells were directly grown on coverslips for at least twenty-four hours prior to fixation in cold methanol for min at - ˚c (hela or hek t cells, respectively). when indicated, a prior permeabilization step was carried out to evidence the strong association of isg to nuclear bodies. in this case, cells were transfected in duplicate and while one aliquot was immediately fixed, the other was first washed in cold wash buffer (pbs, . % tween ) and further permeabilized in cold psb containing . % triton-x for min on ice. cells were then fixed and incubated with the indicated antibodies (see relevant section above). dapi fluoromount g mounting medium was used ( , southern biotech). images were acquired using a spectral zeiss lsm or lsm confocal microscopes and analyzed using the fiji software. for p bodies analyses, cells were fixed first in % paraformaldehyde, then in % methanol ( min each), followed by permeabilization in cold psb containing . % triton-x for min on ice. cells either stably or transiently expressing isg were lysed in pre-cooled buffer sd- ( mm nacl, mm tris ph . , . % triton x , . mm mncl and × roche protease inhibitor cocktail), sonicated and the soluble supernatants were incubated with pre-washed anti-flag antibody-conjugated agarose beads (a , sigma, μl per -cm plate dish) for hours at ˚c with constant nutation. beads were then extensively washed in the same buffer and either analyzed by wb or incubated with . - μg of exogenously added nucleic acids for minutes at ˚c in a total volume of μl of sd- buffer, prior to agarose gel migration and densitometric quantification by multi gauge v . software (fujifilm). sequences of the complementary dna/rna oligonucleotides are as follows: dna oligonucleotides d and d were respectively: acatgtacaggatgcatttg and tacaggatgcatttg. rna oligonucleotides r u, r d and r d were respectively: caaaugcauccuguacau gu, acauguacaggaugcauuug, uacaggaugcauuug. hybrids were obtained by mixing the desired oligonucleotides at a concentration of μg in sd- buffer, prior to denaturation at ˚c and annealing at ˚c for minutes each. poly i:c and yeast trnas were purchased from invivogen and sigma, respectively. ssrna was a luciferase based mrna generated by in vitro transcription of approximatively nucleotides. ssdna and dsdna were respectively a long chemically-synthesized oligonucleotide available in the laboratory for other purposes (eurogentec, cgcggaa ttcaccatggattacaaggatgacgacga taagggtggtggttcagccccactggatgccgccctccac) and double-stranded pcdna plasmid (invitrogen) linearized with psti. the levels of type i ifns secreted in the supernatant of cells expressing isg was determined according to a well established procedure using hl reporter cells [ ] . briefly, isg -stable u cells were stimulated for hours with doxycyclin to induce isg expression, then supernatant was harvested, filtered and added to hl cells in serial dilution ( . cells in -well plate). twenty hours afterwards, cells were lysed and the extent of luciferase accumulation was measured (see above, luciferase assays section). hl cells stimulated with serial dilutions of ifnα were used in parallel to ensure the linearity of the assay. the limit of detection of this assay is in our hands of u/ml. rnas were extracted by the trizol reagent ( , ambion), or by nucleospin rna ( , macherey-nagel) according to the manufacturers' instructions. when indicated, cytoplasmic and nuclear rnas were separated and purified, as previously described (ricci ep, pmid: ) . the extracted rna was treated with rq dnase to remove contaminant dna (m , promega) and then reverse-transcribed by qscript cdna supermix using the manufacturer's instructions and either oligo-dt or random primers (quanta biosciences; the enzyme was instead omitted in the no-rt controls). when indicated, the amount of transfected dna was quantified after the addition of rq -dnase to the cell culture media to remove extracellular dna (twice for two hours at ˚c at u/ ml). after extensive cell washing, cells were lysed with the following solution ( mm kcl, mm tris hcl ph . , . mm mgcl , . % np- , . % tween ) in the presence of μg/ml of proteinase k (p , sigma) for one hour at ˚c. dna was then purified through phenol/chloroform extraction and dna precipitation. qpcrs were performed on a stepone plus real-time pcr system (applied biosystems) using the faststart universal sybr green master mix (roche). primers were as follows (forward-f and reverse-r from ' cytoplasmic and nuclear fractions were obtained from transfected hek t cells. briefly, . - . cells were collected and resuspended in . ml of cold rnla buffer ( mm of tris hcl ph . , mm nacl, mm mgcl , . % np- , mm dtt and u/ml of rnasin plus-promega) for minutes on ice, then centrifugated at . g for minutes at ˚c. the supernatant (cytoplasmic fraction) was transferred to a new tube, while the pellet (nuclear fraction) was washed twice with . ml of cold rnla buffer to eliminate residual cytoplasm and discarded. the pellet was then resuspended with . ml of cold rnla buffer, sonicated on ice for seconds. an aliquot of each fraction was stored for wb analysis, while the rest was used for rna extraction (trizol, thermo fisher scientific). results are presented on normalized cytoplasmic and nuclear fractions. ribosomal fractions were purified as described [ ] . briefly, cells at less than % confluency were washed in cold pbs, gently pelleted and lysed for minutes in hypothonic buffer ( mm kcl, . mm mgcl , mm tris-hcl ph . ). nuclei were pelleted by centrifugation at g for minutes and the supernatant was then transferred centrifuged again to remove mitochondria ( . g for minutes). supernatant was then harvested, adjusted to . m of kcl and placed onto a sucrose cushion ( . % w/v) prior to ultracentrifugation at rpm on a tl ultracentrifuge (for two hours at ˚c). after ultracentrifugation, the supernatant constitutes the cytoplasmic fraction remaining after migration of the ribosomes at the bottom of the tube. the replication-competent vsv-gfp (indiana serotype, [ ] ) contains an additional viral transcriptional unit coding gfp between m and g and was kindly provided by joanna brunel and denis gerlier (ciri, ens-lyon). infections of isg expressing cells were carried out generally mois comprised between . and . , twenty-four hours after dox-induction of isg . the percentage of gfp-positive cells was determined at different time points postinfection by fluorescence-activated cell sorting (facs). isg -knockout mice were generated by clustered regularly interspaced short palindromic repeats (crispr)-associated systems (crispr/cas ). briefly, a single isg sgrna was designed to target the mouse isg orf ( '-gatcactaatacgactcactataggtggg cctcaaagggtgaggttttagagctagaaat- '). in vitro transcription t kit ( , takara) was used for transcription of both sgrna and cas , the rna products were purified and injected into c bl/ mice zygotes [ ] . screening of knockout mice was carried out by pcr on genomic dna from f and f mice followed by pcr products cloning and sequencing (ta cloning kit, takara: f: tttctgagggtcgccaa and r, tgtacttgtcataca ggact) for in vivo vsv replication studies, age-and sex-matched groups of littermate mice were intraperitoneally infected with plaque-forming units (pfu) of wt-vsv (indiana serotype, but in this case lacking gfp, [ ] ). mice were then either sacrificed twelve hours after infection for tissue analysis and in the case of lung sections for staining with hematoxylin and eosin that marks preferentially inflammatory cells or kept for prolonged periods of time for survival studies. pems were harvested by peritoneal washes from mice treated days before via an intraperitoneal injection of ml of % sterile thioglycollate (sigma t ). the cell suspension was filtered through a -μm cell strainer (falcon ) to remove cell clumps and plated at . million per well in -well plate in complete medium to force cell adhesion to plastics and then cells were extensively washed to remove non-adherent cells. vsv infections were performed the next day at an moi of . prior to flow cytometry analysis twenty-four hours later. primary blood cells were obtained from the blood of healthy donors (efs-lyon) as discarded "leukopacks". these were obtained anonymously so that gender, race, age of donors, inclusion of women, minorities or children cannot be determined. this research is exempt from approval and was therefore not submitted to institutional review board approval, although written informed consent was obtained from blood donors to allow use of their cells for research purposes. all animal experiments described in this study were performed according to the regulations of the association for assessment and accreditation of laboratory animal care in shanghai and the animal investigation committee of east china normal university (document approval n˚m ). c bl/ wild-type mice were purchased from the shanghai laboratory animal company. mice were housed in a temperature and humidity-controlled room ( ˚c +/- ˚c and % +/- %, respectively) with a -hour light/ -hour dark cycle. throughout the protocol, animals were weighed and observed twice daily for clinical signs of infection. animals that reached the end of the experiment or lost more than % of initial body weight were sacrificed. supporting information s fig. expression levels and cytotoxicity of isg . a) the levels of isg mrnas were quantified by rt-qpcr in the different cell types indicated. monocyte-derived macrophages and dendritic cells (mdm and dcs, respectively) were generated upon incubation of primary blood monocytes with m-csf or gm-csf and il , respectively for to days as described in [ ] . when indicated, cells were incubated for twenty-four hours with . u/ml of interferon α, prior to cell lysis. the expression levels of isg obtained in dox-inducible stable cell lines generated upon retroviral mediated gene transduction are also shown (dox concentrations of , and μg/ml). b) the cytotoxicity of isg was evaluated in stably transduced cell lines in which isg induction is dox-sensitive. cells were counted daily upon induction of isg with dox concentrations of , and μg/ml. the graph presents data obtained from three to four independent experiments, while the wb panels present typical results obtained. fig a. � , p� . according to a student t test comparing control and isg conditions. (tif) s fig. exonuclease activities and specificities of isg in vitro and in cells. a and b) hek t cells transiently transfected in cm plates with isg coding dna were lysed and isg immunoprecipitated via anti-flag antibodies conjugated to agarose beads. after washing, beads bound material was incubated with the indicated nucleic acids ( μg), prior to loading on agarose gels and densitometry quantification. nucleic acids were as follows: ssrna, in vitro transcribed single-stranded rna, length of approximatively nucleotides; ssdna, single-stranded dna, oligonucleotide of nucleotides; dsdna, double stranded pcdna plasmid (invitrogen) linearized with psti; poly i:c and yeast trnas, self-explicatory. in b, the indicated rna and dna forms were generated as described in the methods section. c) hek t cells transiently transfected as above were directly lysed and the amount of small nuclear rnas (u , u , u and u ) or of total rna was evaluated by rt-qpcr and agarose migration, respectively. d) as in c but cells were also transfected with ng of in vitro transcribed mrna containing the ' utr of the hepatitis c virus (hcv) and bearing the firefly luciferase. upon cell lysis the amount of transfected hcv-luc mrna was determined by rt-qpcr. a schematic representation of the rna target and the position of the pcr amplicon is provided. the graphs present means and sem of independent experiments (n = to depending on target for b; n = for c; n = for d). the panels present typical results obtained. � , p� . , following a student t test. hek t cells transiently transfected with isg and gfp coding plasmids (as in fig b) were analyzed by wb twenty-four hours afterwards. to better appreciate the linearity and magnitude of the defect in gfp accumulation by wb, the control sample was serially diluted. c) as above, but cells were analyzed by flow cytometry to appreciate the decrease in the percentage of gfp-positive cells (displayed in the panels), as well as their mfi. d) hek t cells were transiently transfected with a fixed dose of gfp reporter and increasing concentrations of isg followed by cell analysis by wb (from : to : ). e) hek t cells were transfected with dnas coding for indicated proteins using either calcium phosphate or lipofectaminebased dna transfection, prior to wb analysis. reporter. the amount of gfp reporter was then determined by densitometry after wb. c) the different isg mutants were immunoprecipitated from transfected hek t cells and incubated with a single-stranded rna oligo (r ). upon migration on an agarose gel, the amount of intact rnas were measured by densitometry. d) correlative analysis between the rnase and antiviral properties of individual isg mutants. the graph present means and sem of four individual experiments (two for panel b). � , p� . following a student t test. e) spatial positioning of the indicated mutations on the crystal structure of isg . (tif) s fig. isg does not affect the production of infectious virion particles of ebv. hone cells containing a latent ebv genome bearing gfp were transfected with isg and ebv reactivated from latency upon tpa/ba treatment as in the legend to fig d. to determine whether isg could affect the translation of the plethora of virion products required for the production of infectious virion particles, supernatants were syringe-filtered and virion infectivity was measured after challenge of target raji cells and flow cytometry analysis three days later. (tif) s fig. isg does not influence nucleocytoplasmic rna transport and is not detectably associated to ribosomes. a) hek t cells were cotransfected with isg along with two constructs coding for a firefly luciferase reporter expressed from a globin promoter and containing or not an intron, prior to cell lysis and luciferase measurement. b) hek t cells were transiently transfected with isg and gfp-coding plasmids and subsequently lysed to obtain nuclear and cytoplasmic fractions. upon normalization of the two fractions by volume, samples were analyzed by wb and rt-qpcr (gfp). the distribution of two small nuclear rnas known to be enriched in the nucleus (u and u by rt-qpcr) was also included as control for fractionation. c) hek t cells transfected with isg were lysed, the ribosomal fraction purified, followed by wb analysis. the graphs present data obtained from two (luc) and five (pcrs) independent experiments and panels present typical results. � , p� . , following a student t test. (tif) s fig. the catalytically inactive m -isg mutant is devoid of translation inhibition activity. hek t cells were transfected with the indicated reporters along with wt or m -isg mutant prior to cell lysis and luciferase assay measurement. the graph presents data obtained from two and four independent experiments. glo, globin; pv, polyomavirus; myc, c-myc. the nomenclature st and nd refers to the position of the cistron in bicistronic vectors. (tif) s fig. isg and ifit inhibit vsv infection. hek t cells transfected with plasmids coding the above-mentioned proteins were challenged twenty-four hours afterwards with vsv-gfp at an moi of . prior to flow cytometry analyses twenty four hours later. the graph presents results obtained with three independent experiments. � , p� . , following a student t test between control cells and the indicated condition. (tif) s fig. isg displays a weak association to nuclear bodies and does not modify the extent of p bodies formation in the cell. a) hek t cells were transiently transfected in duplicate with plasmids coding isg (routine transfection rates � %). twenty-four hours afterwards, one aliquot was immediately fixed while the second was first permeabilized with detergent prior to fixation. both were then similarly processed and analyzed by confocal microscopy using antibodies specific for isg (flag), as well as for the nuclear speckles marker sc . this procedure is commonly used to study nuclear bodies and associated proteins that resist detergent extraction prior to fixation. representative pictures and relative distributions are show here (> cells scored per condition). b) control cells were analyzed by confocal microscopy with the endogenous p bodies markers tnrc a and ddx (isg -expressing cells and a portion of the overlay of control cells depicted here are presented in fig a) . molecular cloning of a new interferon-induced pml nuclear body-associated protein the exonuclease isg mainly localizes in the nucleolus and the cajal (coiled) bodies and is associated with nuclear smn protein-containing complexes isg , a new interferoninduced rnase specific for single-stranded rna, defines an alternative antiviral pathway against rna genomic viruses exoribonuclease superfamilies: structural analysis and phylogenetic distribution crystal structure of human isg , an interferon-induced antiviral ribonuclease the human interferon-and estrogen-regulated isg / hem gene product degrades single-stranded rna and dna in vitro identification and characterization of interferon-induced proteins that inhibit alphavirus replication identification of three interferon-inducible cellular enzymes that inhibit the replication of hepatitis c virus identification of five interferon-induced cellular proteins that inhibit west nile virus and dengue virus infections antiviral activities of isg in positivestrand rna virus infections the interferon-induced exonuclease isg exerts antiviral activity through upregulation of type i interferon response proteins. msphere interferon-induced exonuclease isg exhibits an antiviral activity against human immunodeficiency virus type interferon-stimulated gene of kda protein (isg ) degrades rna of hepatitis b virus to impede the replication of hbv in vitro and in vivo influenza a virus-induced expression of isg inhibits viral replication by interacting with nucleoprotein interferon-inducible ribonuclease isg inhibits hepatitis b virus replication through directly binding to the epsilon stem-loop structure of viral rna a robust cell culture system supporting the complete life cycle of hepatitis b virus interferon-stimulated gene (isg)-expression screening reveals the specific antibunyaviral activity of isg cell-type-specific growth restriction of vesicular stomatitis virus polr mutants is linked to defective viral polymerase function the epstein-barr virus nuclear antigen- reprograms transcription by mimicry of high mobility group a proteins altered histone h stoichiometry and an absence of nucleosome positioning on transfected dna p-bodies react to stress and nonsense movement of eukaryotic mrnas between polysomes and cytoplasmic processing bodies processing bodies require rna for assembly and contain nontranslating mrnas quantifying mrna targeting to p-bodies in living human cells reveals their dual role in mrna decay and storage p-bodies and stress granules: possible roles in the control of translation and mrna degradation p-bodies: composition, properties, and functions p-body purification reveals the condensation of repressed mrna regulons p-body formation is a consequence, not the cause, of rna-mediated gene silencing adenosine methylation as a molecular imprint defining the fate of rna chemical modifications in the life of an mrna transcript micrococcal nuclease digestion of nuclei reveals extended nucleosome ladders having anomalous dna lengths for chromatin assembled on non-replicating plasmids in transfected cells chromatin profiling of epstein-barr virus latency control region phosphorylation of initiation factor elf- and the control of reticulocyte protein synthesis regulation of protein synthesis: activation by double-stranded rna of a protein kinase that phosphorylates eukaryotic initiation factor apobec a is a specific inhibitor of the early phases of hiv- infection in myeloid cells inactivation of the human immunodeficiency virus type inhibitory elements allows rev-independent expression of gag and gag/protease and particle formation molecular characterization of human argonaute-containing ribonucleoprotein complexes and their bound target mrnas mirna repression of translation in vitro takes place during s ribosomal scanning protein kinase ck phosphorylation of eb regulates its function in the production of epstein-barr virus infectious viral particles the enhancer factor r of epstein-barr virus (ebv) is a sequence-specific dna binding protein propagation and recovery of intact, infectious epstein-barr virus from prokaryotic to human cells inquiring into the differential action of interferons (ifns): an ifn-alpha mutant with enhanced affinity to ifnar is functionally similar to ifn-beta extracellular udp and p y function as a danger signal to protect mice from vesicular stomatitis virus infection through an increase in ifn-beta production purification of ribosomes from human cell lines we thank serge bouaziz (university paris descartes, paris), rosa bernardi (division of experimental oncology, irccs san raffaele scientific institute) and emiliano ricci (ens-lyon) for counseling on structure analyses, murine cells and ribosome purification, respectively. we are indebted to denis gerlier, joanna brunel and to fabrice mure (ciri, france, lyon) for providing the vsv-gfp virus and intron/less globin luciferase reporters, respectively. we also acknowledge ju-tao guo from the baruch s. blumberg institute, doylestown, pennsylvania, usa for sharing material. key: cord- -s ddek f authors: kim, ye ji; kim, eui tae; kim, young-eui; lee, myoung kyu; kwon, ki mun; kim, keun il; stamminger, thomas; ahn, jin-hyun title: consecutive inhibition of isg expression and isgylation by cytomegalovirus regulators date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: s ddek f interferon-stimulated gene (isg ) encodes an ubiquitin-like protein that covalently conjugates protein. protein modification by isg (isgylation) is known to inhibit the replication of many viruses. however, studies on the viral targets and viral strategies to regulate isgylation-mediated antiviral responses are limited. in this study, we show that human cytomegalovirus (hcmv) replication is inhibited by isgylation, but the virus has evolved multiple countermeasures. hcmv-induced isg expression was mitigated by ie , a viral inhibitor of interferon signaling, however, isgylation was still strongly upregulated during virus infection. rna interference of ube l (e ), ubch (e ), herc (e ), and ubp (isg protease) revealed that isgylation inhibits hcmv growth by downregulating viral gene expression and virion release in a manner that is more prominent at low multiplicity of infection. a viral regulator pul was found to interact with isg , ube l, and herc , and be isgylated. isgylation of pul regulated its stability and inhibited its activities to suppress nf-κb signaling and complement the growth of ul -null mutant virus. moreover, pul reciprocally suppressed virus-induced isgylation independent of its own isgylation. consistently, isgylation was more pronounced in infections with the ul -deleted mutant virus, whose growth was more sensitive to ifnβ treatment than that of the wild-type virus. therefore, pul is a viral isg target that also counteracts isgylation. our results demonstrate that isgylation inhibits hcmv growth at multiple steps and that hcmv has evolved countermeasures to suppress isg transcription and protein isgylation, highlighting the importance of the interplay between virus and isgylation in productive viral infection. type i interferons (ifns) are multifunctional cytokines that represent crucial components of the innate immune response to viral infection. recognition of viral infection by host cells induces the synthesis of type i ifns. secreted ifns interact with ifn receptors on target cells, triggering a signaling cascade that involves janus kinase (jak) and signal transducer and activator of transcription (stat) families. activated stat and stat heterodimerize and bind to ifn regulatory factor (irf ) to form a complex called ifn-stimulated gene factor (isgf ). this complex translocates into the nucleus and induces isgs with diverse antiviral activities by binding to ifnstimulated response elements (isres) in their promoters (for review [ ] ). isg was identified as an ifn-inducible ubiquitin homolog. like ubiquitin, its carboxy-terminal lrlrgg motif is required both for recognition by processing enzymes and covalent conjugation to lysine residues of target proteins. isg modification (also termed isgylation) is an ifnstimulated and -regulated process that is found only in higher vertebrate animals and appears to modulate the function of target proteins (for review [ ] ). ube l is the e activating enzyme for isg [ ] , and ubch , an ubiquitin e conjugating enzyme, also acts as the isg e conjugating enzyme [ , ] . herc domain and rcc -like domain containing protein (herc ), estrogenresponsive finger protein (efp), and human homolog of drosophila ariadne (hhari) have been identified as e ligases for isgylation in human cells [ ] [ ] [ ] [ ] . isg , ube l, ubch , and herc are ifn-inducible [ , , , ] . conjugated isg s are removed by an isg -specific protease, ubp (also known as usp ) [ ] . interestingly, ubp is also ifn-inducible [ , ] and acts as a negative regulator of innate immune responses independent of its protease activity but dependent on its direct interaction with ifnar , a subunit of the type i ifn receptor [ ] . antiviral responses involving protein isgylation have been reported against diverse viruses. several cellular proteins involved in antiviral signaling, including rig-i, mda- , stat , jak , irf , pkr, mx , and rnase l, were also identified or suggested as substrates for isgylation [ ] [ ] [ ] [ ] [ ] [ ] . isgylation suppressed replication of diverse viruses, such as influenza virus (type a and b) [ ] [ ] [ ] [ ] , human immune deficiency virus (hiv) [ , ] , hepatitis c virus (hcv) [ ] [ ] [ ] , japanese encephalitis virus [ ] , sindbis virus [ , , ] , ebola vp virus-like particle [ , ] , herpes simplex virus type- [ ] , murine γ-herpesvirus [ ] , vaccinia virus [ ] , dengue and west nile viruses [ ] , porcine reproductive and respiratory syndrome virus [ ] , kaposi's sarcoma-associated herpesvirus (kshv) [ ] , and respiratory syncytial virus [ ] . however, the antiviral mechanism of isgylation against specific viruses is poorly understood. herc associates with polyribosomes, and isgylation appears to be restricted largely to newly synthesized proteins, suggesting that newly synthesized viral proteins may be primary targets of isg [ ] . isgylation of ns a in influenza a virus disrupted its association with importin-α, which mediates the nuclear import of ns a, thus inhibiting viral replication [ ] . isgylation also suppressed the release of retrovirus particles by disrupting the budding process-related protein complex (for review [ ] ). an isgylation-independent antiviral effect of isg was also demonstrated in chikungunya virus infection [ ] . although several studies have suggested a general role for isg as an antiviral molecule, proviral effects of isgylation have also been reported for certain viruses. in newcastle disease virus (ndv) infection, isgylation of rig-i reduced ifn responses as a negative feedback regulation mechanism [ ] , whereas isgylation of irf stabilized irf [ ] . in addition, enhanced isgylation by isg overexpression promoted hcv production, while reduced isgylation by ube lor isg -knockdown inhibited hcv production [ , ] . therefore, the effects of global protein isgylation appear to vary among the different viruses. in addition, isg is secreted to the extracellular space as a free unconjugated form [ , ] . while secretion of isg in granulocytes is shown to activate t cells and natural killer cells to produce ifnγ in mycobacterial infection [ , ] , the role of secreted isg in viral infection is not clear. furthermore, a role of free isg as a negative regulator that prevents ifnα/β overamplification and auto-inflammation by sustaining ubp levels has been suggested in humans but not in mice [ , ] . human cytomegalovirus (hcmv) is an opportunistic pathogen that causes severe disease complications and pathologies in newborns and immunocompromised individuals [ ] . during productive infection, hcmv gene expression occurs sequentially in three phases: immediateearly (ie), early, and late. ie proteins and virion-associated tegument proteins play key roles in initiating viral gene expression and modulating host cell functions. hcmv employs several mechanisms to counteract ifn production and subsequent isg activation. ie and pp inhibit ifn production [ ] [ ] [ ] , whereas ie suppresses the ifn response by directly binding to stat [ ] [ ] [ ] and pml [ , ] . hcmv infection results in a decrease in the levels of jak and p , two components of the type i ifn signaling pathway [ , ] . modulation of the stability and phosphorylation of stat proteins during hcmv infection was also reported [ , ] . isg transcription is induced in hcmv infection; however, its regulation during infection, the role of isgylation in viral growth, and viral targets of isg have not been characterized. in this study, we show that isg expression and isgylation are initially induced after hcmv infection but later suppressed by viral responses, and that ie , a viral inhibitor of stat signaling, plays an important role in reducing isg transcription. by silencing the expression of e , e , and e isgylation enzymes and of an isg protease, we also demonstrate that isgylation inhibits hcmv growth at multiple steps, including viral gene expression and virion release. furthermore, we show that pul , a viral tegument protein, interacts with isg , e , and e and is modified by isg , which inhibits pul activity to promote viral growth. moreover, we reveal that expression of pul is able to suppress isgylation induced by virus infection. our results indicate that hcmv has evolved countermeasures to suppress isg transcription and protein isgylation, highlighting the important of the interplay between virus and isg signaling during virus infection. the time course of isg expression and protein isgylation during hcmv infection were investigated with different multiplicity of infections (mois). in human fibroblast (hf) cells infected with hcmv (towne), the levels of isg and protein isgylation were elevated by h at all mois tested (mois of . to ) (fig a, lanes - ) . at and h after infection, even greater levels of isg expression and protein isgylation were observed at relatively low mois h. cells were fixed with methanol and double-label ifa was performed with antibodies for isg and ul - . hoechst stain was used to stain cell nuclei. the images were obtained by confocal microscopy. ( . , . , and ) (fig a, lanes - and - ) ; however, the levels of free isg and isg conjugates at high mois ( and ) were much lower than those at low mois ( fig a, lanes - and - ) . the time course of isg expression and protein isgylation was also examined in cells infected with uv-inactivated virus (uv-hcmv). in uv-hcmv infection, the levels of isg and isg conjugates were elevated at and h and correlated proportionally with moi ( fig a, lanes - and - ) . levels of free isg at h induced by uv-hcmv were lower than those at h, probably due to the termination of signaling ( fig a, compare lanes - and - ) . the lack of viral gene expression in uv-hcmv infection was verified by the absence of viral ie protein expression. collectively, these results comparing hcmv and uv-hcmv infection demonstrate that isg expression and protein isgylation are initially induced by hcmv infection, but are subsequently suppressed in a manner dependent on viral gene expression. notably, we observed a greater induction of isg and protein isgylation with hcmv than with uv-hcmv at low mois (mois of . , . , and ) (fig a, compare lanes - and - ). we hypothesized that this was due to the induction of isg at different level in uninfected cells that surround infected cells at low mois. to test this hypothesis and to examine the effect of hcmv infection on isg expression at a single cell level, hf cells were infected with hcmv or uv-hcmv at a low moi ( . ) and stained for isg and viral ul - proteins. we found that isg expression was reduced in hcmv-infected cells, in which ul - viral replication proteins were expressed at high level; however, it was markedly increased in neighboring uninfected cells compared to mock-infected cells. we also found that the levels of isg in uninfected neighboring cells were higher in hcmv infection than in uv-hcmv infection ( fig b) . this result suggests that an indirect effect of virus infection on neighboring uninfected cells is responsible for the greater induction of isg and protein isgylation by hcmv than by uv-hcmv at low mois. since hcmv ie inhibits the activation of isre-containing promoters by sequestering stat [ ] [ ] [ ] and pml [ ] , ie expression may be responsible for the suppression of free isg and isg conjugate levels during hcmv infection. to test this hypothesis, we first compared the effects of wild-type hcmv, uv-hcmv, and ie -deleted mutant virus (cr ) infection (moi of ) on isg transcription by rt-pcr. the cr virus exhibited an moi-dependent growth pattern with showing a severe growth defect in hf cells at low mois but normal growth at high mois [ ] . all viruses increased isg mrna levels h after infection; however, isg induction was terminated earlier for hcmv than uv-hcmv, and not appreciably terminated for cr , which continued to produce high levels of isg transcripts even at a late stage of infection ( h) (fig a) . isg transcription induced by uv-hcmv infection might be gradually decreased due to the termination of the ifn signaling through several negative regulatory mechanisms. however, when cells were infected with cr at this high moi, the replication of virus without ie appeared to lead to a robust activation of ifn signaling. this result indicates that ie indeed plays an important role in reducing isg transcription during hcmv infection. we also compared the levels of isg and isg conjugates in hcmv, uv-hcmv, and cr infection. when cells were infected with viruses at an moi of , free isg levels were elevated at h by all viruses, whereas the level of isg conjugates markedly increased after h (fig b) . the delayed induction of isgylation is similar to what was observed in ifnβtreated cells [ ] and may result from the delayed induction of the isgylation machinery. consistent with the results shown in fig a, uv-hcmv induced more isg conjugates than wild-type virus at this moi ( fig b, compare lanes - and - ). importantly, cr also resulted in greater isgylation than wild-type virus (fig c. compare lanes - and - ), indicating that ie is required for the suppression of isg expression and isgylation during hcmv infection. immunoblot analysis demonstrated that ie and ie failed to be expressed in uv-hcmv infection and that ie failed to be expressed and ie levels were reduced in cr infection under these experimental conditions (fig b and c ). although our results show that ie is largely responsible for the suppression of isg transcription, it is notable that cr infection resulted in slightly lower levels of isgylation compared to uv-hcmv infection (fig b, compare lanes and ) . considering that isg transcript levels remained high up to h in cr -infected cells (fig a) , this finding suggests that other viral processes, besides those mediated by ie , may also be implicated in the downregulation of protein isgylation. . cell lysates were also prepared and analyzed by immunoblotting as in fig a (b) . (c) control and ie -expressing hf cells produced by retroviral vectors were mock-infected or infected with wild-type hcmv, uv-hcmv, or cr virus at an moi of , or treated with ifnβ ( , u/ml) for h. immunoblotting was performed with antibodies for isg , ie /ie , p (encoded by ul ) and β-actin. (d) t cells were co-transfected with plasmids expressing myc-isg (wild-type, isg gg , or isg aa ), ha-ube l (e ), flag-ubch (e ), or ha-herc (e ) as indicated. at h after transfection, cell lysates were prepared by boiling the cell pellets in sodium dodecyl sulfate (sds) loading buffer and immunoblotted with anti-myc and anti-β-actin (a loading control) antibodies. (e and f) co-transfection/isgylation assays were performed in t cells with or without increasing amounts of plasmid expressing ie (e) or in control and ie -expressing hf cells (f). immunoblotting was performed with anti-myc, anti-ie , and anti-β-actin antibodies. the inhibition of isg expression by ie was further investigated using ie -overexpressing hf cells generated using retroviral vectors. control and ie -overexpressing hf cells were infected with hcmv, uv-hcmv, or cr . the results of immunoblot analysis showed that ie overexpression suppressed the induction of isg and isg conjugates by both virus infection and ifnβ treatment (as a control) (fig c, compare lanes - and - ), further supporting the critical role of ie in reducing isg expression. although reduction of protein isgylation during hcmv infection may be largely attributed to suppression of isg transcription by ie , it cannot be ruled out that ie also affects the isgylation reaction. therefore, we studied whether ie directly affects isg conjugation reactions using co-transfection/isgylation assays. to set up co-transfection/isgylation assays, two different isg forms, an active form with a stop codon immediately after the c-terminal double glycine residues (isg gg ) and an inactive form with the double glycine residues substituted with alanine residues (isg aa ), were employed. in cells transiently co-transfected with isg , ube l (e ), ubch (e ), and herc (e ), intact isg and isg gg (an active form), but not isg aa (an inactive form), were conjugated to proteins (fig d) . when co-transfection/isgylation assays were performed with or without ie overexpression, ie expression by co-transfection or retroviral transduction did not inhibit levels of isg conjugates, indicating that ie does not inhibit the enzymatic cascade of reactions required for protein isgylation (fig e and f ). the role of protein isgylation in hcmv infection was investigated by silencing expression of isgylation enzymes using rna interference. hf cells expressing control shrna or shrna for ubp , an isg -specific protease, were generated using lentiviral vectors. ifnα treatment of normal and control shrna (shc)-expressing hf cells induced the expression of ubp proteins; however, ubp -specific shrnas (shubp - and shubp - ) efficiently suppressed this induction (fig a) . ubp knockdown enhanced protein isgylation in cells infected with uv-hcmv that stimulates interferon signaling (fig b) , but reduced the production of progeny virions in hcmv-infected cells to nearly % of that in control cells (fig c) , suggesting that enhanced isgylation by ubp knockdown attenuates hcmv growth. however, ubp is also known to negatively regulate ifn signaling by downregulating jak/stat signaling [ , ] . consistently, we found that ubp knockdown enhanced stat phosphorylation in hcmv-infected cells ( fig d) . therefore, it is possible that the reduction of hcmv growth in ubp knockdown cells is also a result of other aspects of the ifn response. the effect of isgylation on hcmv growth was further assessed by depleting herc , a major isg e ligase in human [ , ] . herc knockdown hf cells were generated using lentiviral vectors expressing shrnas. qrt-pcr assays confirmed the efficient reduction of herc transcript levels in cells expressing herc -specific shrnas (shherc - and shherc - ) compared to in cells expressing control shrna (shc) after uv-hcmv infection ( fig e) . we found that herc knockdown markedly reduced protein isgylation as expected (fig f) , but increased virus titers by -to -fold compared to in control cells (fig g) , demonstrating that the reduction of isgylation by herc knockdown facilitates hcmv growth. similar enhancement of hcmv growth was observed in hf cells depleted of ube l (e ) or ubch (e ) (s fig). collectively, our results with ubp , herc , ube l, and ubch knockdown cells demonstrate an inverse relationship between isgylation and hcmv growth, indicating a general inhibitory role of isgylation in hcmv infection. we also investigated whether enhanced isgylation affects hcmv growth by ectopically expressing isg together with isgylation enzymes. hf cells co-transfected with plasmids expressing myc-isg gg or myc-isg aa and isgylation enzymes were infected with hcmv and the production of progeny virions was compared. the results showed that the expression of myc-isg gg and isgylation enzymes induced higher levels of isg conjugates and reduced progeny virus titers to % of control, whereas the expression of isg aa did not significantly affect the levels of isg conjugates or progeny virions (s fig) . these results are also consistent with our results using shrna-expressing cells in which we showed an inverse relationship between levels of isg conjugates and hcmv growth. to investigate the mechanisms by which isgylation inhibits hcmv growth, we first compared the expression profile of viral proteins in control and herc knockdown cells. when cells were infected with an moi of . , levels of major ie (ie and ie ), early (p ), and late (pp ) viral proteins produced at , , , and h were higher in herc knockdown cells than in control cells ( fig a, left panels) . this effect of knocking down herc diminished when cells were infected at higher mois. at an moi of , ie expression was similar between control and herc knockdown cells, although the levels of ie , p , and pp were slightly increased in herc knockdown cells (fig a, center panels) . at an moi of , levels of viral proteins were comparable between control and herc knockdown cells (fig a, right panels) . progeny virion titers measured in the culture supernatant correlated with the levels of expressed viral proteins ( fig b) . these results demonstrate that reduced isgylation by silencing herc promotes viral gene expression, an effect that is more evident at lower mois. we further investigated whether isgylation affects the activation of viral promoters using reporter assays (s fig). hf cells co-transfected with isg , ube l, ubch , and herc exhibited substantially increased protein isgylation, whereas cells co-transfected without herc did not. in hf cells exhibiting enhanced isgylation by co-transfection, the activity of viral mie promoter was repressed to % of that observed in control cells. similarly, ie -mediated activation of viral early [ul - and ul (pol)] and late [ul (pp )] promoters was also suppressed in cells showing enhanced isgylation. in control experiments, ectopic expression of herc alone was not sufficient to increase isgylation and therefore did not affect viral promoter activation. these results demonstrate that enhanced isgylation inhibits the activation of viral promoters. we also assessed whether isgylation inhibits hcmv virion release as is the case in hiv infection [ ] . control and herc knockdown cells were infected with hcmv at an moi of or and the levels of cell-associated and extracellular progeny virions produced were compared. the results showed that although more progeny virions were produced in herc knockdown cells than in control cells (fig b) , the percentages of cell-associated virions to total virions were lower in herc -knockdown cells, . % (moi ) and . % (moi ), than in control cells, . % (moi ) and % (moi ) (fig c) . these results indicate that, as observed in hiv infection, isgylation inhibits hcmv virion release. to identify potential hcmv proteins that interact with the isg pathway, we screened the hcmv orf library [ ] for isg -or ube l-interacting proteins using yeast two-hybrid assays. twenty hcmv proteins were identified as potential isg -interacting proteins, and five viral proteins (encoded from rl , ul , ul a, ul , and ul ) were found to interact with both isg and ube l. most of these isg binding and all of ube l binding in yeast assays were also detected by co-immunoprecipitation (co-ip) assays (s fig and s table) . among them, the ul gene encodes the tegument proteins, p and p , which are produced using two in-frame start codons and are shown to regulate viral gene expression, nf-κb signaling, and virion stability [ ] [ ] [ ] [ ] . since ul interacted with both isg and ube l and its role in viral growth was relatively well reported compared to others, we further investigated the interaction of pul with the isg pathway. in co-ip assays, ul -p interacted with isg aa , demonstrating that isg can non-covalently interact with ul ( fig a) . in similar co-ip assays, ul -p also interacted with ube l and herc , but not ubch (fig b- d ). when co-ip assays were performed using hcmv (towne)-infected cell lysates, immunoprecipitation with an anti-ul antibody co-precipitated unconjugated free isg (fig e) , and in a reciprocal experiment, immunoprecipitation with an anti-isg antibody co-precipitated ul -p (fig f) , indicating that ul -p interacts with isg during virus infection. it has been suggested that newly synthesized viral proteins may be broadly modified by isg during virus infection [ ] . therefore, we also investigated whether ul is covalently conjugated by isg during hcmv infection. cell lysates prepared from hcmv (towne)infected cells were boiled in sds-containing buffer and then immunoprecipitated with an anti-ul antibody. immunoblotting of the sample with anti-isg antibody revealed a band that is consistent with an isg -modified form of ul -p (fig g) . to further investigate these non-covalent and covalent interaction of ul proteins with isg , we generated a recombinant hcmv (toledo) expressing ul proteins tagged at their carboxyl termini with an ha tag (s fig). compared to the wild-type virus, the recombinant virus produced equivalent amounts of viral major ie (ie and ie ) and ul proteins, and progeny virions (fig h and i ). when co-ip assays were performed with lysates from ul -ha virus infected cells, immunoprecipitation of ul proteins with an anti-ha antibody co-precipitated free isg (fig j) . when ha-ul virus-infected cells were subjected to co-ip assays to detect isgylated proteins, bands that are consistent with isg -modified forms of ul proteins were detected (fig k) . since isg can modify ubiquitin, forming isg -ubiquitin mixed chains [ ] and a single lysine reside can be poly-isgylated [ , ] , the smear bands of isgylated ul appear to be ul proteins that contain isg -ubiquitin mixed chains or poly-isg chains. taken together, our data suggest that the ul -encoded proteins non-covalently interact with isg and are also covalently modified by isg . in a control experiment, we found that some viral proteins, which did not interact with isg , ube l, and herc in co-ip assays, could be isgylated in co-transfection/isgylation assays (s fig), supporting the concept that viral proteins may be broadly isgylated during infection due to ifn-upregulated expression of herc (e ) in polyribosomes [ ] . the ul orf from the towne strain contains three lysine residues (k , k , and k ) and k and k are conserved in human cmvs (fig a) . to determine the isgylation sites of ul proteins, we performed co-transfection/isgylation assays using wild-type ul -p or its mutants in which lysine residues were replaced with arginines. the results showed that the k r, k r, and k r mutants were still isgylated; however, the k / r double mutant was not isgylated, indicating that k and k are the major isgylation sites (fig b) . when hf cells expressing wild-type ul -p or k / r mutant were generated by retroviral vectors, expression level of the k / r mutant protein was higher than that of the wild-type protein (fig c) , suggesting that isgylation of ul proteins may regulate protein stability. the sumo fusion proteins were often used to study the function of sumo modification of proteins [ ] [ ] [ ] [ ] [ ] . to investigate the effect of ul isgylation on its activity, we used the ul -p (k / r)-isg aa fusion protein as a surrogate for the isg -modified form of ul -p . we produced the isg fusion to the lysine mutant form of ul to compare the activities of isgylation-defective ul and its isg fusion form. hf cells expressing the k / r mutant or the ul -p (k / r)-isg aa fusion protein were generated by retroviral vectors (fig d) . ul proteins have been shown to inhibit tnfα-induced nf-κb activation [ ] . ul -p (k / r) moderately inhibited tnfα-induced nf-κb activation but ul -p (k / r)-isg aa did not, suggesting that isgylation of ul inhibits its activity to downregulate nf-κb signaling (fig e) . we further investigated the effect of ul isgylation on its role in viral growth. when control and ul -expressing cells were infected with the ul -deleted mutant hcmv (ad ) [ ] and the levels of progeny virus titers were compared, pre-expression of the k / r mutant significantly increased the growth of mutant virus but its isg aa fusion protein did not (fig f) . together, these results indicate that isgylation of ul inhibits its activities to suppress nf-κb signaling and promote the growth of ul -deleted mutant virus. since influenza virus ns b antagonizes isgylation in human cells via direct binding to isg [ , ] , we tested whether ul can regulate protein isgylation. we found that expression of ul -p reduced the levels of isg conjugates in cells co-transfected with ube l (e ), ubch (e ), herc (e ), and isg gg , an active form of isg (fig a) . in a similar assay, isgylation of charged multivesicular body protein (chmp) , a component of the endosomal sorting complex required for transport (escrt) machinery, was inhibited by ul -p expression (fig b) . when control and ul -p -expressing hf cells were infected with uv-hcmv, the levels of isg conjugates were significantly reduced in ul -p -expressing cells compared to control cells (fig c) . this effect of ul was also found with the k / r mutant protein (fig d) , which still interacted with isg aa in co-ip assays (fig e) , suggesting that the inhibitory effect of ul on isgylation is not dependent on its own isgylation. in control experiments, viral proteins such as pul , pul , and ie did not inhibit isgylation of cellular proteins in co-transfection/isgylation assays (s and s figs) . to further investigate the inhibitory effect of ul on isgylation during hcmv infection, we compared isgylation levels between cells infected with wild-type and ul -deleted mutant viruses. with an moi of . , we found that the isgylation levels were initially increased until h but gradually decreased at h and h, and that the ul -deleted virus infection expression levels of myc-isg , srt-chmp , and β-actin in whole cell lysates were also determined by immunoblotting. (c) control and ul -p -expressing hf cells produced by retroviral vectors were infected with uv-hcmv at an moi of . the samples were prepared at the indicated time points and the levels of isg , ul and β-actin were determined by immunoblotting. the relative levels of isgylated ul protein over unmodified protein (normalized with β-actin) are also shown as graphs. (d) control or ul (wild-type or mutant)-expressing hf cells were infected with uv-hcmv and immunoblotting was performed as described in (c). (e) t cells were co-transfected with plasmids expressing srt-ul -p (wild-type or k / r) or myc-isg aa as indicated. at h after transfection, cell lysates were immunoprecipitated with anti-srt antibody. immunoprecipitated samples and whole cell lysates were detected by immunoblotting. (f) hf cells expressing control shrna (shc) or shrna specific for ie (shie ) produced by retroviral transduction were infected with wild-type or Δul mutant virus at an moi of for the indicated time points. cell lysates were immunoblotted with antibodies for isg , viral proteins (ie , ie , and ul ), and β-actin. (g) hf cells were pre-treated or not with ifnβ ( u/ml) for h and then showed only minimally increased levels of isgylation at late times of infection compared to wild-type virus infection (s fig, compare lanes - and - ). we reasoned this minimal effect of ul to the suppression of isg transcription by ie . therefore, to minimize the effect of ie expression we performed a similar experiment in cells expressing shrna for ie (shie ). control shrna (shc) and shie -expressing hf cells, which were produced by retroviral vectors, were infected with wild-type and ul -deleted mutant viruses at an moi of . the results of immunoblotting showed that the expression of shie substantially reduced the ie protein accumulation compared to control cells (fig f, compare lanes - and - ) , and that in shie -expressing cells the isgylation levels were substantially increased at h after infection and gradually decreased at h and h under these experimental conditions (fig f, lanes - ) . notably, we found that the ul -deleted virus less effectively reduced the isgylation levels than wild-type virus at the late phase of infection (fig f, compare lanes - and - ). these results demonstrate an inhibitory effect of ul on isgylation during virus infection. since hcmv growth was suppressed by ifn-induced isgylation and ul inhibited isgylation, we tested whether ul -deleted mutant virus is more susceptible to type i ifn treatment than wild-type virus. we found that while the titers of wild-type virus were reduced by -fold by ifnβ pre-treatment, those of ul -deleted virus were reduced by -fold, indicating that ul -deleted virus less effectively overcomes the ifnβ-mediated anti-viral responses than wild-type virus (fig g) . overall, our results demonstrate that, in addition to ie that suppresses isg transcription, ul plays an important role in evading the isg -associated antiviral responses by inhibiting protein isgylation (fig h) . our analysis with uv-hcmv and ie -deleted mutant virus demonstrates that isgylation is induced by hcmv infection and that ie plays a central role in downregulating isgylation by reducing isg transcription. the latter is consistent with previous findings that ie represses transcription of isgs by sequestrating stat [ ] [ ] [ ] and pml [ , ] . notably, although ie effectively reduced isg expression, the level of isgylation during hcmv infection largely depended on the moi. our data provide evidence for the antiviral roles of isgylation during hcmv infection. to discern the effects of free isg expression and protein isgylation on virus replication, we used hf cells in which a specific isgylation enzyme was depleted by shrna. the data consistently showed an inverse relationship between the level of isg conjugates and hcmv growth; i.e., enhanced isgylation by ubp knockdown decreased viral growth, while reduced isgylation by depletion of ube l, ubch , or herc increased viral growth. therefore, we conclude that protein isgylation in general inhibits hcmv growth. free isg has also been shown to inhibit the replication of certain viruses [ , , ] . in our analysis, however, overexpression of isg gg with ube l, ubch , and herc led to a mild reduction of hcmv growth, whereas isg aa , an inactive form, did not significantly affect viral growth, suggesting that expression of free isg prior to hcmv infection may minimally affect viral growth in cultured cells. it should be noted that given the role of free isg in stabilizing ubp (or usp ), a negative regulator of ifn signaling [ ] , overexpression or depletion of isg might affect both positive and negative activities of isg to viral growth. infected with wild-type or Δul mutant virus at an moi of . the production of progeny virions in the culture supernatant at days after infection was measured by infectious center assays. the results shown are the mean values and standard errors of four independent experiments. (h) summary for the hcmv strategy that consecutively inhibits isg transcription and protein isgylation. ie inhibits isg transcription, while ul inhibits protein isgylation. doi: . /journal.ppat. .g hcmv replication was inhibited by isgylation at multiple steps. first, the expression of viral genes was inhibited under conditions where the level of isg conjugates was increased. viral regulators that promote viral gene expression may be a direct target of isgylation. however, we could not observe isgylation of ie , which is responsible for the activation of mie promoters, or ie , a strong transactivator of viral early and late genes. isgylation of cellular proteins that in particular play a role in innate immune responses may affect viral gene expression. notably, isgylation of irf increases its stability and enhances irf -mediated transcriptional activation during sendai virus infection [ , ] and isgylation of ehp, an mrna ' cap structure-binding translation suppressor, plays a role in ifn-induced innate immune response [ ] . second, hcmv virion release was inhibited by isg conjugation. the inhibitory role of isg expression in the budding process of enveloped viruses has been demonstrated in retrovirus infection. isg expression inhibits ubiquitination of hiv gag and tumor susceptibility gene- (tsg ) proteins, leading to disruption of their interaction [ ] . isg conjugation of chmp , a, and in the escrt machinery causes release of vacuolar proteinsorting (vps ) from the membrane, leading to inhibition of virion release [ ] . the escrt machinery seems to be involved in the process of hsv- and hcmv maturation [ ] [ ] [ ] [ ] . therefore, isgylation may affect the hcmv maturation process by targeting components involved in the escrt machinery. although the antiviral role of isg expression and isgylation has been demonstrated in several viruses, studies on viral targets for isgylation and viral strategies that interfere with the isg -mediated antiviral functions are limited to a few examples. isgylation of ns a of influenza a virus inhibits virus replication by interfering with ns a nuclear import [ ] . kshv virf is isgylated but it role on virf function is not known [ ] . in the present study, we demonstrated that hcmv pul is a target for isgylation. ul isgylation appears to regulate protein stability by competing with ubiquitination. our analysis using the ul -p -isg fusion protein demonstrated that isgylation inhibits the activities of ul -p to downregulate the tnfα-mediated nf-κb activation and to complement the growth defect of ul -deleted virus. therefore, isgylation of pul is thought to inactivate its function, suppressing hcmv growth. given the notion that newly synthesized proteins are targeted extremely broadly, perhaps stochastically, by herc in polyribosomes [ ] , several other hcmv proteins may be isgylated during infection. we indeed observed that some viral proteins, which did not interact with isg , ube l, and herc in co-ip assays, were isgylated in our co-transfection/isgylation assays. however, we think that isgylation occurs in a manner dependent on the context of each protein. furthermore, since several hcmv proteins were bound to isg and/or ube l in yeast and co-ip assays, it is also likely that other viral proteins besides pul may affect isgylation. identification of more hcmv proteins that interact with the isgylation system and studies on their functional relevance are warranted. a few examples for viral regulation of isgylation have been described. the ns protein of influenza b virus non-covalently binds to isg and inhibits isgylation [ ] . similarly, the vaccinia virus e protein interacts with isg and disrupts its antiviral activity [ ] . nairoviruses and arteriviruses have shown to encode ovarian tumor domain (otu)-containing proteases that hydrolyze isg from target proteins [ ] and severe acute respiratory syndrome (sars) coronavirus encoded the papain-like protease that cleaves both ubiquitin and isg [ ] . recently, kshv-encoded virf was shown to interact with herc [ ] . in this study, we demonstrated that hcmv ul -p is able to non-covalently bind to isg , ube l, and herc , and inhibit isgylation. whether binding all of isg , ube l, and herc is critical for pul to inhibit isgylation is not clear and needs to be further investigated. comparative analysis of wild-type and ul -deleted mutant viruses demonstrate that de novo expression of ul in virus-infected cells correlated with reduced accumulation of isg conjugates. in addition to ie , the presence of additional viral functions that downregulate protein isgylation was prompted by our observation that uv-hcmv infection resulted in a higher level of isg conjugates than ie -deleted mutant virus at late times (fig b) . ul deletion mutant virus shows a moderate growth defect at low mois [ , ] . notably, the ul virus (in ad strain) showed a reduced plaque formation at low mois and this defect could be rescued by ie overexpression [ ] . more importantly, like ie -deleted virus [ ] [ ] [ ] , the growth of ul -deleted virus was more sensitive to pre-treatment of type i ifns [ ] (and this study), suggesting a role of ul in antagonizing type i ifn response. it is likely that a common isg -targeting mechanism is shared by influenza b virus, vaccinia virus, and hcmv. hcmv also encodes a tegument protein pul that contains a deubiquitinating protease domain; however, its activity was specific for ubiquitin and did not cleave isg [ ] . in this study, we demonstrate that the cellular isgylation system is a critical part of cellular innate immune response against hcmv infection. we also provide evidence that hcmv has developed several strategies to disarm the isgylation-mediated antiviral activity; ie reduces isg transcription and ul inhibits protein isgylation. this interplay of hcmv with the cellular isgylation system may be critical for the virus to successfully establish a persistent infection. human foreskin fibroblast (hf) (atcc) and human embryonic kidney (hek) t cells (atcc) were grown in dulbecco's modified eagle's medium supplemented with % fetal bovine serum, penicillin ( u/ml), and streptomycin ( μg/ml). dna transfection of t cells was performed using the n,n-bis-( -hydroxyethyl)- -aminoethanesulfonic acid-buffered saline (bbs) version of the calcium phosphate procedure. electroporation of hf cells was conducted using a microporator mp- (digital bio), as described previously [ ] . stocks for the parent towne virus and the cr mutant virus with ie -deleted were prepared in ihfie . cells as described previously [ ] . the hcmv (towne strain)-gfp virus was grown in hf cells after electroporation with the bacmid dnas. wild-type and ul -deleted hcmvs (ad strain) were previously described [ ] and grown in ul -expressing hf cells. wild-type and ul -ha-expressing hcmvs (toledo strain) generated in this study were grown in hf cells. to produce uv-inactivated hcmv (uv-hcmv), the virus stock was irradiated with uv light three times at . j/cm using a cl- crosslinker (uvp). mammalian expression plasmids for ha-ie (pdjk ) and ha-ie (pdjk ) were cloned using the psg vector and plasmid for myc-isg (pok ) was cloned using the pcs -mt (with a hexa-myc tag) vector using gateway technology as previously described [ ] . plasmids for myc-isg gg (pyj ), an active form of isg with a termination codon added immediately after the double glycine residues, or myc-isg aa (pyj ), a conjugation-defective mutant in which the double glycine residues are replaced with alanine residues, were produced using the stratagene quickchange site-directed mutagenesis protocol. the psg -driven plasmids expressing flag-ubch (pyj ) and ha-herc (pyj ) were produced using gateway technology. plasmids for srt-ul -p (pse ) and myc-ul -p (pse ) were cloned using the pcdna (life technologies) vector and pcs -mt vector, respectively, using gateway technology. for the srt-ul -p expression plasmid used in isgylation assays, two lysine residues on the linker between the srt tag and the ul orf were changed to alanines to block their possible isgylation, resulting in pyj . site-directed mutagenesis was performed on the pyj background to produce plasmids expressing the lysine to arginine mutant versions of srt-ul -p ; k r (pyj ), k r (pyj ), k / r (pyj ), and k / / r (pyj ). plasmids for ha-hube l (pcaggs-ha-hube l) and flag-hubch (pflagcmv -ubch ) and plasmids for s-tagged herc (pci-neo-s -herc ) were kindly provided by dong-er zhang (moores cancer center, university of california, san diego, la jolla, ca , usa). yeast ah (mata) cells were transformed with plasmid expressing the gal -dna-binding (db)-isg (trp + ) or gal -db-ube l (trp + ) fusion protein. y (matα) cells were transformed with plasmid expressing the gal -activation domain (a)-hcmv orf (leu + ) fusion proteins. each transformant was selected on plates lacking tryptophan (sc-trp) or leucine (sc-leu). trp + and leu + transformants were mated with each other on ypd plates. diploid cells (a/α) were selected on plates lacking both tryptophan and leucine (sc-trpleu). trp + leu + colonies were tested for their growth on plates that lack tryptophan, leucine and histidine (sc-trpleuhis). cells expressing bait and prey that interact with each other grow on sc-trpleuhis. cells expressing both gal -db-isg and gal -a-ube l were used as a positive control, whereas cells expressing gal -db-isg and gal -a only were used as a negative control. the toledo-bac clone encoding ul proteins with a c-terminal ha tag was produced by using a counter-selection bac modification kit (gene bridges). the scheme for bacmid mutagenesis is described in s fig and the lmv primers used for mutagenesis are listed in s table. retroviral vectors expressing ie (pyh ) or ul (pyj ), ul (k / r) (pyj ), and ul (k / / r) (pyj ) were produced on the background of pmin (murine leukemia virus-based retroviral vector) using gateway technology as previously described [ ] . retroviral vectors expressing shrna for ubch (pmscvpuro-shubch ) was previously described [ ] . to produce retroviral vectors expressing shrna for ube l (pmscvpuro-shu-be l- ), short hairpin rna (shrna) for ube l was amplified with u promoter by pcr with primers -tttggatcccaaggtcgggcaggaagagggcctatttcc- and -tttgaattcaaaaaggatgatgacagcaacttctctcttgaagaagttgctgtc atcatccggtgtttcgtcctttccacaagatatataa- (target sequence underlined). the pcr product was digested with bamhi and ecori and ligated to mscv-pgkpuro (bd biosciences clontech) digested with bglii and ecori. recombinant retroviruses were prepared in t cells after co-transfection with retroviral vectors together with the packaging plasmids phit (gag-pol) and pmd-g expressing the envelope g protein of vesicular stomatitis virus (vsv) [ ] using metafectene reagents (biotex). viral supernatants were collected at h after transfection. hf cells were transduced by retroviruses in the presence of polybrene ( . μg/ml). cells were selected with g ( . mg/ml) (calbiochem) and maintained in a medium containing g ( . mg/ml). lentiviral vector plko. -trc control expressing a non-hairpin control rna (shc) was purchased from addgene. plko. -based lentiviral vectors expressing shrna for ubp (shubp - : trcn and shubp - : trcn ) and herc (shherc - : trcn and shherc - : trcn ) were purchased from open biosystems. to produce lentiviruses, t cells were transfected with lentiviral vectors together with plasmids pcmv-dr . expressing the gag-pol, tat, and rev proteins of human immunodeficiency virus (hiv) and pmd-g. at h, the viral supernatants were collected and used to transduce hf cells in the presence of polybrene ( . μg/ml). the transduced cells were selected with puromycin ( μg/ml) and maintained in a medium containing puromycin ( . μg/ml). mouse monoclonal antibody (mab) r, which detects epitopes present in both ie and ie , was purchased from chemicon. mouse mabs against ul (p ) and ul (pp ) were obtained from virusys. anti-β-actin and anti-α-tubulin mouse mabs were purchased from sigma. anti-ha rat mab f and anti-myc mouse mab e , conjugated with peroxidase or labeled with fluorescein isothiocyanate (fitc), were purchased from roche. anti-isg (f- ) and anti-stat mouse mabs were obtained from santa cruz. mouse mab against srt epitope was previously described [ ] . ubp antibody was previously described [ ] . rabbit polyclonal ab (pab) for stat (c- ) and stat phosphorylated at tyr were purchased from santa cruz and upstate, respectively. rabbit pab for isg was kindly provided by chin ha chung (seoul national university, seoul, republic of korea). for immunoblot analysis, cells were washed with phosphate-buffered saline (pbs) and total cell lysates were prepared by boiling the cell pellets in sodium dodecyl sulfate (sds) loading buffer. equal amounts of the clarified cell extracts were separated on a sds-polyacrylamide gel or gradi-gel ii (elpis biotech, republic of korea) and electroblotted onto nitrocellulose membranes. the blots were blocked by incubation for min at room temperature with pbs plus . % tween (pbst) containing % nonfat dry milk. after being washed with pbst three times, the blots were incubated with the appropriate antibodies in pbst for h at room temperature. after three min washes with pbst, the blots were incubated with horseradish peroxidase-conjugated goat anti-mouse igg or anti-rabbit igg (amersham) for h at room temperature. the blots were then washed three times with pbst, and the protein bands were visualized with enhanced chemiluminescence system (amersham). for ifa, cells were fixed in ice-cold methanol for min and rehydrated in cold pbs. then, the cells were incubated with appropriate primary antibodies in pbs at °c for h, followed by incubation with appropriate secondary antibodies at °c for h. the mounting solution containing hoechst and anti-fade reagent (molecular probes) was used. for double-labeling, two different antibodies were incubated together. slides were examined and with a carl zeiss lsm meta confocal microscope system. the diluted samples were used to inoculate a monolayer of × hf cells in a -well plate. at h post infection, cells were fixed with μl of cold methanol for min. the cells were then washed three times in phosphate-buffered saline (pbs) and incubated with anti-ie rabbit polyclonal antibody in pbs at °c for h, followed by incubation with phosphatase-conjugated anti-rabbit immunoglobulin g (igg) antibody in phosphate-buffered saline (pbs) at °c for h. finally, the cells were gently washed in pbs and treated with μl of developing solution (nitroblue tetrazolium/ -bromo- -chloro- -indolylphosphate) at room temperature for h. the positively stained cells were counted for at least three to five separate fields per well under a light microscope (× magnification). co-transfected t cells ( × ) or virus-infected hf cells were harvested and sonicated in . ml co-ip buffer [ mm tris-cl (ph . ), mm naf, mm sodium phosphate, . % triton x- , containing protease inhibitors (sigma)] by a microtip probe (vibra-cell; sonics and materials, inc., usa) for s (pulse on: l s, pulse off: s). cell lysates were incubated with appropriate antibodies. after incubation for h at °c, μl of a % slurry of protein a-and g-sepharose (amersham) were added and then the mixture was incubated for h at °c to allow adsorption. the mixture was then pelleted and washed times with co-ip buffer. the beads were resuspended and boiled for min in loading buffer. each sample was analyzed by sds-page and immunoblotting with appropriate antibodies. for co-transfection/isgylation assays, t cells were co-transfected with plasmids expressing target protein and isgylation enzymes. co-transfected or virus-infected cells were treated with . mm nem (n-ethylmaleimide) for min before they were harvested. cell pellets were resuspended with % sds lysis buffer containing protease inhibitors and boiled for min. cell lysates were diluted -fold with co-ip buffer ( mm tris-cl [ph . ], mm naf, mm sodium pyrophosphate, containing protease inhibitors) and sonicated by using a microtip probe (vibra cell; sonics and materials, inc.). the clarified cell lysates were incubated with appropriate antibody for h and then with μl of a % slurry of protein g for h. the mixture was pelleted and washed seven times with co-ip buffer. the bound proteins were boiled and analyzed by sds-page followed by immunoblot assays reverse transcription-pcr (rt-pcr) and quantitative real-time rt-pcr (qrt-pcr) total rnas were isolated from × cells using trizol reagent (invitrogen) and maxtract high density (qiagen). first-strand cdna was synthesized by using the random hexamer primers in the superscript iii system (invitrogen). quantitative real-time tr-pcr (qrt-pcr) was performed using the applied biosystems abi prism sds software and the following primers: for isg , -gctgggacctgacggtg- (sense) and -ttagctccgcccgcca g- (anti-sense); for ube l, -aggtggccaagaacttggtt- (sense) and -cacca cctggaagtccaaca- (anti-sense); for ubch , -aacctgtccagcgatgatgc- (sense) and -tggtgcaaggcttccagttc- (anti-sense); for herc , -gggatgaa agtgctgaggag- (sense) and -cattttctgaagcgtccaca- (anti-sense); for β-actin, -agcgggaaatcgtgcgtg- (sense) and -cagggtacatggtggtgcc- (anti-sense). statistical significances were determined using the student's t-test and are indicated by à p< . , Ãà p< . , or ÃÃà p< . . [ ] was a gift from hua zhu (umdnj-new jersey medical school, newark, nj, usa). the rpsl-neo cassettes were pcr-amplified using lmv / primers containing homology arms consisting of nucleotides upstream and downstream of the target region plus nucleotides homologous to the rpsl-neo cassette. the amplified rpslneo fragments with homology arms were purified and introduced into e. coli gs containing wild-type toledo-bac for recombination by electroporation using a gene pulser ii (bio-rad). the intermediate toledo-bac constructs containing the rpsl-neo cassette were selected on luria broth (lb) plates containing kanamycin. next, the rpsl-neo cassette was replaced by annealed oligo dnas (lmv / ) consisting of only homology arms ( nucleotides upstream and downstream of the target region). the Δul toledo-bac was selected on lb plates containing streptomycin. the mutated regions were amplified by pcr and sequenced to verify the desired mutation. the toledo-bac encoding ul -ha was generated from the mutant toledo-bac. first, the rps-neo cassettes flanked by homology arms were inserted again into the mutant toledo-bac. next, dna fragments containing the wild-type ul gene with a ha tag at its cterminus were pcr amplified by -steps using lmv / and lmv / . the amplified ul -ha gene was then inserted into the toledo-bac containing the rps-neo cassette by homologous recombination. the lmv primers used for mutagenesis are listed in s table. (b) the regions containing the ul orf from wt, Δul , and ul -ha bacmid dnas were pcr amplified with lmv / . (c) wt, Δul , and ul -ha bacmid dnas were digested with bglii and the digestion patterns were compared via agarose gel electrophoresis. the bands corresponding to , and , bp from wild-type and ul -ha bacmids, respectively, and a band of , bp from Δul bacmid were indicated as arrowheads. comparative co-transfection/isgylation assays for ul and ie were performed in t cells with or without increasing amounts of plasmids expressing srt-ul -p or srt-ie ie as in fig d. cell lysates were prepared and immunoprecipitated with anti-srt antibody, followed by immunoblotting with anti-myc antibody. whole cell lysates were immunoblotted with anti-srt antibody to determine the expression levels of ul -p and ie , or with anti-myc antibody to determine the effect of ul -p or ie expression on isgylation. (tif) s fig. comparison of isgylation between wild-type and Δul virus infected cells. hf cells were mock-infected or infected with wild-type or Δul mutant virus (ad ) at an moi of . . cell lysates were immunoblotted at the indicated time points with antibodies for isg , viral proteins (ie , ie , and ul ), and β-actin. (tif) s table. pcr primers used for bacmid mutagenesis. (tif) s table. summary of the hcmv proteins that interacted with isg and ube l in yeast two-hybrid assays and co-ip assays. (tif) interferon-inducible antiviral effectors isg and immune diseases influenza b virus ns protein inhibits conjugation of the interferon (ifn)-induced ubiquitin-like isg protein the ubch ubiquitin e enzyme is also the e enzyme for isg , an ifn-alpha/beta-induced ubiquitin-like protein interferon-inducible ubiquitin e , ubc , is a conjugating enzyme for protein isgylation herc , an interferon-induced hect e enzyme, is required for conjugation of isg in human cells herc is an ifn-induced hect-type e protein ligase that mediates type i ifn-induced isgylation of protein targets the interferon-inducible ubiquitin-protein isopeptide ligase (e ) efp also functions as an isg e ligase isg modification of the eif e cognate ehp enhances cap structure-binding activity of ehp interferon induces a -kilodalton protein exhibiting marked homology to ubiquitin proteome analysis reveals ubiquitinconjugating enzymes to be a new family of interferon-alpha-regulated genes lipopolysaccharide activates the expression of isg -specific protease ubp via interferon regulatory factor rnase-l-dependent destabilization of interferon-induced mrnas. a role for the - a system in attenuation of the interferon response cloning and characterization of human ubiquitin-processing protease- from terminally differentiated human melanoma cells using a rapid subtraction hybridization protocol rash ubp is a novel regulator of interferon signaling independent of its isg isopeptidase activity positive regulation of interferon regulatory factor activation by herc via isg modification ubch regulates ubiquitin and isg conjugation to rig-i proteomic identification of proteins conjugated to isg in mouse and human cells high-throughput immunoblotting. ubiquitiin-like protein isg modifies key regulators of signal transduction human isg conjugation targets both ifninduced and constitutively expressed proteins functioning in diverse cellular pathways isg enhances the innate antiviral response by inhibition of irf- degradation interferon-induced isg conjugation inhibits influenza a virus gene expression and replication in human cells ifn-stimulated gene functions as a critical antiviral molecule against influenza, herpes, and sindbis viruses herc attenuates influenza a virus by catalyzing isgylation of viral ns protein mice lacking the isg e enzyme ube l demonstrate increased susceptibility to both mouse-adapted and nonmouse-adapted influenza b virus infection innate antiviral response targets hiv- release by the induction of ubiquitin-like protein isg role of interferon-stimulated gene isg- in the interferon-omega-mediated inhibition of human immunodeficiency virus replication modulation of alpha interferon anti-hepatitis c virus activity by isg inhibition of hepatitis c virus replication by ifn-mediated isgylation of hcv-ns a inhibition of hepatitis c virus rna replication by isg does not require its conjugation to protein substrates by the herc e ligase isg over-expression inhibits replication of the japanese encephalitis virus in human medulloblastoma cells isg arg and the isg -conjugating enzyme ube l are important for innate immune control of sindbis virus identification of interferon-stimulated gene as an antiviral molecule during sindbis virus infection in vivo isg inhibits nedd ubiquitin e activity and enhances the innate antiviral response isg inhibits ebola vp vlp budding in an l-domain-dependent manner by blocking nedd ligase activity vaccinia virus e protein prevents the antiviral action of isg isg facilitates cellular antiviral response to dengue and west nile virus infection in vitro nonstructural protein of porcine reproductive and respiratory syndrome virus inhibits the antiviral function of interferon-stimulated gene kaposi's sarcoma-associated herpesvirus viral interferon regulatory factor interacts with a member of the interferon-stimulated gene pathway isg is upregulated in respiratory syncytial virus infection and reduces virus growth through protein isgylation the isg conjugation system broadly targets newly synthesized proteins: implications for the antiviral function of isg isg conjugation system targets the viral ns protein in influenza a virus-infected cells budding of enveloped viruses: interferon-induced isg -antivirus mechanisms targeting the release process isg is critical in the control of chikungunya virus infection independent of ube l mediated conjugation negative feedback regulation of rig-i-mediated antiviral signaling by interferon-induced isg conjugation isg , a ubiquitin-like interferonstimulated gene, promotes hepatitis c virus production in vitro: implications for chronic infection and response to treatment the interferon stimulated gene functions as a proviral factor for the hepatitis c virus and as a regulator of the ifn response isg : leading a double life as a secreted molecule emerging roles for immunomodulatory functions of free isg mycobacterial disease and impaired ifn-gamma immunity in humans with inherited isg deficiency fighting mycobacteria through isgylation human intracellular isg prevents interferon-alpha/beta over-amplification and auto-inflammation isg deficiency and increased viral resistance in humans but not mice fields virology human cytomegalovirus ul -coded pp virion protein inhibits antiviral gene expression in infected cells human cytomegalovirus immediate-early gene expression blocks virusinduced beta interferon production major human cytomegalovirus structural protein pp (ppul ) prevents interferon response factor activation in the interferon response a human cytomegalovirus antagonist of type i ifn-dependent signal transducer and activator of transcription signaling binding stat by the acidic domain of human cytomegalovirus ie promotes viral growth and is negatively regulated by sumo physical requirements and functional consequences of complex formation between the cytomegalovirus ie protein and human stat positive role of promyelocytic leukemia protein in type i interferon response and its regulation by human cytomegalovirus characterization of recombinant human cytomegaloviruses encoding ie mutants l p and - reveals that viral targeting of pml bodies perturbs both intrinsic and innate immune responses human cytomegalovirus inhibits major histocompatibility complex class ii expression by disruption of the jak/stat pathway human cytomegalovirus inhibits ifn-alphastimulated antiviral and immunoregulatory responses by blocking multiple levels of ifn-alpha signal transduction human cytomegalovirus interferes with signal transducer and activator of transcription (stat) protein stability and tyrosine phosphorylation human cytomegalovirus ie protein elicits a type ii interferon-like host cell response that depends on activated stat but not interferon-gamma defective growth correlates with reduced accumulation of a viral dna replication protein after low-multiplicity infection by a human cytomegalovirus ie mutant the interferon-inducible -kda ubiquitin homolog conjugates to intracellular proteins protein isgylation modulates the jak-stat signaling pathway analysis of human cytomegalovirus-encoded sumo targets and temporal regulation of sumoylation of the immediate-early proteins ie and ie during infection open reading frame ul of human cytomegalovirus encodes a novel tegument protein that contains a strong transcriptional activation domain deletion of open reading frame ul from the human cytomegalovirus genome results in reduced viral growth, which involves impaired stability of viral particles distinct domains within the human cytomegalovirus u(l) protein are important for wildtype viral replication and virion stability the human cytomegalovirus ul protein antagonizes nf-kappab activation identification and characterization of a novel isg -ubiquitin mixed chain and its role in regulating protein homeostasis negative regulation of isg e ligase efp through its autoisgylation role of noncovalent sumo binding by the human cytomegalovirus ie transactivator in lytic growth the pias homologue siz regulates perinuclear telomere position and telomerase activity in budding yeast sumoylation regulates telomere length homeostasis by targeting cdc . nature structural & molecular biology sumoylation of atrip potentiates dna damage signaling by boosting multiple protein interactions in the atr pathway tpz tpp sumoylation reveals evolutionary conservation of sumo-dependent stn telomere association different roles for two ubiquitin-like domains of isg in protein modification human cytomegalovirus exploits escrt machinery in the process of virion maturation viral and host control of cytomegalovirus maturation intracellular trafficking and maturation of herpes simplex virus type gb and virus egress require functional biogenesis of multivesicular bodies herpes simplex virus type cytoplasmic envelopment requires functional vps ovarian tumor domain-containing viral proteases evade ubiquitin-and isg -dependent innate immune responses selectivity in isg and ubiquitin recognition by the sars coronavirus papain-like protease ul -deficient human cytomegalovirus produces virions with hypophosphorylated pp tegument protein that is unstable within newly infected cells cleavage specificity of the ul deubiquitinating protease activity of human cytomegalovirus and the growth of an active-site mutant virus in cultured cells pias enhances sumo- modification and the transactivation activity of the major immediate-early ie protein of human cytomegalovirus ubp (usp ) specifically removes isg from conjugated proteins coding potential of laboratory and clinical strains of human cytomegalovirus we thank dong-er zhang and chin ha chung for providing plasmids and antibodies. we also thank gary s. hayward for sharing the hcmv orf library, edward s. mocarski for providing the cr virus, and hua zhu for providing the hcmv (toledo) bacmid. conceptualization: yjk etk jha. key: cord- - xy s authors: hu, dan; zhu, zhongyu; li, shun; deng, yongqiang; wu, yanling; zhang, nana; puri, vinita; wang, chunyu; zou, peng; lei, cheng; tian, xiaolong; wang, yulu; zhao, qi; li, wei; prabakaran, ponraj; feng, yang; cardosa, jane; qin, chengfeng; zhou, xiaohui; dimitrov, dimiter s.; ying, tianlei title: a broadly neutralizing germline-like human monoclonal antibody against dengue virus envelope domain iii date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: xy s dengue is the most widespread vector-borne viral disease caused by dengue virus (denv) for which there are no safe, effective drugs approved for clinical use. here, by using sequential antigen panning of a yeast antibody library derived from healthy donors against the denv envelop protein domain iii (diii) combined with depletion by an entry defective diii mutant, we identified a cross-reactive human monoclonal antibody (mab), m . , which bound with high affinity to denv diii from all four denv serotypes. immunogenetic analysis indicated that m . is a germline-like mab with very few somatic mutations from the closest vh and vλ germline genes. importantly, we demonstrated that it potently neutralized denv both in vitro and in the mouse models of denv infection without detectable antibody-dependent enhancement (ade) effect. the epitope of m . was mapped to the highly conserved regions on diii, which may guide the design of effective dengue vaccine immunogens. furthermore, as the first germline-like mab derived from a naïve antibody library that could neutralize all four denv serotypes, the m . can be a tool for exploring mechanisms of denv infection, and is a promising therapeutic candidate. a a a a a dengue virus (denv) causes the most prevalent mosquito-borne viral disease. over . billion people are at risk for infection in over countries, - million are infected with symptoms, and up to , die from dengue hemorrhagic fever (dhf) and dengue shock syndrome (dss) each year [ , ] . no specific antiviral drug has been available against denv infection; the only approved vaccine, dengvaxia, has caused considerable controversy regarding its safety and potential benefits [ ] [ ] [ ] [ ] . for decades, anti-denv vaccine and biological drugs development has been hampered by the high sequence divergence ( - %) among the four denv serotypes [ , ] . such divergence leads to the fact that one antibody may not be sufficient to neutralize all denv infection. instead, the induced humoral immune response to one denv infection can enhance the infection and disease processes brought by a subsequent infection with another denv serotype [ ] [ ] [ ] . these findings suggest that the development of new and broadly neutralization antibodies against all the serotypes of denv could be promising candidate anti-denv agents, and may also guide the design of effective and safe vaccine immunogens. the denv envelope glycoprotein (e protein), which mediates virus entry into cells, is the major neutralizing target of antibodies [ ] [ ] [ ] [ ] [ ] . e protein is a type ii fusion protein and consists of three domains: di, dii, and diii of which diii has been proposed to contain a receptor binding domain [ ] [ ] [ ] [ ] . recent studies revealed that cross-reactive conserved epitopes exist on dii as well as diii of the denv e protein [ , [ ] [ ] [ ] . during the naturally-occurring primary denv infection, a large fraction of the antibody repertoire consists of dii-specific antibodies which are, unfortunately, typically poor in neutralization and may increase the likelihood of severe disease upon subsequent infection through a mechanism known as antibody-dependent enhancement (ade) [ ] [ ] [ ] . in contrast, antibodies targeting diii have proven to be the most potent neutralizing antibodies, but very few could be elicited in naturally infected individuals [ , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . despite this, previous studies indicated that anti-denv diii serotype-specific and cross-reactive antibodies could be elicited using denv diii as vaccine immunogen [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] and in infected humans [ ] [ ] [ ] [ ] . it has also been demonstrated that the lysine at position on diii is the critical residue in the cross-reactive epitope [ ] . therefore, the conserved epitope on diii represents an attractive target for the development of broadly neutralizing denv antibodies. here, we report the isolation of a potent denv diii-specific human monoclonal antibody (mab), designated as m . , from a large naïve antibody library constructed by the blood of healthy adult donors. a competitive sorting strategy using a diii mutant as competitor was applied to identify antibodies precisely targeting the conserved neutralizing epitope. to our knowledge, m . is the first human mab isolated from a naïve antibody library which could neutralize all the four serotypes denv viruses. importantly, both heavy and light chain genes of m . are very close to their putative germline predecessors. its fully human origin, the germline-like nature, combined with high-affinity and broad neutralizing activity toward all denv serotypes, suggest that m . is a promising candidate antiviral agent and may also provide a unique template for designing effective dengue vaccine immunogens. we previously prepared some large naïve antibody libraries using peripheral blood b lymphocytes of non-immunized healthy donors and used them for panning/screening against viral and cancer targets [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . in this study, we used a competitive library sorting strategy to isolate broadly neutralizing antibodies against denv - ( fig a and b) . the yeast-displayed naïve single chain antibody fragment (scfv) library was used to screen against the biotinylated denv diii, and, importantly, ten times concentration unbiotinylated diii k e mutant was used as the competitor. the yeast cells were selected to present the antibody-expressing cells that could bind well to the wild-type diii instead of the diii mutant, resulting in the isolation of antibodies that can target the cross-reactive neutralizing epitopes covering the residue lysine [ ] . potent enrichment was achieved after four rounds of sorting, and a panel of antibodies were identified ( fig b) . two antibodies, designated as m and m , bound potently to denv diiis. their scfv gene were fused with human igg fc for protein expression, and surface plasmon resonance (spr) experiments were used to evaluate the antigens binding. the equilibrium dissociation constant (k d ) of m for the denv - diiis were . nm, . nm, . nm and . nm, respectively. the mab m displayed a broader binding profile compared with that of m , with the k d of . nm, . nm, . nm and nm to denv - , respectively (table , s and s figs). to further improve the affinity of m and m with the four denv serotypes, we constructed a mutant library using the error-prone pcr technologies. following three cycles of mutagenesis and selection, two clones were identified from the enriched pool of yeast sorting, designated as m . and m . . biacore analysis showed that the cross-reactive binding activities of m . and m . to all diiis were preserved after the affinity maturation process. the k d of m . for the denv - diiis were . nm, pm, . pm and nm, respectively (s fig). although the binding to denv - diiis was improved, the m . had only slightly increased binding affinity to denv diii compared to its parental mab m . notably, the m . exhibited high affinity to all the denv diiis. the k d of m . for the denv - were . nm, . nm, . nm, and . nm respectively, which demonstrated that m . could bind to all the four serotype denv viruses with high avidity (table , fig ) . we also assessed the binding specificity of m . by elisa, and the results showed that m . had weak cross-reactivity with zika virus (zikv) diii and no binding with other irrelevant antigens (s fig). next, we assessed the neutralization capacity of m . and m . against the four denv serotypes using a denv luciferase reporter viral particle (rvp) neutralization assay. we used denv rvps against the four dengue serotypes that are common strains in denv research: denv- (westpac ), denv- (s ), denv- (ch ), and denv- (tvp ). the luminescent reporter expression was proportional to the number of rvps added to bhk dc-sign cells, confirming the linear correlation between the extent of rvp infection and reporter gene expression. in consistent with the biacore binding results, both m . and m . could neutralize all the four serotype denv, and m . displayed better neutralization than m . , with the % neutralization titers (ic to further evaluate the neutralization breadth of m . igg against the four denv serotypes, a standard plaque reduction neutralization assay (prnt) on bhk- cells was performed using denv - live viruses, including denv- (genbank fj ), denv- gz / (s fig, isolated from a denv- infected patient in guangzhou, china), denv- (genbank af ), denv- - (genbank af ), and denv- b (genbank af ). an irrelevant human mab g was used as the negative control [ ] , and a g , a broadly neutralizing mab against all the four denv serotypes, was used as the positive control [ , ] . as shown in fig , m . igg could neutralize all the four denv serotypes. the % neutralization titers (ic ) of m . against denv - was . , . , . , and . μg/ml respectively ( table ). we next used a well-established ade assay to detect the in vitro ade effect of m . igg. a mutated form of m . igg was also generated containing the leucine to alanine substitutions at positions and (m . igg-lala), which lacked binding to fcγ receptors. the ade effects of denv- or denv- by m . igg, m . igg-lala, as well as a g were measured. interestingly, neither m . igg nor m . igg-lala presented any ade effect against different serotypes of denv ( fig f, s fig) . in contrast, potent ade effects were observed for the dii-specific mab a g . these results showed that m . igg is a denv diii-specific mab without detectable ade effect. we further analyzed the sequences of mabs using the imgt tool to identify their closest vh and vλ germline genes. the results indicated that m . and m . originated from different b-cell lineages ( table ). the m . vh gene was derived from the ighv - and the vλ gene was from iglv - . in contrast, the m . vh gene was derived from the ighv - and the vλ gene was from iglv - . interestingly, we found that the encoding genes of both m . and m . closely resembled their corresponding germline gene segments. notably, m . vh and vλ gene shared . % and . % sequence identities with the ighv - � and iglv - � germlines respectively (fig a and b ). these results indicated that the mab m . is a germline-like antibody, which, in general, could show better drug properties and lower immunogenicity compared to somatically hypermutated antibodies [ ] . to further investigate the immunogenetic characteristics of m . -like antibodies, we analyzed in detail the ighv - recombination frequencies with specific ighd and ighj genes families from naïve immunoglobulin m (igm) repertoires of health adult donors and neonatal igm repertoires of newborn babies, using next-generation sequencing data previously generated from our antibodyome studies [ ] . by querying the m . sequence from the igm repertoires, sequences were found to display m . -like v(d)j recombination from the genes ighv- - , ighd , and ighj out of a total of , , sequences from healthy adult igm repertoires. in igm repertoires of newborn babies, a similar recombination frequency was also observed, in which sequences with m . -like v(d)j recombination were found from , , sequences. our analysis showed that ighv - is one of the most frequently used ighv genes, and identified that many of those sequences sharing a significant degree of resemblance to m . ( fig c) . in brief, analysis of these data showed the potential of eliciting robust immune responses with the m . -like germline antibodies by vaccination. to determine whether m . can protect denv infections in vivo, we firstly used a lethal denv - infection suckling mouse model. the mice were challenged with denv - at pfu/mouse via intracranial injection. four hours later, the mice were treated intracranial with a single dose ( μg) of m . igg, m . igg-lala mutant and g (unrelated antibody control). these animals were monitored for morbidity and mortality daily. as shown in fig , all the mice in control groups died from denv infection, and most of them died within the first two weeks of viral challenge. interestingly, there was no significant difference in therapeutic efficacy against denv - infection between m . and the lala-mutated m . . the m . igg protected % denv- , denv- , denv- and % denv- infection whereas (c) germline-rooted circular phylogenetic tree of m . -like antibody sequences found in igm libraries derived from healthy human adults and neonates. the m . and a sequence showed highest similarity to m . were shown in red. sequence id started with cb represents sequence derived from the neonates, and that started with hh represents sequence derived from the healthy adults. the phylogenetic tree was constructed by the neighbor-joining method. https://doi.org/ . /journal.ppat. .g lala-mutated m . protected % denv- , denv- and % denv- , denv- respectively. therefore, m . has no detectable ade as confirmed in both in vitro and in vivo experiments. we also used the ag (types-i and -ii ifn receptor deficient) mice to test the therapeutic effect of m . against denv- (s fig). the results showed that all the mice in the control antibody treatment group died while the survival rate of mice can reach % in m . treatment group, indicating that the antibody can also protect the lethal infection of denv- in ag mice. taken together, these results indicated that m . can protect denv - infections in vivo. to map the epitope of the germline-like mab m . and identify in greater detail the structural basis of denv neutralization, we employed multiple approaches (fig ) . sequence alignment of different denv genotypes and mapping of the conserved amino acid residues of denv diii showed that four serotypes denv diiis amino acid residues were different from one another between amino acids - ( fig a) . subsequently, serotype derived diii consensus gene was randomly mutated to construct a yeast-displayed mutant library. two rounds of sorting of those yeast cells showing expression on surface but lacking the binding to m . was performed. a total of binding escape mutants were aligned with the serotype consensus protein sequence. mutation frequency at each position was plotted against the residue position number. similarly, unique diii sequences derived from naturally isolated serotype dengue viruses from genebank were also aligned with the consensus sequence (fig b and c) . the superimposed profiles of the two set of sequences showed that many of the escaped mutations located in the well-conserved area, indicating the broad crossreactivity of m . to naturally isolated dengue viruses. besides, the epitope mapping shows that the m . epitope is at close to or partially overlaps the dimerization interface between domains ii and iii. these results may explain why m . is a potent cross-reactivity antibody to all the four denv serotypes. furthermore, computational docking of denv diii-m . antibody complex was performed using zdock method. we selected the three top scored docked complexes that contained the key residues identified from an experimental epitope mapping approach. one of the top scored docked models exhibited minimum clashes with appropriate protein interface parameters and was used to demonstrate the lactation the potential epitopes and their interactions with m . antibody, which might shed light on the molecular mechanisms of broadly cross-reactive neutralization. fig d showed the docking model of the diii-m . antibody complex in which these epitopes are highlighted in green surfaces. the docking model revealed a different orientation of antibody binding as compared to the diii complex structure with fab a d- that was previously determined [ ] . the epitope comprised of three structurally proximal regions, residues - in green, , and in dark green, and - at the c-terminal in lime. one of the key residues, k , contacts the cdr-l of m . which has a germline mutation. in env-diii-fab- a d- complex structure, the residue k contacts the cdr-h . the hydrophobic residues, ile and trp, of cdr-h contact the center part of the epitope, and other loops h , h , l and l also involve in the binding. the surface area of the interface between diii and m . antibody in the model complex is Å , a typical of antibody-antigen interactions. there are six hydrogen bonds likely to form and no salt bridges at the interface. in brief, the binding regions of the m . may be close to or partially overlaps the dimerization interface between domains ii and iii, which might indicate the broad crossreactivity of m . to the four serotypes of denv. dengue is a disease with a complex immune response orchestrated by host cells partially due to the presence of four serotypes of denv. importantly, after a primary denv infection, one can be protected against or aggravate of a secondary infection with a different serotype, which bring many difficulties to develop an effective vaccine. thus, it is very urgent to develop an effective and cross-reactive antiviral therapy against denv infection. monoclonal antibodies (mabs) are of growing importance for protective and pathogenic immune responses to viruses. at present, there are many therapeutic antibodies to treat viral infections under development, such as antibodies for hiv- , sars-cov, mers-cov, nipah and hendra viruses, and h n influenza virus [ , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] . fortunately, screening antibodies from the large naïve libraries has enabled the rapid development of high-affinity human mabs, especially for the rapid response to the outbreak of emerging viruses and diseases. we recently successfully identified two human germline-like mabs against mers-cov and h n influenza virus from the naïve library, named m and m , respectively [ , ] . they both can naturally exist with very low level of somatic hypermutation in the naïve library with which they have potent binding activity against the envelop proteins of mers-cov and h n influenza virus. most importantly, m and m all showed highly therapeutic effective in the animal models. therefore, the naïve library screening can be quickly used to isolate germline-like antibodies that effectively bind to complex protein targets like those in denv viruses. how to increase the neutralization breadth is a key issue in developing anti-denv antibodies. previous studies revealed two classes of broadly neutralizing antibodies to flaviviruses, including antibodies targeting the conserved epitopes in dii or diii [ ] [ ] [ ] ]. while the conserved fusion loop epitope (fle) in dii is the immunodominant epitope in e protein, unfortunately, this epitope frequently induced poorly neutralizing and strongly infectionenhancing antibodies via ade [ ] [ ] [ ] . therefore, diii represents the ideal target for neutralizing antibodies. in this study, we applied a highly efficient yeast-display-based sorting strategy by using the highly diverse denv diiis as antigen and the competitive sorting technique. by applying this method, we quickly and efficiently identified two human germline-like broadspectrum anti-denv mabs (m and m ) from the naïve scfv yeast library using the diii antigen that make them as promising candidate therapeutics as well as the template for vaccine development. another class of highly efficient broadly neutralizing antibodies that target the envelope dimer epitopes (ede) from the secondary acute denv infection plasmablasts has been identified by dejnirattisai et al. [ ] . these antibodies may especially get through with high somatic mutations from the secondary virus infection. compared with the highly somatically mutated antibodies, germline-like antibodies typically have better safety and drug-related property [ ] . importantly, the hendra and nipah antibody m . is a near germline antibody and exhibited a very good drugability, which was from a similar library that was also used to isolate our m and m -like antibodies. m . was successful as a candidate therapeutic mab in animal models and was also completed the phase i clinical trial (actrn ) without side effects [ ] . to further improve the affinity of m with the four serotypes denv diiis, we subjected m to affinity maturation process, and named it as m . . subsequently, we analyzed m . sequence using the imgt tool to identify its closest vh and vλ germline genes. interestingly, we found that m . is still a germline-like antibody although it went through the mutation process in vitro, with over % identities of its vh and vλ genes to the ighv - � and iglv - � germline respectively. in order to evaluate the neutralization effect of the m . igg, we used a standard plaque reduction neutralization with bhk cells to measure denv infection and neutralization. the m . igg showed broadly neutralization towards the four serotypes denv as well as a recent denv isolate from clinical samples. more importantly, m . did not present any ade effects in different serotypes of denv. the in vivo study results demonstrated the therapeutic potential of m . against severe denv - infections. in brief, the m . could neutralize the four serotypes denv in vitro and protect the denv infection mouse model in vivo without detectable ade effects. we therefore expect that m . has a likeness drugability of m . and could be developed as a candidate therapeutic in the future. we have also localized the m . epitope by using a combination of computational structural modeling, display-based antigen mutagenesis, and sequence-based analysis of mutants. the epitope appears to overlap with the epitope previously explored as targets for cross-reactive murine mabs and close to or partially overlaps the dimerization interface between domains ii and iii. this further indicates that this epitope could be an important component of vaccine immunogens intended to elicit cross-reactive neutralizing antibodies. in progress are our experiments to crystallize the complex of m . with denv diii that would allow precise determination of the m . epitope. the major result of this study is the identification of a germline-like human mab, m . , from a naïve yeast antibody library which binds with high (picomolar) affinity to diiis from all serotypes and neutralizes the four denv serotypes. there are two major implications from this finding: ) m . is a potential candidate therapeutic which could be further developed in preclinical and clinical settings. ) the epitope of the germline-like mab m . could guide the design of effective candidate vaccine immunogens capable of eliciting m . and/or m . -like antibodies. bhk cells were cultivated in dulbecco's modified eagle medium (dmem) supplemented with % fetal bovine serum (fbs) (biowest). mosquito cells c / were cultured in rpmi- medium supplemented with % fbs. all cells were maintained in a humidified atmosphere of % co at ˚c incubator, except for c / cells, which were cultivated at ˚c. denv- (genbank fj ), denv- gz / (isolated from denv- infected patient in guangzhou), denv- (genbank af ), denv- - (genbank af ), and denv- b (genbank af ) were propagated in c / cells by using rpmi medium and the titers were measured by standard plaque forming assay in bhk cells. denv diii genes from all serotypes were synthesized by genescript, inc (nanjing, china), fused with igg fc and a c-terminal avi-tag, and cloned into psectag expression vector. the diii. (serotype ) k e mutant was generated through overlapping pcr. for the conversion of igg from scfv, the heavy and light chains of scfv were amplified and recloned into the ptt-igg vector. the plasmids were transfected into expi cells (thermo fisher) for transient expression, and purified using protein g column (ge healthcare, piscataway, nj) according to the manufacturer's instructions. the purified protein was biotinylated by mixing with biotinylation reagents in pbs for min on ice, according to the manufacturer's instructions (pierce). a large yeast-displayed scfv library was used for antibody screening, and the screening protocols were essentially carried out as described previously [ ] . briefly, μg of binotinylated diii. -fc and cells of the initial naïve library were mixed and washed by pbsa, and incubated with μl streptavidin conjugated microbeads (miltenyi biotec, auburn, ca) before loading onto the automacs system (miltenyi biotec) for sorting. after three rounds of sorting, the downsized library was further sorted against binotinylated diii. -fc ( μg/ml) but also using unbiotinylated k e mutant ( μg/ml) as the competitor. to generate the m scfv and m scfv mutant libraries, random mutagenesis of the scfv genes were performed through error-prone pcr by using a genemorph ii kit (stratagene) following the manufacturer's instructions with minor modifications. to further diversify the mutation profile, um of each of the two nucleotide analogues ( -oxo-deoxyguanosine triphosphate and '-deoxy-p-nucleoside- '-triphosphate) was mixed in the pcr reaction mixture. for the second and third cycle library constructions, an extra step of dna shuffling pcr was inserted into the regular pcr cycles to combine the beneficial mutations obtained from previous maturation process. dna shuffling pcr step was performed as following: cycles of denature at ˚c for seconds followed by annealing/extension at ˚c for second on the bio-rad mycycler. binding affinities of m scfv, m scfv, m . scfv, and m . scfv to the denv diiis were analyzed by surface plasmon resonance technology using a biacore x instrument (ge healthcare). the antibodies were covalently immobilized onto a sensor chip (cm ) using carbodiimide coupling chemistry. a control reference surface was prepared for nonspecific binding and refractive index changes. for analysis of the kinetics of interactions, varying concentrations of antigens were injected at flow rate of μl/min using running buffer containing mm hepes, mm nacl, mm edta, and . % surfactant p- (ph . ). the association and dissociation phase data were fitted simultaneously to a : langumir global model by using the nonlinear data analysis program biaevaluation . . all the experiments were done at ˚c. neutralizing activity of mabs was measured using a standard plaque reduction neutralization with bhk cells as previously described [ ] . briefly, -fold serial dilutions of mabs were added to approximately pfu of a variety of dengue virus strains and incubated for h at ˚c. then, the mixture was added to bhk cell monolayers in a -well plate in duplicate and incubated for h at ˚c. the mixture was removed, and ml of . % (w/v) lmp agarose (promega) in dmem plus % (v/v) fbs was layered onto the infected cells. after further incubation at ˚c for days, the wells were stained with % (w/v) crystal violet dissolved in % (v/v) formaldehyde to visualize the plaques. prnt values were determined using non-linear regression analysis. prnt data were calculated by doing a non-linear regression analysis using sigmaplot (version . , systat software, inc., ca) as previously described [ ] . denv rvps from all four serotypes were pre-incubated with an equal volume of serially diluted antibodies ( μg/ml to . μg/ml pre-dilution or . μg/ml to . μg/ml predilution, as measured based on the dilution of antibody prior to combining with rvps) in dmem infection media for h at room temperature and transferred to wells of a -well plate. an equal volume of denv rvps were added to each well followed by slow agitation for h at room temperature. bhk dc-sign cells were added to each well at a density of , cells per well followed by incubation at ˚c in % co for h. cells were subsequently fixed in lysed and analyzed for luminescent reporter expression using the wallac victor. the percent infection for each concentration of mab or serum was calculated, and the raw data was expressed as percent infection versus log of the mab concentration or the reciprocal serum dilution. the data were fit to a sigmoidal dose-response curve using prism (graphpad software, la jolla, ca) to determine the titer of antibody that achieved a % reduction in infection. maximum infection was determined in the absence of antibodies. the in vitro ade assay was performed using k cells [ ] . briefly, serial -fold dilutions of antibodies under concentrations ranging from to . μg/ml were mixed with denv- or denv- , and incubated for h at ˚c. mixtures were then added to × k cells at multiplicity of infection of . ~ . for h in -well plates. the cells were subsequently washed times with serum free rpmi- medium. after collecting cells by centrifugation, the cell pellets were re-suspended with rpmi- medium containing % fbs and added to -well plates, then incubated for days at ˚c with % co . the titer of viruses in the supernatant was then measured using a plaque assay. the ade effect was calculated as different viral yields in the supernatant after infection in the presence of the added antibodies. the epitope mapping of m . was performed using previously described protocols [ ] . briefly, random mutagenesis of the denv diii. gene was performed using a genemorph ii kit (stratagene). as described above, the yeast-displayed mutant library was mixed with biotinylated m . scfv-fc, washed, and stained by mouse anti-c-myc antibody (roche), alexa- conjugated goat-anti-mouse antibody (invitrogen), and pe-conjugated streptavidin (invitrogen). after two rounds of sorting on a facsaria ii cell sorter (bd biocsiences, san jose, ca), the sorted cells were amplified and their plasmids were prepared and sequenced. homology modeling of variable regions of heavy (v h ) and light (v l ) chains for m . scfv antibody was carried out using the swiss-model workspace [ ] by selecting the closest template structures (pdb codes: qos for heavy chain and dd for light chain), whose sequence similarities were % and % respectively. the v h -v l orientation of m . scfv structure was assigned similar with one of the templates (pdb code: dd ) that showed minimum steric clash for creating the final m . scfv model. the crystal structure of denv diii serotype (pdb code: r ) was used for docking with the modeled scfv antibody m . . docking of scfv m . to the dengue env-iii was performed by zdock server (http://zdock.bu.edu) that uses a fast fourier transform (fft)-based rigid-body protein docking algorithm with scoring functions combining pairwise shape complementarity, desolvation and electrostatic energies. based on the escape mutants that led to the loss of epitopes and available crystal structure of denv diii, we selected a list of residues as biological constrains, , , , , , and , on the surface of env-diii as potential contacting residues for docking constraints. similarly, one or two residues from each of cdr-h , h and l loops were chosen at the docking interface. cdr-h and h loops had dominant hydrophobic residues whereas cdr-l had a germline mutation, and they all had high antigen-contacting propensities [ ] . results from the top zdock predictions were filtered using the userdefined residues and a angstrom distance cutoff. three predicted complexes were only kept as all residues selected come together at the interface and were further examined by pdbepisa (protein interfaces, surfaces and assemblies). pymol was used for the analysis of docked model and graphical illustration [ ] . the suckling mice were purchased from b&k universal group limited (shanghai, china) and housed under specific pathogen-free conditions at the animal facilities of the shanghai public health clinical center, fudan university (shanghai, china). before infection, the mice were transferred to the animal biosafety level (bsl- ) laboratory (shanghai, china). one day mice were used for all experiments. all mice were intracerebrally injected with pfu of denv - . at h post infection, mice were passively transferred a single dose of μg antibody m . igg, m . igg lala mutant or g igg as the negative control via intracerebrally injection. survival rates and disease sings were monitored daily. the ag mice (type i and type ii interferon receptor-deficient) were purchased from b&k universal group limited (shanghai, china) and housed under specific pathogen-free conditions at the animal facilities of the shanghai public health clinical center, fudan university (shanghai, china). before infection, the mice were transferred to the bsl- laboratory (shanghai, china). groups of mixed-sex -to -week-old mice were used for all experiments. all mice were intraperitoneally injected with x pfu of denv- in a volume of μl. at h post infection, mice were passively transferred a single dose of μg antibody m . igg-lala, or g antibody as the control via i.p. injection. survival rates, weight loss, and disease sings were monitored daily. specific-pathogen-free ag mice ( - weeks old) and suckling mice were used for all experiments. all experimental protocols were reviewed and approved by the institutional committee of the who dengue classification and case definitions: time for a reassessment global spread and persistence of dengue the risks behind dengvaxia recommendation dengvaxia: age as surrogate for serostatus. the lancet infectious diseases : dengvaxia controversy: impact on vaccine hesitancy ade and dengue vaccination cross-reacting antibodies enhance dengue virus infection in humans the growth and potential of human antiviral monoclonal antibody therapeutics variable surface epitopes in the crystal structure of dengue virus type envelope glycoprotein monoclonal antibody mapping of the envelope glycoprotein of the dengue virus diversity and junction residues as hotspots of binding energy in an antibody neutralizing the dengue virus mapping of a dengue virus neutralizing epitope critical for the infectivity of all serotypes: insight into the neutralization mechanism characterization of the interaction of domain iii of the envelope protein of dengue virus with putative receptors from cho cells monoclonal antibodies that bind to domain iii of dengue virus e glycoprotein are the most efficient blockers of virus adsorption to vero cells structural insights into the mechanisms of antibody-mediated neutralization of flavivirus infection: implications for vaccine development the development of therapeutic antibodies against dengue virus antibodies to envelope glycoprotein of dengue virus during the natural course of infection are predominantly cross-reactive and recognize epitopes containing highly conserved residues at the fusion loop of domain ii antibodies induced by dengue virus type and envelope domain iii recombinant proteins in monkeys neutralize strains with different genotypes vaccines and immunization strategies for dengue prevention serotype-specificity of recombinant fusion proteins containing domain iii of dengue virus evaluation of the protective efficacy of a recombinant dengue envelope b domain fusion protein against dengue virus infection in mice short report: antibody responses of mice immunized with a tetravalent dengue recombinant protein subunit vaccine mice immunized with a dengue type virus e and ns fusion protein made in escherichia coli are protected against lethal dengue virus infection an in-depth analysis of original antigenic sin in dengue virus infection in-depth analysis of the antibody response of individuals exposed to primary dengue virus infection dengue: defining protective versus pathologic immunity dengue virus neutralization by human immune sera: role of envelope protein domain iii-reactive antibody a potent germline-like human monoclonal antibody targets a ph-sensitive epitope on h n influenza hemagglutinin exceptionally potent neutralization of middle east respiratory syndrome coronavirus by human monoclonal antibodies junctional and allele-specific residues are critical for mers-cov neutralization by an exceptionally potent germline-like antibody eradication of tumors through simultaneous ablation of cd /b -h -positive tumor cells and tumor vasculature cd -targeted car t cells induce remission in b-all that is naive or resistant to cd -targeted car immunotherapy human monoclonal antibodies as candidate therapeutics against emerging viruses highly efficient selection of epitope specific antibody through competitive yeast display library sorting a human monoclonal antibody against small envelope protein of hepatitis b virus with potent neutralization effect a bispecific antibody effectively neutralizes all four serotypes of dengue virus by simultaneous blocking virus attachment and fusion a broadly flavivirus cross-neutralizing monoclonal antibody that recognizes a novel epitope within the fusion loop of e protein therapeutic antibodies, vaccines and antibodyomes broad and potent hiv- neutralization by a human antibody that binds the gp -gp interface broad and potent neutralization of hiv- by a gp -specific human antibody structural basis for broad and potent neutralization of hiv- by antibody vrc human infection with a novel avian-origin influenza a (h n ) virus therapeutic treatment of nipah virus infection in nonhuman primates with a neutralizing human monoclonal antibody potent cross-reactive neutralization of sars coronavirus isolates by human monoclonal antibodies nonneutralizing antibodies induced by the hiv- gp nhr domain gain neutralizing activity in the presence of the hiv fusion inhibitor enfuvirtide: a potential therapeutic vaccine strategy a new class of highly potent, broadly neutralizing antibodies isolated from viremic patients infected with dengue virus swiss-model: modelling protein tertiary and quaternary structure using evolutionary information antibody-antigen interactions: contact analysis and binding site topography simplifying and enhancing the use of pymol with horizontal scripts we thank professor dane wittrup from mit for providing the yeast display vector and yeast strain. key: cord- -lhhjax s authors: pickering, suzanne; betancor, gilberto; galão, rui pedro; merrick, blair; signell, adrian w.; wilson, harry d.; kia ik, mark tan; seow, jeffrey; graham, carl; acors, sam; kouphou, neophytos; steel, kathryn j. a.; hemmings, oliver; patel, amita; nebbia, gaia; douthwaite, sam; o’connell, lorcan; luptak, jakub; mccoy, laura e.; brouwer, philip; van gils, marit j.; sanders, rogier w.; martinez nunez, rocio; bisnauthsing, karen; o’hara, geraldine; macmahon, eithne; batra, rahul; malim, michael h.; neil, stuart j. d.; doores, katie j.; edgeworth, jonathan d. title: comparative assessment of multiple covid- serological technologies supports continued evaluation of point-of-care lateral flow assays in hospital and community healthcare settings date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: lhhjax s there is a clear requirement for an accurate sars-cov- antibody test, both as a complement to existing diagnostic capabilities and for determining community seroprevalence. we therefore evaluated the performance of a variety of antibody testing technologies and their potential use as diagnostic tools. highly specific in-house elisas were developed for the detection of anti-spike (s), -receptor binding domain (rbd) and -nucleocapsid (n) antibodies and used for the cross-comparison of ten commercial serological assays—a chemiluminescence-based platform, two elisas and seven colloidal gold lateral flow immunoassays (lfias)—on an identical panel of sars-cov- -positive samples and pre-pandemic negatives. there was a wide variation in the performance of the different platforms, with specificity ranging from % to %, and overall sensitivity from . % to . %. however, the head-to-head comparison of multiple sero-diagnostic assays on identical sample sets revealed that performance is highly dependent on the time of sampling, with sensitivities of over % seen in several tests when assessing samples from more than days post onset of symptoms. furthermore, these analyses identified clear outlying samples that were negative in all tests, but were later shown to be from individuals with mildest disease presentation. rigorous comparison of antibody testing platforms will inform the deployment of point-of-care technologies in healthcare settings and their use in the monitoring of sars-cov- infections. a a a a a as of the st of june , over million cases of sars-cov- have been confirmed worldwide, accounting for more than , deaths (https://covid .who.int/). lack of treatments or vaccines have forced governments to adopt strict quarantine strategies in an attempt to control the spread of the virus, causing major economical disturbances as well as adversely affecting quality of life and healthcare provision. current guidelines by leading health bodies, including the centers for disease control and prevention (cdc) in the us and public health england (phe) in the uk, recommend sars-cov- diagnosis from upper or lower respiratory specimens (including nasopharyngeal swabs and bronchoalveolar lavage) using real-time rt-pcr, typically targeting the nucleocapsid (n) or rna-dependent rna polymerase (rdrp) genes [ , ] . these tests are highly sensitivecapable of detecting vestigial viral rna levels-and are optimal for the early detection of the virus. however, the performance of the test is dependent on the time the sample is collected, with viral load declining after the first week of symptoms [ , ] . there is, therefore, a clear requirement for accurate serology testing as a companion diagnostic to pcr-based testing. this is highlighted by the recent appearance of clusters of paediatric inflammatory multisystem syndrome (pims) and other hyperimmune reactions associated with sars-cov- infection [ ] [ ] [ ] . presentation is delayed relative to active viral infection, with the detection of antibody responses being key to clinical diagnosis. in addition, monitoring population seroprevalence will be central to future public health planning based on disease susceptibility and herd immunity [ ] . for this to be meaningful, it is imperative that antibody detection methods are affordable, reliable, and readily accessible. however, with an incomplete knowledge of the immunology of covid- , evaluating tests with the assumption that antibodies 'should' be there, and comparatively to rt-pcr, is problematic. head-to-head comparisons of multiple sero-diagnostic assays on identical samples therefore provides a robust assessment of individual assay performance. accordingly, we developed a highly specific semi-quantitative elisa for the detection of anti-spike (s), -s receptor binding domain (rbd) and -n antibodies, and used this to cross-evaluate ten commercial antibody tests (seven lateral flow immunoassays (lfias), one chemiluminescent assay and two elisas) on a collection of serum samples from confirmed rna positive patients, and pre-pandemic samples from march . our results demonstrate a wide variation in the performance of the different platforms, ranging from . % to . % sensitivity and from % to % specificity. as expected, performance is highly dependent on the time the sample was taken post onset of symptoms (pos) and disease severity. results obtained in this work have enabled the diagnostic-grade validation for one of the lfias evaluated for pilot clinical use for adult and paediatric patients with a range of clinically-suspected covid- inflammatory syndromes at guy's and st thomas' hospitals. serum samples collected from individuals between the th of march and st of april at st thomas' hospital were used to compare a panel of serological assays. at the time of study, uk government guidelines limited sars-cov- testing to individuals requiring hospitalisation, and all individuals had rt-pcr-confirmed sars-cov- infection. samples were representative of typical hospital admissions during the period, with a spectrum of clinical severities from mild (requiring no respiratory support) to critical (requiring extra-corporeal membrane oxygenation (ecmo)), and a range of time points after self-reported onset of symptoms ( to days) ( table ). in-house elisas were developed to measure antibody responses against the full-length s, the rbd and n. recombinant s and rbd were expressed in hek f cells and purified by affinity and size exclusion chromatography. n was expressed in and purified from e. coli. a total of pre-pandemic serum samples from several cohorts were used to determine the lower limit of the assay, including sera from individuals attending st thomas' hospital in march , sera from vaccination studies, cancer patients, healthy volunteers, and individuals with acute ebv infection (s table) . samples from hospital patients with confirmed sars-cov- infection (from the described serum samples in table ) were used as positive controls. all serum samples from pcr positive icu patients taken at least days post symptoms showed strong igg binding to s, rbd and n (fig , s table) . in contrast, although high igm reactivity was also observed to s and rbd in some individuals, only of the negative control samples showed igg reactivity to s or rbd (fig , s table) . high igm and igg reactivity was observed in the pre-pandemic samples against n (fig ) suggestive of potential cross-reaction with seasonal coronaviruses. of note, all individuals with acute ebv infection had high igm reactivity to n and rbd. importantly, none of the pre-covid sera had detectable igg binding to n and s or n and rbd. taking into account the reactivity of negative control samples in this elisa, % specificity could be reached using a cut-off where igg against n or s both have od values at least -fold above the wells containing secondary antibody only (fig b, s table) . all serological assays were evaluated with the same set of serum samples from confirmed sars-cov- -positive individuals. each of the samples was tested: for anti-sars-cov- igm by in-house elisa and seven lfias; for iga by commercial elisa (fig ) ; and for igg by inhouse elisa, seven lfias, a commercial elisa and for total antibody (igg, igm and iga) using a chemiluminescent assay (fig ) . the commercial elisas detect anti-s antibodies; for the lfias this is undisclosed proprietary information, but for some of the tests is known to be s. with no existing standardised diagnostic test for the assessment of the serological response to sars-cov- , we started by comparing commercial serological assays with the configuration of the in-house elisa most likely to represent antibodies detected by the commercial tests (detection of anti-s igm and igg antibodies), and that had high specificity and sensitivity (s table) . for the purpose of illustration, the intensity of bands shown in a positive lfia test, or signal strength in commercial elisa or chemiluminescent assay, is reproduced as a heatmap. however, the visible detection of a band or result above a given manufacturer's threshold scored as a positive result regardless of classification. for samples giving a strong response by comparison of serological assays for detection of sars-cov- antibodies comparison of serological assays for detection of sars-cov- antibodies elisa (> -fold), the majority of the commercial assays show a consensus positive result. weaker antibody responses yielded mixed results from the lfias, with a clear pattern of increasing detection of antibodies seen across all tests with increasing time pos. samples from days - gave an extremely mixed picture, indicative of an early evolving immune response. for the detection of igm, of the samples from days or more pos were negative by anti-s elisa. of these samples were positive, in some cases only weakly, for at least two lfias and/or iga. the remaining were negative in at least lfia tests and for iga (indicated with a yellow circle in fig ) . these same samples were negative for igg by in-house and commercial elisa, in all lfias and by chemiluminescent assay (indicated with a yellow circle in fig ) . importantly, all samples came from individuals with a disease severity score of . later time points were obtained for three of these individuals (both of the day samples and the sixth day sample) and the next available samples were found to be strongly positive for igg and igm by lfia (days , and pos, respectively). further investigation into the nature of the negative day sample revealed that it was an error in self-reporting the time of symptom onset: this individual had covid- -compatible symptoms for days prior to sampling, yet tested negative for rna days pos. days pos the individual tested positive for rna, therefore the more likely time of sampling was between and days pos. samples were not re-classified, as they are representative of the real-time analyses being performed during the peak of a pandemic. however, the day sample was omitted from sensitivity calculations. overall, with the exception of genbody and watmind, all tests gave cross-assay agreements between . and . % (fig and s fig) . the highest level of agreement was seen between the in-house elisa, surescreen, accu-tell, spring and euroimmun tests. interestingly, the in-house elisa igm and euroimmun iga results showed particularly good agreement ( . %), although the euroimmun detected iga more frequently in early samples compared with the igm detected by in-house elisa (fig ) . results from the sars-cov- -positive samples and an identical set of pre-pandemic negative samples were used to evaluate assay sensitivities and specificities, with extended specificity assessments using larger sample numbers performed on selected tests (fig a, s fig and s -s tables). cross comparison of overall specificities and sensitivities led to the shortlisting of six tests with the highest specificity and sensitivity (elisa igm and igg, surescreen, accu-tell, spring and euroimmun iga). these were the same tests that gave the best agreement in the cross-assay comparisons. notably, sensitivity increased for all tests with increasing days pos, with antibodies being variably detected at early times ( fig b) . deep blue, accu-tell, surescreen and spring also displayed the highest levels of sensitivity at less than days. in sum, deep blue, accu-tell, surescreen, spring, biohit, medomics, euroimmun (iga and igg) and in-house elisas (igm and igg) all had sensitivities above % for samples taken � days pos. sample-by-sample analyses of multiple serological assays showed a trend for increasing detection of antibodies with increasing days pos (up to ). we also observed that individuals with � . to < are positive, and � are strong positive. samples are grouped according to days post onset of covid- symptoms, and squares aligned in columns under each bar of the graph show results for a single serum sample. yellow circles indicate samples from days or more pos that were negative by elisa and in at least other tests, as detailed in the text. https://doi.org/ . /journal.ppat. .g comparison of serological assays for detection of sars-cov- antibodies comparison of serological assays for detection of sars-cov- antibodies severe disease had a more readily detectable antibody response, particularly compared to those who had a brief hospital stay with minimal intervention. samples were grouped according to days pos and clinical severity, and compared for anti-s igm and igg by in-house elisa. significant differences in antibody levels were observed with increases in both days and severity, although there was no association between days and severity themselves (fig a) . to assess the development of the antibody response in sequential samples from the same individual, and to evaluate the ability of lfias to detect nuances in antibody response, longitudinal samples (from individuals with disease severity scores of ) were tested by one of the best performing lfias (surescreen) and in-house elisa (fig b and c) . a similar pattern of detection was seen for both types of assay, with igm detectable earlier than igg, and both tests showing consensus for the strength and timing of the response. comparison of serological assays for detection of sars-cov- antibodies this study describes the development of six in-house elisa configurations for the detection of igm and igg against sars-cov- s, rbd and n. anti-s and -n igg attained specificities of up to %- % when results for both targets are combined-and sensitivities of up to %. we used this semi-quantitative platform to cross-evaluate seven lfias, a chemiluminescent test and two commercial elisas. importantly, the availability of sequential serum samples from patients admitted from the start of the outbreak under an existing ethics agreement for storage and analysis of surplus amounts of routinely collected clinical samples, enabled us to conduct this detailed study including examining assay sensitivity with respect to time pos. our analysis demonstrates a broad range of performance across the different platforms, with several commercial tests performing above % specificity. we found that all platforms showed highest sensitivity, with narrowest confidence limits, in samples taken days pos, with most tests reaching a value of over % (fig b) . when all commercial tests were compared, accu-tell, surescreen and spring demonstrated highest sensitivity at earlier time points, while maintaining specificities of % or above. these tests also gave the best comparison of serological assays for detection of sars-cov- antibodies comparison of serological assays for detection of sars-cov- antibodies cross-assay agreements with each other and with the in-house elisa. in the best-performing tests, we also observed that signal strength aligned with that seen by elisa; this was further supported by the sequential signal increase seen in longitudinal samples from five individuals. we approached this study with the intention that an unbiased and transparent comparison will be of broad value to the scientific and infection diagnostics communities, both in terms of naming and comparing the kits, and in the nature of the samples likely to be encountered in hospitals during a sars-cov- outbreak. few studies have been published to date in which multiple lfias and elisas have been evaluated side-by-side with named kits [ ] [ ] ; and in those that have, only one test overlaps with our study, deep blue [ ] . early reports stating that lfias have insufficient sensitivity may have been due to testing samples from mixed time points and disease severities, or tests that differ from those evaluated here [ ] . the strength of our study is the head-to-head evaluation of multiple tests on identical serum samples. the sample set was not compiled retrospectively for the purpose of evaluation, but was part of an ongoing process to deploy a serological assay to broaden diagnostic capability at guy's and st thomas' hospitals during the peak of the sars-cov- epidemic in london. these samples are therefore entirely representative of the type that will be encountered by hospital laboratories, and the challenges associated with variable time to seroconversion and errors in self-reporting onset of symptoms are real. the cross-evaluation of multiple tests enabled the identification of samples that were negative in every test performed, despite the fact that samples were taken at days pos or later. while this could highlight a characteristic of covid- where a subset of patients do not produce a detectable antibody response, we have evidence that in three cases (the two day samples and sixth day ) where later samples were available ( , and days pos, respectively; the only next available samples) both igm and igg antibodies were detected in several lfias. in another case (sample at day pos), we uncovered a likely error in self-reported symptom onset and the sample could actually range anywhere between and days pos. it is important to note that all of the six unexpectedly negative samples were from covid- cases classified as severity level . the fact that several post-day samples were negative in all serological tests has practical implications for the use of such assays in diagnostic settings, and thought should be given to the meaning of a negative result. it also implies that serological surveys are likely to underestimate the level of exposure to sars-cov- , and the wide variation in the detection of antibodies, both in terms of time and disease severity, casts into doubt the utility of "immunity passports". however, although it is unclear at present whether detection of antibodies to sars-cov- indicates protection against future infection, measurement of antibodies to s, rather than n, is likely to better predict neutralisation function. in agreement with previous studies demonstrating a relationship between disease severity and antibody titres [ ] [ ] [ ] , we observed a significant increase in detection of antibodies with increased severity of symptoms ( fig a) . importantly, this correlation is not explained by a concomitant increase in the days pos of the sample. therefore, before deployment in situations where the pre-test prevalence is likely to be low, such as seroprevalence studies, outpatient assessment or pre-admission screening for operations, these assays will require further evaluation with known sars-cov- asymptomatic and ambulatory cases, alongside an extended set of pre-pandemic samples. this is a priority as countries navigate their way out of lockdown and move towards living with the ongoing threat of sars-cov- , and seroprevalence studies will be important in the implementation and management of safe public health policies. to maintain the high specificity of our in-house elisa in community cohorts where pcr status is unknown, we would recommend determining seropositivity based on igg to both n and s. sequential or alternate detection of igm, iga and igg may also provide information on the history of infection. in the only iga test that we evaluated (euroimmun), comparison of serological assays for detection of sars-cov- antibodies specificity was high while also showing a strong signal in early samples and a good overall sensitivity. detection of iga in serum, and potentially even earlier by mucosal sampling, may be a useful diagnostic tool. next generation antibody tests may improve on those currently being trialled, but our results demonstrate that lfias may have utility in a hospital setting as of now, particularly if deployed where a rapid result could aide a clinical pathway or decision in real time, such as ward location or prioritisation of further diagnostics and follow up. the ease of use and affordability of the lfias weighs heavily in their favour, especially for potential in resourcepoor settings or as point-of-care solutions in hospitals. choosing the tests with the highest specificity will translate to confidence in a positive test result in the clinic; negative or borderline cases may be tested serially or used together with elisa testing [ ] . combination testing, in parallel with rt-pcr, and serial or sequential testing, would provide diagnostic solutions to the delayed-onset syndromes such as pims that are increasingly being reported post-peak pandemic [ ] [ ] [ ] . further evaluations of candidate tests should be performed prior to use in clinical settings, ideally tailored to the intended usage and likely pre-test prevalence. a limitation of our study was the restricted number of pre-pandemic negative and confounding samples used to cross-validate the commercial kits, and further specificity and sensitivity studies with a shortlisted group of commercial tests are currently in progress. it will be particularly important to evaluate samples from individuals infected with other human coronaviruses or respiratory viruses for potential cross-reacting antibodies. a further consideration that should be given for healthcare service deployment in the hospital or community setting is consistency of use. although the lfias are marketed as home testing kits, in our experience user assiduity is essential to their optimal performance, particularly when scoring borderline cases and considering need for two independent readers. there will also likely be need to evaluate alternative sources of blood collection, particularly pin-prick collection, and even different samples such as saliva, all of which should be fully evaluated before deployment. in summary, our study compares the performance of commercially available platforms and several combinations of in-house methods for the detection of anti-sars-cov- antibodies in serum samples. although lfias lack the semi-quantitative information provided by elisa tests, they have a clear utility advantage over elisa or chemiluminescence-based technologies. shortlisted tests, combined with confirmatory reflex testing using our in-house elisa, are now being taken forward into extended validations as part of a pilot clinical service at guy's and st thomas' hospitals. these incremental steps keeping different technologies in scope, whilst multiple different use-cases are still being defined, will help determine the clinical utility and cost-effectiveness of covid- serological testing in healthcare settings both in the hospital and the community. for the st thomas' hospital samples, surplus serum was retrieved from the routine biochemistry laboratory at point of discard, and then aliquoted, stored and linked with a limited clinical dataset by the direct care team, before anonymisation under an existing ethics framework (rec reference /nw/ ) and with expedited r&d approval. serum/plasma samples used as negative controls in the in-house elisa development were obtained from the kcl infec comparison of serological assays for detection of sars-cov- antibodies patient overview and sample origin individual venous serum samples collected at st thomas' hospital, london from patients diagnosed as sars-cov- positive via real-time rt-pcr, were obtained for serological analysis. samples ranged from to days after onset of self-reported symptoms. for the longitudinal study serum samples ( - days after symptoms onset) were obtained from patients ( - samples each) with confirmed covid- diagnosis. two patients overlapped between the longitudinal study and validation study meaning in total there are unique patients between both studies. patient information is given in table . patients diagnosed with covid- were classified as follows: -asymptomatic or no requirement for supplemental oxygen. -requirement for supplemental oxygen (fio < . ) for at least hrs. -requirement for supplemental oxygen (fio � . ) for at least hrs. -requirement for non-invasive ventilation (niv)/ continuous positive airways pressure (cpap) or proning or supplemental oxygen (fio > . ) for at least hrs and not a candidate for escalation above level care. -requirement for intubation and mechanical ventilation or supplemental oxygen (fio > . ) and peripheral oxygen saturations < % (with no history of type respiratory failure (t rf)) or < % (with known t rf) for at least hrs. -requirement for ecmo. all sera/plasma was heat-inactivated at ˚c for mins before use in the in-house elisa. high-binding elisa plates (corning, ) were coated with antigen (n, s or rbd) at μg/ ml ( μl per well) in pbs, either overnight at ˚c or hr at ˚c. wells were washed with pbs-t (pbs with . % tween- ) and then blocked with μl % milk in pbs-t for hr at room temperature. wells were emptied and sera and plasma diluted at : and : respectively in milk were added and incubated for hr at room temperature. control reagents included cr ( μg/ml), cr ( . μg/ml), negative control plasma ( : dilution), positive control plasma ( : ) and blank wells. wells were washed with pbs-t. secondary antibody was added and incubated for hr at room temperature. igm was detected using goatanti-human-igm-hrp ( : , ) (sigma: a ) and igg was detected using goat-anti- comparison of serological assays for detection of sars-cov- antibodies human-fc-ap ( : , ) (jackson: - - -jir). wells were washed with pbs-t and either ap substrate (sigma) was added and read at nm (ap) or -step tmb substrate (thermo scientific) was added and quenched with . m h s before reading at nm (hrp). n protein was obtained from the james lab at lmb, cambridge. the n protein used is a truncated construct of the sars-cov- n protein comprising residues - (both ordered domains with the native linker) with an n terminal uncleavable hexahistidine tag. n was expressed in e. coli using autoinducing media for h at ˚c and purified using immobilised metal affinity chromatography (imac), size exclusion and heparin chromatography. s protein consists of a pre-fusion s ectodomain residues - with proline substitutions at amino acid positions and , a gggg substitution at the furin cleavage site (amino acids - ) and an n terminal t trimerisation domain followed by a strep-tag ii [ ] . the protein was expressed in l hek- f cells (invitrogen) grown in suspension at a density of . million cells/ml. the culture was transfected with μg of dna using pei-max ( mg/ml, polysciences) at a : ratio. supernatant was harvested after days and purified using streptactinxt superflow high capacity % suspension according to the manufacturer's protocol by gravity flow (iba life sciences). the rbd plasmid was obtained from florian krammer at mount sinai university [ ] . here the natural n-terminal signal peptide of s is fused to the rbd sequence ( to ) and joined to a c-terminal hexahistidine tag. this protein was expressed in ml hek- f cells (invitrogen) at a density of . million cells/ml. the culture was transfected with μg of dna using pei-max ( mg/ml, polysciences) at a : ratio. supernatant was harvested after days and purified using ni-nta agarose beads. we tested seven point-of-care colloidal-gold-based lfias detecting igg and igm antibodies against sars-cov- , full details of which are shown in table . with the exception of medomics, all lfias were ce ivd marked. target antigens were undisclosed proprietary information, but for several of them were known to be s. lfias were run according to manufacturer's instructions. typically, - μl of serum was added to the lfia membrane start point, followed by - drops of supplied buffer. kits were run at room temperature for minutes and then immediately scored using a -point scale (negative, borderline, positive, strong positive) for both igm and igg. scoring was performed independently by two individuals. comparison of serological assays for detection of sars-cov- antibodies the sars-cov- ab diagnostic test kit (shenzen watmind medical co., ltd.) detecting total antibody against sars-cov- was run on the chemical luminescence immunity analyzer mf (shenzen watmind medical co., ltd). the platform was calibrated with a supplied control cartridge daily prior to testing. panel samples were analysed according to manufacturer's instructions. results equal to and below . au (arbitrary units) /ml were negative, scores above . au/ml were deemed positive. for comparison to other immunoassays, scores between < au/ml were deemed positive, scores > au/ml were deemed a strong positive. expected binomial exact % confidence intervals were calculated on prism . using wilson/ brown statistical analysis. results for each test were either categorised according to whether the serum sample was from < , � , � , or � days pos, or severity of illness, with indicating mild illness (requiring no respiratory support) and indicating critical (requiring ecmo) (see materials and methods for full classification). % confidence intervals are shown for each assay in all panels (wilson/brown expected binomial). (tif) s table. specificity and sensitivity of in-house elisas during development phase. specificity and sensitivity (%) were determined for each configuration of the in-house elisa (detection of igm and igg to n, s and rbd) during initial development. pre-pandemic samples from several cohorts were used as negative controls for specificity calculations, and rt-pcr-confirmed sars-cov- positive samples were used as positive controls for sensitivity calculations. % cis are shown for each calculation. (docx) nhs. guidance and standard operating procedure covid- virus testing in nhs laboratories centers for disease control and prevention. interim guidelines for collecting, handling, and testing clinical specimens for covid- temporal profiles of viral load in posterior oropharyngeal saliva samples and serum antibody responses during infection by sars-cov- : an observational cohort study sars-cov- viral load in upper respiratory specimens of infected patients an outbreak of severe kawasaki-like disease at the italian epicentre of the sars-cov- epidemic: an observational cohort study european centre for dieases prevention and control. paediatric inflammatory multisystem syndrome and sars-cov- infection in children clinical characteristics of children with a pediatric inflammatory multisystem syndrome temporally associated with sars-cov- herd immunity: understanding covid- test performance evaluation of sars-cov- serological assays evaluation of nine commercial sars-cov- immunoassays antibody testing for covid- : a report from the national covid scientific advisory panel antibody responses to sars-cov- in patients of novel coronavirus disease viral kinetics and antibody responses in patients with antibody responses to sars-cov- in patients with covid- recommendations for verification and validation methodology and sample sets for evaluation of assays for sars-cov- (covid- adjuvented influenza-h n vaccination reveals lymphoid signatures of age-dependent early responses and of clinical adverse events potent neutralizing antibodies from covid- patients define multiple targets of vulnerability a serological assay to detect sars-cov- seroconversion in humans thank you to dr terry wong for his support in acquiring test kits. thank you to surescreen diagnostics for their engagement and technical assistance.thank you to bindi patel, nicola varatharajah, abayomi fatola and amelia moore for laboratory assistance.thank you to florian krammer for provision of the rbd expression plasmid, and leo james and leo kiss for the provision of purified n protein.we thank king's college london infectious diseases biobank for provision of pre-covid- vaccine samples, all patients and control volunteers who participated in this study and to all clinical staff who helped with recruitment, including those working with the tapb project at the royal free hospital. we thank the maini lab at the division of infection and immunity for providing pre-covid pandemic healthy control samples.we are extremely grateful to all patients and staff at st thomas' hospital who participated in this study. comparison of serological assays for detection of sars-cov- antibodies comparison of serological assays for detection of sars-cov- antibodies key: cord- -njvalb authors: lau, susanna k. p.; woo, patrick c. y.; li, kenneth s. m.; zhang, hao-ji; fan, rachel y. y.; zhang, anna j. x.; chan, brandon c. c.; lam, carol s. f.; yip, cyril c. y.; yuen, ming-chi; chan, kwok-hung; chen, zhi-wei; yuen, kwok-yung title: identification of novel rosavirus species that infects diverse rodent species and causes multisystemic dissemination in mouse model date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: njvalb while novel picornaviruses are being discovered in rodents, their host range and pathogenicity are largely unknown. we identified two novel picornaviruses, rosavirus b from the street rat, norway rat, and rosavirus c from five different wild rat species (chestnut spiny rat, greater bandicoot rat, indochinese forest rat, roof rat and coxing's white-bellied rat) in china. analysis of complete genome sequences showed that “rosavirus b” and “rosavirus c” represent two potentially novel picornavirus species infecting different rodents. though being most closely related to rosavirus a, rosavirus b and c possessed distinct protease cleavage sites and variations in yn-xm-aug sequence in ’utr and myristylation site in vp . anti-rosavirus b vp antibodies were detected in norway rats, whereas anti-rosavirus c vp and neutralizing antibodies were detected in indochinese forest rats and coxing's white-bellied rats. while the highest prevalence was observed in coxing's white-bellied rats by rt-pcr, the detection of rosavirus c from different rat species suggests potential interspecies transmission. rosavirus c isolated from t cells causes multisystemic diseases in a mouse model, with high viral loads and positive viral antigen expression in organs of infected mice after oral or intracerebral inoculation. histological examination revealed alveolar fluid exudation, interstitial infiltration, alveolar fluid exudate and wall thickening in lungs, and hepatocyte degeneration and lymphocytic/monocytic inflammatory infiltrates with giant cell formation in liver sections of sacrificed mice. since rosavirus a has been detected in fecal samples of children, further studies should elucidate the pathogenicity and emergence potential of different rosaviruses. picornaviruses are positive-sense, single-stranded rna viruses with icosahedral capsids. they infect various animals and human, causing various respiratory, cardiac, hepatic, neurological, mucocutaneous and systemic diseases [ , ] . based on genotypic and serological characterization, the family picornaviridae is currently divided into genera with at least species. among the various picornaviruses belonging to nine genera that are able to infect humans, poliovirus and human enterovirus a are best known for their neurotropism and ability to cause mass epidemics with high morbidities and mortalities [ , ] . picornaviruses are also known for their potential for mutations and recombination, which may allow the generation of new variants to emerge [ ] [ ] [ ] [ ] [ ] [ ] . emerging infectious diseases like avian influenza and coronaviruses have highlighted the impact of animal viruses after overcoming the inter-species barrier [ ] [ ] [ ] [ ] [ ] . as a result, there has been growing interest to understand the diversity and evolution of animal and zoonotic viruses. for picornaviruses, numerous novel human and animal picornaviruses have been discovered in the past decade [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . we have also discovered a novel picornavirus, canine picodicistrovirus (cpdv), with two internal ribosome entry site (ires) elements, which represents a unique feature among picornaviridae [ ] . moreover, novel picronaviruses were identified in previously unknown animal hosts such as cats, bats and camels [ ] [ ] [ ] , reflecting our slim knowledge on the diversity and host range of picornaviruses. the discovery and characterization of novel picornaviruses is important for better understanding of their evolution, pathogenicity and emergence potential. although rodents can be infected by several picornaviruses, the picornaviral diversity is probably underestimated, given the enormous species diversity of rodents. moreover, little is known about the pathogenicity of the recently discovered rodent pricornaviruses, such as rodent stool-associated picornavirus (rosavirus) a , mouse stool-associated picornavirus (mosavirus) a , norway rat hunnivirus and rat-borne virus (rabovirus a) [ , ] . in this report, we explored the diversity of picornaviruses among rodents in china and discovered two potentially novel picornaviruses, "rosavirus b" and "rosavirus c". while rosavirus b was detected in the street rat, norway rats, rosavirus c was detected in five different wild rat species, suggesting potential interspecies transmission. their complete genome sequences were determined, which showed that "rosavirus b" and "rosavirus c" represent two novel picornavirus species distinct from rosavirus a. rosavirus c isolated from cell culture causes multisystemic diseases in a mouse model, with histopathological changes and positive viral antigen expression in lungs and liver of infected mice. a total of complete genomes from samples of four different wild rodent species (chestnut spiny rat, coxing's white-bellied rat, roof rat and indochinese forest rat) positive for "rosavirus c" and one street rodent species (norway rat) positive for "rosavirus b" were sequenced directly from the positive respiratory or alimentary samples and characterized. these strains were selected because they were detected from different rodent species or geographical locations (hong kong, hunan and guangxi) to allow comparison between host species-or geographically distinct strains. the g + c contents of the three rosavirus b and rosavirus c genomes range from to %, with genome size to bases, after excluding the polyadenylated tract (table ) . however, the genome sizes of some strains may be larger, as further sequencing of the ends may have been hampered by secondary structures. they share similar genome organization typical of picornaviruses, with utr at both ' ( - bases) and ' ( - bases) ends, and a large open reading frame of - bases, which encodes potential polyprotein precursors of - aa known to be cleaved by virus-encoded proteases. the predicted protease cleavage sites at p (encoding capsid proteins) p and p (both encoding non-structural proteins) are shown in table . notably, "rosavirus b" differed from phylogenetic trees constructed using the aa sequences of p , p (excluding a) and p (excluding a) of rosavirus b and c are shown in fig and the corresponding pairwise aa identities are shown in table . a and a regions were excluded to avoid bias due to poor sequence comparison of genome features of rosavirus b and c to those of rosavirus a is summarized in table . the conserved sequence yn-xm-aug is present in the 'utr of rosavirus b and c. while y -x -aug is found in rosavirus a, the number of y ( or ) and x ( - ) varies among rosavirus b and c. the putative translation initiation sites were contained by an optimal kozak context (rnnaugg), with in frame aug at position to . the 'utr of many picornaviruses possesses an internal ribosomal entry site/segment (ires) which is responsible for directing the initiation of translation in a cap-independent manner, and requires both canonical translation initiation and ires trans-acting factors [ , ] . similar to rosavirus a [ ] , rosavirus b and c also contained a type ii-like ires with stem loops, major domains [ , , [ ] [ ] [ ] [ ] [ ] [ ] and conserved motifs (fig ) . however, domain e was only present in rosavirus c strains, rask f, ratlc a, nfsm f and rasm a, and rosavirus b strain rnyl r, but not other strains. the pyrimidine-rich region was located near ' end of 'utr. one to three stem-loop structures were present upstream of the start codon and/or between the pyrimidine-rich region and start codon of the polyprotein [ , ] . the predicted "vp " of rosavirus b and c are probably cleaved into vp and vp based on sequence alignment [ ] . in contrast to rosavirus a of which the vp possessed the myristylation site, gxxx [st] , involved in capsid assembly or virus entry [ ] , such myristylation site is absent table . coding potential and putative proteins of "rosavirus b" and "rosavirus c" compared to rosavirus a. in rosavirus c and variably present in rosavirus b (present in strains rncw r and rncw r but absent in strain rnyl r). the predicted a of rosavirus b and c exhibited . % aa identities to that of rosavirus a, possessed the conserved h-box/nc involved in cell proliferation control, but not asn-pro-gly-pro (npgp) motifs [ , ] . their predicted c possessed gxxgxgks motif for ntp-binding [ ] and ddlxq motif for putative helicase activity [ ] . their predicted c pro contained the catalytic triad h-d-c [ ] , conserved gxcg motif in the protease active site and gxh motif [ , ] . their predicted d pol contained conserved kde[li]r, gg[lmn]psg, ygdd and flkr motifs [ ] , although the second gly was replaced by ala in gg[lmn]psg. although rosavirus c were detected in five different rodent species from hong kong, hunan and guangxi, no major distinct genome features were identified between strains from different rodent species or geographical locations. yet, the two strains from coxing's white-bellied rat from hunan (nchn io) and guangxi (ncgx in), were always clustered together in the p , p and p trees, suggesting that geographically distinct strains may be genetically closely related (fig ) . these two strains from mainland china possessed a total of unique nucleotide substitutions over the entire genomes compared to the other eight rosavirus c strains from hong kong. viral sequences belonging to "rosavirus b" were only detected from the street rodent species, norway rat (rattus norvegicus), whereas sequences belonging to "rosavirus c" were detected from five different wild rodent species, greater bandicoot rat (bandicota indica), chestnut spiny rat (niviventer fulvescens), roof rat (rattus rattus) and indochinese forest rat (rattus andamanensis) from hong kong, and using available rosavirus b and c genome sequences for analysis, the ka/ks ratios for various coding regions were estimated ( table ). the ka/ks ratios for most coding regions were low, supporting purifying selection. of the various cell lines inoculated with the rodent samples positive for rosavirus b (three samples) or rosavirus c (eight samples), viral replication was detected by rt-pcr in the lysates of t cells infected by rosavirus c strain rasm a, with viral load of . × copies/ml ( . × tcid ) at day . cytopathic effect (cpe), mainly in the form of rounded and refractile cells rapidly detaching from the monolayer, was also observed in infected t cells five days after inoculation, which showed viral vp expression by immunofluorescence in % of cells (fig ) . electron microscopy of ultracentrifuged cell culture extracts from infected t cells showed the presence of non-enveloped viral particles of around - nm in diameter compatible with those described for members of the family of picornaviridae (fig ) . to determine the seroprevalence of rosavirus b and c among different rodent species, western blot analysis was performed on available rodent serum samples to test for specific antibodies against rosavirus b or c recombinant vp protein. the purity of the recombinant vp proteins was confirmed by the dominant band observed at the predicted size of kda upon sds polyacrylamide gel electrophoresis. anti-rosavirus b antibodies were detected in two ( . %) of norway rats from hong kong whereas anti-rosavirus c antibodies were detected in three ( . %) of indochinese forest rats from hong kong and three ( . %) of coxing's whitebellied rats from hunan province. however, the antibodies from norway rats against rosavirus b were likely of low levels, as reflected by the relatively weak band observed (table and fig ) . using sera with anti-rosavirus b antibodies against rosavirus c recombinant vp protein and novel rosavirus in rodents sera with anti-rosavirus c antibodies against rosavirus b recombinant vp protein, no cross reactivities were observed between the two proteins. neutralization assays showed that five of the six rats with anti-rosavirus c antibodies by western blot analyses were positive for neutralizing antibodies against rosavirus c rasm a with titer : to : . we attempted to study the pathogenicity in mice challenged with rosavirus c rasm a isolated from infected t cells. to mimick the fecal-oral route of transmission typical of many picornavirus infections, oral inoculation of rosavirus c rasm a was performed on fourday-old suckling mice. one of the suckling mice was eaten by its mother on day one post-challenge. all the remaining suckling mice survived after viral challenge till sacrifice, but some mice exhibit transient roughening of hair two to three days after challenge. among the nine mice sacrificed on day post-challenge, rosavirus c rasm a was detected in the intestine and lung of all nine mice, kidney of one mouse, and spleen and liver of three and four mice respectively by rt-pcr. among the five mice sacrificed on day post-challenge, rosavirus c rasm a was detected in the intestine of all five mice, liver and lung of four mice, and spleen and kidney of two and one mice respectively by rt-pcr. among the three mice sacrificed on day post-challenge, rosavirus c rasm a was detected in the lung of one mouse by rt-pcr. among the three mice sacrificed on day post-challenge, rosavirus c rasm a was detected in the lung of one mouse by rt-pcr. anti-rosavirus c vp antibody was detected in none of the mice sacrificed on day and , two of the five mice sacrificed on day , and one of the three mice sacrificed on day by western blot assay (table ). qrt-pcr of tissues positive by rt-pcr showed high levels of mean viral rna copies in lung ( . × copies/g) and intestine ( . × copies/g) tissues of mice sacrificed on day (fig ) . since some picornaviruses have been associated with neurovirulence, intracerebral inoculation was also performed on another group of one-day old suckling mice. one of the suckling mice was eaten by its mother on day one post-challenge. all the remaining suckling mice survived after viral challenge till sacrifice. among the nine mice sacrificed on day post- novel rosavirus in rodents challenge, rosavirus c rasm a was detected in the lung, liver, brain and spleen of eight mice, and intestine and kidney of four mice by rt-pcr. among the five mice sacrificed on day post-challenge, rosavirus c rasm a was detected in the lung of three mice, brain and liver of five mice, intestine of two mice, and spleen and kidney of four mice by rt-pcr. among the three mice sacrificed on day post-challenge, rosavirus c rasm a was detected in the brain, intestine, liver and lung of one mouse by rt-pcr. among the three mice sacrificed on day post-challenge, rosavirus c rasm a was detected in the lung of one mouse by rt-pcr. anti-rosavirus c vp antibody was detected in none of mice sacrificed on day , four of the five mice sacrificed on day , and two of three mice sacrificed on day and by western blot assay (table ). qrt-pcr of tissues positive by rt-pcr showed high levels of mean viral rna copies in various tissues ( . to . × copies/g) of mice sacrificed on day (fig ) . histological examination of various organs revealed alveolar fluid exudation, interstitial infiltration, alveolar fluid exudate and wall thickening in lung sections of mice sacrificed on day after oral or intracerebral inoculation. moreover, hepatocyte degeneration and lymphocytic/monocytic inflammatory infiltrates with giant cell formation were observed in liver sections of mice sacrificed on day after oral or intracerebral inoculation (fig ) . immunohistochemical staining with guinea pig anti-serum against rosavirus c vp protein antibody revealed viral antigen expression in bronchiolar and bronchial epithelial cells in lung sections, and hepatocytes in liver sections (fig ) . we report the discovery of two novel rodent picornaviruses, rosavirus b and c, from six rodent species in southern china. though being phylogenetically most closely related to rosavirus a, "rosavirus b" and "rosavirus c" should represent two novel species distinct from rosavirus a under the genus rosavirus, according to the criteria for international committee on taxonomy of viruses species demarcation for different members of picornaviridae [ ] . rosavirus b and c also exhibited different genome features when compared to rosavirus a. notably, the absence of myristylation site in vp of rosavirus c and its varying presence in rosavirus b is intriguing. the vp myristylation site has been shown to play a role in localization of the capsid protein table . rt-pcr and western blot analysis of mice challenged with rosavirus c rasm a. day at which mice were sacrificed (total no. of mice) for cellular entry and permeability [ , ] . its absence in some rosavirus strains suggests that rosaviruses may utilize alternative strategies for capsid localization on cellular targets. in addition, the variations in yn-xm-aug sequence in 'utr may also suggest different translational dynamics among rosaviruses. besides phylogenetic and genomic evidence, the two viruses infect different host, with "rosavirus b" infecting norway rats (a street rat) and "rosavirus c" known to infect different rodents [ , , ] . rosavirus a was first discovered in a wild canyon mouse (peromyscus crinintus) in california [ ] . subsequently, a variant, rosavirus , was detected in the fecal specimens of children in the gambia [ ] , which prompted further studies to investigate potential transmission of rosavirus from rodents to humans. in this study, the observed low ka/ks ratios in various coding regions supported that norway rats (street rats) in hong kong, and wild rats across hong kong and mainland china, are natural reservoirs of rosavirus b and c respectively. in particular, coxing's white-bellied rats appeared to be an important host for rosavirus c. the relatively low seroprevalence of rosavirus c, as compared to rt-pcr detection rate, among tested coxing's white-bellied rats ( . %) may be due to delayed antibody response during acute infection when the animals were still shedding viruses. together with the ability of rosavirus c in infecting house mouse (mus musculus), our findings provided evidence for the interspecies transmission potential of rosavirus c among different rodent species. this is in line with the ability of bat picornaviruses group to in infecting bats of different genera or species [ ] . encephalomyocarditis virus (emcv), of which swine is the major reservoir, can also infect different animals including rodents, elephants, boars, macaques and humans [ ] . further investigations are warranted to elucidate the ability of rosaviruses to cross species barrier and emerge in other animals or human. the present results suggest that rosaviruses can be pathogenic to their hosts. although rosavirus a has been detected in rodents and human previously [ , ] , no virus isolate was available for pathogenicity studies. a few picornaviruses, such as emcv (cardiovirus a) and parechovirus, were also known to cause systemic infections in infected rodents. in particular, theiler's murine encephalomyelitis virus (tmev), which primarily causes asymptomatic enteric infections in mice, has been intensively studied because of its ability to cause myocarditis, type diabetes and acute or persistent demyelinating infections mimicking multiple sclerosis [ , ] . on the other hand, rodents experimentally infected with emcv may develop type diabetes mellitus, encephalomyelitis, myocarditis, orchitis and sialodacryoadenitis [ ] . interestingly, lv, which may cause type diabetes and fetal deaths in infected rodents, has been recently found in human intrauterine fetal death and sudden infant death syndrome [ ] [ ] [ ] [ ] . however, the pathogenicity of other rodent picornaviruses, such as rosavirus a , mosavirus a , murine kobuvirus , norway rat hunnivirus and rabovirus a, was less clear [ , ] . the ability of rosavirus c in causing multisystemic infection in mice with high viral loads in infected organs suggested that rosaviruses may cause severe infections in their host. further experimental studies using other rosaviruses and rodent species may help to better understand the pathogenicity of members of the genus rosavirus in different rodents and human. rodents are the largest order of mammals on earth, accounting for % of the approximately , living mammalian species. they are widely distributed, being found in all habitats except the oceans. the order, rodentia, with around species under families, is further classified into five suborders: anomaluromorpha, castorimorpha, hystricomorpha, myomorpha and sciuromorpha. [ , ] . viruses of at least families, including adenoviridae, arenaviridae, arteriviridae, astroviridae, bunyaviridae, caliciviridae, circoviridae, coronaviridae, flaviviridae, hepadnaviridae, hepeviridae, herpesviridae, papillomaviridae, paramyxoviridae, parvoviridae, picobirnaviridae, polyomaviridae, reoviridae, rhabdoviridae, togaviridae, picornaviridae and poxviridae, are known to infect rodents [ , ] . rodent pathogens may infect human either by direct contact such as bites and inhalation of aerosolized animal excreta, or indirectly through vectors such as ticks and fleas. urban rodents may pose particular risk to human health, as in the case of hantavirus and lymphocytic choriomeningitis virus infections. more epidemiological studies should be performed to explore the diversity of rodent picornaviruses and their potential risks to human. a total of wild and street rodents, belonging to eight different species, were captured from various locations in both rural and urban areas of hong kong, hunan province and guangxi province of china over a five-year period (september to august ) ( table ) . samples from hong kong were provided by the agriculture, fisheries and conservation department (afcd) and food, environment and hygiene department (fehd), the government of the hong kong special administrative region (hksar), as part of a surveillance program on local rodents. all rodents were individually trapped and samples were collected from each rodent using procedures described previously [ , ] . to prevent cross contamination, collection of samples were performed using disposable swabs with protective gloves changed for each rodent. wild rodents in rural areas of hong kong were released back to nature after sample collection. samples from street rodents in urban areas of hong kong and rodents from china were collected immediately after euthanasia as routine policies for disposal of captured rodents. all samples were placed in viral transport medium (earle's balanced salt solution, . % glucose, . % sodium bicarbonate, . % bovine serum albumin, mg/ml amikacin, mg/ml vancomycin, u/ml nystatin) to inhibit bacterial and fungal overgrowth, and stored at - °c before rna extraction. viral rna was directly extracted from the respiratory and alimentary samples in viral transport medium using viral rna mini kit (qiagen, hilden, germany). the rna was eluted in μl of rnase-free water and was used as the template for rt-pcr. rt-pcr of d pol gene of picornaviruses using conserved primers and dna sequencing initial picornavirus screening was performed by amplifying a -bp fragment of the d pol gene of picornaviruses by rt-pcr using conserved primers ( '-ggcggytngayggygcs atgccgt- ' and '-ccgaccarcacrtcrtcrccrta- ') and previously described protocols [ , , ] . the primers were designed by multiple alignment of the nucleotide sequences of the d pol genes of all known picornaviruses, based on the conserved d pol motifs, gg[lmn]psg and ygdd. all samples positive by rt-pcr were confirmed by sequencing. briefly, reverse transcription was performed using the superscript iii kit (invitrogen, san diego, ca, usa) and the reaction mixture ( μl) contained rna, first-strand buffer ( mm tris-hcl ph . , mm kcl, mm mgcl ), mm dtt, ng random hexamers, μm of each dntps and u superscript iii reverse transcriptase. the mixtures were incubated at °c for min, followed by °c for min and °c for min. the pcr mixture ( μl) contained cdna, pcr buffer ( mm tris-hcl ph . , mm kcl, mm mgcl and . % gelatin), μm of each dntps and . u taq polymerase (applied biosystem, foster city, ca, usa). the mixtures were amplified in cycles of °c for min, °c for min and °c for min and a final extension at °c for min in an automated thermal cycler (applied biosystem, foster city, ca, usa). standard precautions were taken to avoid pcr contamination and no false-positive was observed in negative controls. all pcr products were gel-purified using the qiaquick gel extraction kit (qiagen, hilden, germany). both strands of the pcr products were sequenced twice with an abi prism xl dna analyzer (applied biosystems, foster city, ca, usa), using the two pcr primers. the sequences of the pcr products were compared with known sequences of the d pol genes of picornaviruses in the genbank database. rt-pcr of d pol gene of novel rodent picornaviruses using specific primers and dna sequencing as initial rt-pcr of the d pol gene revealed at least two potential novel picornavirus species in respiratory and alimentary tract samples, all the respiratory and alimentary tract samples were re-tested using specific rt-pcr assays to enhance the sensitivities for detection of these novel picornaviruses. primers were designed by multiple alignment of the d pol gene sequences obtained during genome sequencing from the initial positive samples. the pcr assays were targeted to a bp ( '-atgctcctgttctcatgctttt - ' and '-gaaaa tctgggtcaggggtgaa - ') fragment and a -bp ( '-tgttctcttgyttytcccag at - ' and '-aaytgcgggtcyggdgtgaa - ') fragment of the d pol gene of the potential novel picornaviruses. the components of the pcr mixtures and the cycling conditions were the same as those described above. purification of the pcr products and dna sequencing were performed as described above, using the corresponding pcr primers. the sequences of the pcr products were compared with known sequences of the d pol genes of picornaviruses in the genbank database. real-time rt-qpcr was performed on samples positive for the novel picornaviruses by rt-pcr as described previously [ , ] . briefly, specific primers targeting a -bp ( '-tgtc agatggtgtcaacagtc aaa- ' and '-tcatggcgcactttcacatt- '), a -bp ( '-acaaatctacagccaa attccaaa- ' and '-gtagggtatgcct ttctggtca a- ') and a -bp ( '-cagccaaattccaaattcagat- ' and '-ccagatcagccatg tttggaa- ') fragment of the c genes were used for rt-qpcr by thermal cyler faststart dna master sybr green i mix reagent kit (roche). cdna was amplified by thermal cycler ht (applied biosystems) with -μl reaction mixtures containing faststart dna master sybr green i mix reagent kit (roche). a plasmid containing the target sequence was used for generating the standard curves. thirteen genomes of the two novel picornavirus species were amplified and sequenced, with rna directly extracted from respiratory or alimentary samples as templates [ , , ] . rna was converted to cdna by a combined sequence-specific-priming,random-priming and oligo (dt) priming strategy. as initial results showed that the two novel picornaviruses are distantly related to known picornaviruses, the cdnas of three initial strains were amplified by '-rapid amplification of cdna ends (race) using the smarter race cdna amplification kit (clontech, usa). the first strand cdna for the ' sequence of the genome was constructed with specific primers designed according to results of the first and subsequent rounds of sequencing and smarter ii a oligonucleotide by smartscribe reverse transcriptase. the ' sequence of the genome is completed by specific primers designed for the ' end from the results of the first and subsequent rounds of sequencing and oligo (dt) primer. sequences were assembled to produce final sequences of the viral genomes. the genomes of the remaining strains were amplified and sequenced by the specific primers designed from the initial three genomes and the ' ends of the viral genomes were confirmed by race using the smarter race cdna amplification kit (clontech, usa). the nucleotide (nt) sequences of the genomes and the deduced amino acid (aa) sequences of the open reading frames (orfs) were compared to those of known picornaviruses. phylogenetic tree construction was performed using maximum-likelihood methods from phyml . program. secondary structure prediction in the 'utr was performed using rnafold [ ] and the ires elements were determined based on sequence alignment with emcv as described previously [ , ] . to estimate the selective pressure in driving viral evolution among different regions of the genomes, the number of synonymous substitutions per synonymous site, ks, and the number of non-synonymous substitutions per non-synonymous site, ka, for each coding region between different strains of rosavirus b and c were calculated using the nei-gojobori method (jukes-cantor) in mega . as described previously [ ] . since the vp is the largest and most surface-exposed protein which contains most of the motifs important for interaction with neutralizing antibodies in picornaviruses, (his) -tagged recombinant vp proteins of rosavirus b strain rncw r from a norway rat and rosavirus c strains, rask f from an indochinese forest rat and ncgx in from a coxing's white-bellied rat, were cloned as described previously [ , ] . briefly, the vp gene was amplified and cloned into the nhei site of expression vector pet- b(+) (novagen, madison, wi, usa) in frame and downstream of the series of six histidine residues. the (his) -tagged recombinant vp polypeptide was expressed and purified using the ni + -loaded hitrap chelating system (ge healthcare, buckinghamshire, uk) according to manufacturer's instructions. western blot analysis was carried out using available rodent sera using the purified recombinant vp protein as described previously [ ] . briefly, the purified (his) -tagged recombinant vp protein was loaded into each well of a sodium dodecyl sulfate (sds)- % polyacrylamide gel and subsequently electroblotted onto a nitrocellulose membrane (bio-rad, hercules, ca, usa). the blot was cut into strips and the strips were incubated separately with serial dilutions of sera collected from different rodent species with for igg detection. antigen-antibody interaction was detected with horse radish peroxidase-conjugated secondary antibodies and ecl fluorescence system (ge healthcare, buckinghamshire, uk). eleven samples tested positive for the novel picornaviruses were subject to virus isolation in various cell lines including vero e (african green monkey kidney; atcc crl- ), crfk (crandell feline kidney; atcc ccl- ), in-house hfl (human embryonic lung fibroblast), t (mouse embryonic fibroblast, atcc ccl- ) cells, rd (human embryo rhabdomyosarcoma; atcc ccl- ), rk e (rat kidney; atcc crl- ) and tcmk (mouse kidney; atcc ccl- ) cells as described previously [ , ] . briefly, after centrifugation, samples were diluted five folds with viral transport medium and filtered. filtrates were inoculated to minimum essential media (mem) and the mixtures were added to -well tissue culture plates by adsorption inoculation. after h of adsorption, excess inoculum was discarded, and the wells were washed twice with phosphate buffered saline and replaced by serum-free mem. cultures were inspected daily by inverted microscopy for cpe. subculturing to fresh cell line was performed from time to time even if there was no cpe and culture lysates were collected for rt-pcr for monitoring viral replication. immunostaining and electron microscopy were performed on samples that were rt-pcr positive. electron microscopy t cells successfully infected by rosavirus c rasm a were subject to negative contrast electron microscopy as described previously [ , ] . tissue culture cell extracts infected with rosavirus c rasm a were centrifuged at g at °c, after which the pellet was resuspended in phosphate-buffered saline and stained with % phosphotungstic acid. samples were examined with a philips em s electron microscope. neutralization assays for rosavirus c rasm a were carried out as described previously with modifications [ ] . briefly, rodent sera serially diluted from : to : were mixed with tcid of rosavirus c rasm a. after incubation for h at °c, the mixture was inoculated in duplicates onto -well plates of t cell cultures. results were recorded after days of incubation at °c. virus stock used to inoculate mice was obtained from at least the th passage of rosavirus c rasm a in t cells. groups of suckling balb/c mice were infected orally ( -day-old) and intracerebrally ( -day-old) as described previously [ ] . approximately μl ( tcid ) of virus suspensions was applied orally and μl ( tcid ) intracerebrally. two mice challenged with culture media from uninfected cells were included as negative controls in both groups. mice were monitored daily for signs of disease. nine/ten, five, three and three mice were sacrificed at day , , and respectively. after euthanasia, necropsies of mice were performed to obtain the following tissues: intestine, spleen, kidney, liver, lung and brain. blood was collected for antibody tests by western blot analysis as described above. to perform immunhistochemical staining on infected cell lines and rodent tissues, guinea pig antiserum against the vp protein of rosavirus c was produced by subcutaneously injecting μg purified recombinant rosavirus c vp protein to three guinea pigs, using an equal volume of complete freund's adjuvant (sigma) as described previously [ ] . incomplete freund's adjuvant (sigma) was used in subsequent immunizations. three inoculations at once every two weeks per guinea pig were administered. two weeks after the last immunization, ml of blood was taken via the lateral saphenous vein of the guinea pigs to obtain the sera. to examine the histopathology and viral replication of rosavirus c rasm a in tissues of challenged mice, necropsy organs of the mice were subject to both viral rna detection by rt-pcr and immunohistological studies as described previously [ ] . tissues for histological examination were fixed in % neutral-buffered formalin, embedded in paraffin, and stained with hematoxylin and eosin (h&e). histopathological changes were observed using nikon i microscope and imaging system. infected cell lines and tissues from challenged mice that were tested positive for rosavirus c rasm a by rt-pcr were subject to viral load studies and immunohistochemical staining for viral vp protein as described previously [ , ] . tissue sections were deparaffinized and rehydrated, followed by blocking endogenous peroxidase with . % h o for min, and then with % bsa/pbs at room temperature for min to minimize non-specific staining. the tissue sections were then pre-treated with streptavidin solution and biotin solution at room temperature for min respectively to avoid high background signals due to the endogenous biotin or biotin-binding proteins in the tissues. the sections were incubated at °c overnight with : dilution of guinea pig anti-vp anti-serum, followed by incubation of min at room temperature with : dilution of biotin-conjugated rabbit anti-guinea pig igg, h & l chain (abcam). streptavidin/peroxidase complex reagent (vector laboratories) was then added and incubated at room temperature for min. sections were counterstained with hematoxylin. cells infected or uninfected by rosavirus c rasm a were included as positive and negative controls respectively in each staining. cells were fixed in chilled acetone at - °c for min before incubation with antibodies for staining. color development was performed using , '-diaminobenzidine and images captured with nikon i imaging system and spot-advance computer software. the . the mice study was carried out in strict compliance with animal welfare regulations. the mice were anesthetized by fetanyl/fluanisone/diazepam during the whole experiment. standard guidelines prescribed in pain and distress in laboratory rodents and lagomorphs, laboratory animals , - ( ) were strictly followed and the well-being of animals were monitored daily with a scoring sheet to ensure minimal pain and distress experienced by the mice. clinical and molecular epidemiology of human rhinovirus c in children and adults in hong kong reveals a possible distinct human rhinovirus c subgroup evolution of virulence in picornaviruses host factors in ev replication comparison of three neurotropic viruses reveals differences in viral dissemination to the central nervous system a distinct group of hepacivirus/pestivirus-like internal ribosomal entry sites in members of diverse picornavirus genera: evidence for modular exchange of functional noncoding rna elements by recombination emergence of enterovirus "doublerecombinant" strains belonging to a novel genotype d originating from southern china: first evidence for combination of intratypic and intertypic recombination events in ev recombinant coxsackievirus a and deaths of children chickens host diverse picornaviruses originated from potential interspecies transmission with recombination complete genome characterization of a novel enterovirus type ev-b isolated in china an insight into recombination with enterovirus species c and nucleotide g- reversion from the viewpoint of neurovirulence of vaccine-derived polioviruses severe acute respiratory syndrome coronavirus-like virus in chinese horseshoe bats isolation and characterization of viruses related to the sars coronavirus from animals in southern china bats are natural reservoirs of sars-like coronaviruses middle east respiratory syndrome coronavirus in dromedary camels: an outbreak investigation genetic characterization of betacoronavirus lineage c viruses in bats reveals marked sequence divergence in the spike protein of pipistrellus bat coronavirus hku in japanese pipistrelle: implications for the origin of the novel middle east respiratory syndrome coronavirus identification of cardioviruses related to theiler's murine encephalomyelitis virus in human infections a highly divergent picornavirus in a marine mammal a highly prevalent and genetically diversified picornaviridae genus in south asian children a novel picornavirus associated with gastroenteritis circulation of lineages of a novel saffold cardiovirus in humans discovery of a novel human picornavirus in a stool sample from a pediatric patient presenting with fever of unknown origin clinical features and complete genome characterization of a distinct human rhinovirus (hrv) genetic cluster, probably representing a previously undetected hrv species, hrv-c, associated with acute respiratory illness in children distinguishing molecular features and clinical characteristics of a putative new rhinovirus species, human rhinovirus c (hrv c) comparative analysis of six genome sequences of three novel picornaviruses, turdiviruses , and , in dead wild birds, and proposal of two novel genera, orthoturdivirus and paraturdivirus, in the family picornaviridae complete genome sequence of a novel picornavirus, canine picornavirus, discovered in dogs discovery of rosavirus , a novel variant of a rodent-associated picornavirus, in children from the gambia klassevirus , a previously undescribed member of the family picornaviridae, is globally widespread natural occurrence and characterization of two internal ribosome entry site elements in a novel virus, canine picodicistrovirus, in the picornavirus-like superfamily complete genome analysis of three novel picornaviruses from diverse bat species identification of a novel feline picornavirus from the domestic cat a novel dromedary camel enterovirus in family picornaviridae from dromedaries in the middle east rosavirus: the prototype of a proposed new genus of the picornaviridae family detection of zoonotic pathogens and characterization of novel viruses carried by commensal rattus norvegicus in new york city re-localization of cellular protein srp during poliovirus infection: bridging a viral ires to the host cell translation apparatus differential susceptibility of sd and cd rats to a novel rat theilovirus isolation of cytopathic small round virus (aichi virus) from pakistani children and japanese travelers from southeast asia candidate porcine kobuvirus prevalence and genetic diversity of aichi virus strains in stool samples from community and hospitalized patients isolation and molecular characterization of aichi viruses from fecal specimens collected in japan, bangladesh, thailand, and vietnam isolation and characterization of a new species of kobuvirus associated with cattle sequence and structural elements that contribute to efficient encephalomyocarditis virus rna translation common conformational changes induced in type picornavirus iress by cognate trans-acting factors myristylation of picornavirus capsid protein vp and its structural significance the a proteins of three diverse picornaviruses are related to each other and to the h-rev family of proteins involved in the control of cell proliferation molecular analysis of duck hepatitis virus type indicates that it should be assigned to a new genus two related superfamilies of putative helicases involved in replication, recombination, repair and expression of dna and rna genomes a new superfamily of putative ntp-binding domains encoded by genomes of small dna and rna viruses viral cysteine proteases are homologous to the trypsin-like family of serine proteases: structural and functional implications cysteine proteases of positive strand rna viruses and chymotrypsin-like serine proteases. a distinct protein superfamily with a common structural fold mutational analysis of the proposed fg loop of poliovirus proteinase c identifies amino acids that are necessary for cd cleavage and might be determinants of a function distinct from proteolytic activity primary structural comparison of rna-dependent polymerases from plant, animal and bacterial viruses virus taxonomy: classification and nomenclature of viruses: ninth report of the international committee on taxonomy of viruses. king amq, adams mj, carstens eb, lefkowitz ej capsid protein vp of human rhinovirus induces membrane permeability by the formation of a size-selective multimeric pore genomic characterization of sebokele virus (sebv ) reveals a new candidate species among the genus parechovirus encephalomyocarditis virus a protein is required for viral pathogenesis and inhibition of apoptosis the genetics of the persistent infection and demyelinating disease caused by theiler's virus experimental encephalomyocarditis virus infection in small laboratory rodents molecular analysis of three ljungan virus isolates reveals a new, close-to-root lineage of the picornaviridae with a cluster of two unrelated a proteins development of type diabetes in wild bank voles associated with islet autoantibodies and the novel ljungan virus association of zoonotic ljungan virus with intrauterine fetal deaths sudden infant death syndrome and ljungan virus rodent phylogeny and a timescale for the evolution of glires: evidence from an extensive taxon sampling using three nuclear novel rosavirus in rodents mammal species of the world rodent-borne diseases and their risks for public health the fecal viral flora of wild rodents identification of novel porcine and bovine parvoviruses closely related to human parvovirus vienna rna secondary structure server mega : molecular evolutionary genetics analysis version . isolation and characterization of a novel betacoronavirus subgroup a coronavirus, rabbit coronavirus hku , from domestic rabbits coronavirus as a possible cause of severe acute respiratory syndrome human enterovirus subgenotype b lacks coxsackievirus a -like neurovirulence in mice infection divergent picornavirus ires elements relevance of rna structure for the activity of picornavirus ires elements putative papain-related thiol proteases of positive-strand rna viruses. identification of rubi-and aphthovirus proteases and delineation of a novel conserved domain associated with proteases of rubi-, alpha-and coronaviruses identification of critical amino acids within the foot-and-mouth disease virus leader protein, a cysteine protease virus-encoded proteinases of the picornavirus super-group the authors have declared that no competing interests exist. key: cord- -e of o authors: kindler, eveline; gil-cruz, cristina; spanier, julia; li, yize; wilhelm, jochen; rabouw, huib h.; züst, roland; hwang, mihyun; v’kovski, philip; stalder, hanspeter; marti, sabrina; habjan, matthias; cervantes-barragan, luisa; elliot, ruth; karl, nadja; gaughan, christina; van kuppeveld, frank j. m.; silverman, robert h.; keller, markus; ludewig, burkhard; bergmann, cornelia c.; ziebuhr, john; weiss, susan r.; kalinke, ulrich; thiel, volker title: early endonuclease-mediated evasion of rna sensing ensures efficient coronavirus replication date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: e of o coronaviruses are of veterinary and medical importance and include highly pathogenic zoonotic viruses, such as sars-cov and mers-cov. they are known to efficiently evade early innate immune responses, manifesting in almost negligible expression of type-i interferons (ifn-i). this evasion strategy suggests an evolutionary conserved viral function that has evolved to prevent rna-based sensing of infection in vertebrate hosts. here we show that the coronavirus endonuclease (endou) activity is key to prevent early induction of double-stranded rna (dsrna) host cell responses. replication of endou-deficient coronaviruses is greatly attenuated in vivo and severely restricted in primary cells even during the early phase of the infection. in macrophages we found immediate induction of ifn-i expression and rnase l-mediated breakdown of ribosomal rna. accordingly, endou-deficient viruses can retain replication only in cells that are deficient in ifn-i expression or sensing, and in cells lacking both rnase l and pkr. collectively our results demonstrate that the coronavirus endou efficiently prevents simultaneous activation of host cell dsrna sensors, such as mda , oas and pkr. the localization of the endou activity at the site of viral rna synthesis–within the replicase complex—suggests that coronaviruses have evolved a viral rna decay pathway to evade early innate and intrinsic antiviral host cell responses. a a a a a host innate immune responses are of particular importance during the early phase of virus infection to restrict virus replication and spread. they rely on the ability to differentiate between immunological "self" and "non-self" in order to swiftly activate diverse antiviral effector mechanisms. conceptually, sensing of virus infection is mainly mediated through recognition of viral nucleic acids, which are considered to comprise pathogen-associated molecular patterns (pamps) that are recognized by specialized host cell pathogen recognition receptors (prrs) [ ] . double-stranded (ds) rna, an obligate replication intermediate of positivestranded rna viruses that is accumulating during replication, is known as an important pamp within the cytoplasm of infected cells. host cell responses to dsrna are versatile and include the expression of ifn-i by activating rig-i like helicases (rlrs), such as rig-i and mda , the inhibition of host cell translation by activating pkr, and the degradation of viral and host cell-derived rna by activating the oas/rnase l pathway [ ] . coronaviruses are positive-stranded rna viruses that replicate in the host cell cytoplasm. they are well known to evade innate immune activation, particularly during the early phase of the infection [ ] [ ] [ ] [ ] . coronavirus innate immune evasion is multifaceted and involves ribose- '-o methylation of viral rna, as well as compartmentalised rna synthesis at virus-induced membrane structures comprised of convoluted membranes and double membrane vesicles [ ] [ ] [ ] . the importance of functions encoded by the cov replicase gene is further exemplified by non-structural protein (nsp) that suppresses host gene expression by mediating host mrna degradation [ , ] , and nsp that contains a papain-like proteinase with deubiquitination activity interfering with ifn-i host cell responses [ , ] . in addition, a number of accessory gene functions, although less conserved, have been described to target downstream events of innate immune activation, such as a phosphodiesterase (pde) activity encoded by some coronavirus strains, which degrades ', '-oligoadenylate messenger molecules essential for rnase l activation [ , ] . here we addressed a possible role of the highly conserved coronavirus endou activity in innate immune evasion. the endou domain is harboured in non-structural protein (nsp) that is considered as an integral component of the coronaviral replicase-transcriptase complex (rtc) [ ] [ ] [ ] [ ] . by using immunofluorescence microscopy analyses in hcov- e-infected cells with a hcov- e-nsp -specific monoclonal antibody the characteristic perinuclear staining pattern known from various other cov nsps was reported [ ] . for mhv-a , a similar study reports mhv-nsp -specific perinuclear puncta that were detected using an mhv-nsp -specific rabbit antiserum that partially overlapped with mhv nucleocapsid staining in mhv-a -infected cells [ , ] . moreover, mhv nsp was shown to co-localize with viral rna and to fractionate in similar fractions as other nsps following mhv infection [ , ] . notably, upon ectopic expression of a fusion protein comprised of the green fluorescent protein (gfp) and mhv-nsp , a pattern of cytoplasmic speckles, distinct from the characteristic pattern of the cov replicase complex was observed, suggesting that the localization of ectopically expressed nsp or gfp-nsp fusion proteins may differ from the localization of nsp that is expressed in the context of the cov polyprotein ab [ ] . the cov endou has uridylate-specific endonucleolytic activity on single-stranded and dsrna [ ] and is related to (i) cellular enzymes prototyped by xendou [ , ] and (ii) viral homologs conserved in all nidoviruses known to infect vertebrate hosts including fish, birds and mammals, suggesting an important role for this enzyme in an ancient cellular pathway. over the past years, a wealth of structural and biochemical information has been obtained for endou. however, the precise role of this virus-encoded nucleolytic activity in coronavirus/nidovirus replication remains enigmatic [ , , [ ] [ ] [ ] . surprisingly, although endou is coexpressed with other key replicative proteins as part of the viral replicase polyprotein, its enzymatic activity is not essential for viral rna synthesis in most cell culture systems [ ] . in this work we illustrate a pronounced impact of the coronavirus endou activity on innate immune evasion. specifically, we show that genetically engineered mutants of mouse hepatitis virus (mhv) and human coronavirus e (hcov- e), respectively, that encode an endou active-site substitution known to abolish nucleolytic activity, were severely attenuated and elicited an immediate and simultaneous activation of host cell dsrna sensors. based on biochemical and structural information on coronavirus endou active-site residues [ , , ] , we constructed endou-deficient mutants of hcov- e and mhv (hcov- e h a and mhv h a ) and assessed their replication characteristics in vitro and in vivo ( fig a) . replication of mhv h a was reduced in l cells, but peak titers almost reached those of wild-type mhv-a , confirming that the coronavirus endou activity is dispensable for virus replication in vitro ( fig b) [ , ] . in sharp contrast, compared to wild-type mhv-a , the endou-deficient mhv h a was severely attenuated in vivo (fig c) . mhv h a replication was not detectable in spleen and liver of c bl/ mice at two days post intraperitoneal infection with plaque-forming units (pfu), demonstrating that the endou activity is required for efficient replication and spread in vivo. notably, replication and spread of mhv h a was partly restored in mice deficient for the ifn-i receptor (ifnar), with viral titers of mhv h a in the spleen and liver of ifnar-deficient mice that did not reach those of mhv-a . interestingly, concerning the role of mda and tlr , which are known as main cytoplasmic and endosomal prrs for coronaviral rna, respectively, mhv h a replication was not restored in mda -deficient, tlr -deficient, or mda -and tlr -deficient mice. this phenotype clearly differs from that described for coronaviruses that lack ribose- '-o methyl-transferase (omt) activity [ ] . thus, in experiments reported previously, we found that replication of omt-deficient mhv (mhv d a ) is restored in mice that are deficient for mda and tlr , suggesting that lack of ribose- '-o methylation is tolerated if these two rna sensors are absent. the lack of any detectable replication of the endou-deficient mhv h a in mda -and tlr -deficient mice therefore indicates that mda -and tlr -mediated ifn-i expression may not exclusively restrict mhv h a replication and that other mechanisms contribute to the observed attenuation of mhv h a replication. the severe attenuation of mhv h a growth in vivo prompted us to assess mhv h a and hcov- e h a replication in primary target cells. as shown in fig a, mhv h a replication in primary murine embryonic fibroblasts (mefs) was comparable to that of mhv-a . data represent two independent experiments, each performed in duplicates. mean and sem are depicted. the % confidence band is highlighted in grey. the differences in peak levels of viral titers were calculated by using the non-linear regression model described in material and methods (peak mhv-a : . , mhv h a : . , p = . , left panel; peak mhv-a : . , mhv h a : . , p = . , right panel) and significance is displayed as * p < . . (c) viral titers of mhv-a and mhv h a in liver and spleen of c bl/ , ifnar -/-, mda -/-, tlr -/-, and mda -/-/tlr -/mice at two days post intraperitoneal infection ( pfu). data represent three to four independent experiments, each based on two to three mice per strain and virus. mean and sem are depicted. data points that show significant differences in a two-sided, unpaired student's t-test are displayed; * p < . , ** < . , *** < . . nd, not detected. until - hours post infection (h.p.i.), but was significantly restricted later during the infection. moreover, replication of mhv h a was even more severely reduced in bone marrow-derived murine macrophages, and accompanied by early induction of ifn-β expression (fig b and c) . notably, levels of ifn-β mrna were only transiently ( to h.p.i.) elevated in mhv h a compared to mhv-a infected macrophages, and declined along with viral titers and viral rna during the late phase of infection. likewise, replication of the endou-deficient hcov- e h a was severely restricted in human blood-derived macrophages (fig d) . we observed significantly elevated ifn-i expression in a panel of human macrophages derived from seven individual donors after infection with hcov- e h a compared to wild-type hcov- e infection (fig d) , consistent with reduced viral replication. next, we addressed if, and to what extent, the growth defects observed for endou-deficient coronaviruses correlate with the induction of ifn-β expression. mda has been described as the main rna sensor of coronavirus infection in murine macrophages [ ] . compared to wild-type macrophages, ifn-β expression was reduced in mda -deficient macrophages following mhv h a infection, as shown by qrt-pcr and ifn-β elisa (fig a and d ). surprisingly, and again in contrast to the phenotype of the omt-deficient mhv d a [ ] , mhv h a replication was not restored in mda -deficient macrophages ( fig a) . similarly, although ifn-β expression was likewise reduced in mavs-deficient macrophages, mhv h a replication was not restored in mavs-deficient macrophages (fig b and d ). even in irf / irf /irf (irf / / -/-) triple-knockout macrophages, that display an almost negligible induction of ifn-β expression, mhv h a replication was not fully restored (fig c and d ). ifn-β protein assessed by ifn-β elisa was below detection in all three macrophage genotypes (mda -/-, mavs -/-, irf / / -/-; fig d) . these results indicate that mhv h a replication is either highly sensitive to already marginal amounts of ifn-i, or that other antiviral host cell responses may contribute to the attenuation of mhv h a . in order to address the sensitivity of endou-deficient coronaviruses to ifn-i, we first assessed mhv h a replication in ifnar-deficient macrophages. as shown in fig a, mhv h a replication was partially restored to levels that almost reached those of wild-type mhv-a replication. importantly, ifn-β expression was elevated in ifnar-deficient macrophages that had been infected with mhv h a compared to those infected with wild-type mhv-a , demonstrating that increased expression of ifn-β can be uncoupled from attenuation of mhv h a (fig b) . we also noted that ifn-β expression was delayed in ifnar-deficient macrophages following mhv h a infection compared to wild-type macrophages. this observation is in agreement with previous reports that suggested a macrophage-specific autocrine ifn-β priming loop in wild-type macrophages enhances cytokine and chemokine expression following mhv infection [ ] . the severe attenuation of mhv h a and hcov- e h a replication in wild-type murine and human macrophages, respectively, precluded the use of primary macrophages to assess the sensitivity of endou-deficient coronaviruses to ifn-i pre-treatment. therefore, we infected murine l cells and human mrc lung fibroblasts with mhv h a and hcov- e h a , respectively, and applied different dosages of ifn-i for hours prior to infection. compared to wild type mhv and hcov- e, respectively, both endou-deficient viruses indeed displayed a pronounced sensitivity to ifn-i pre-treatment ( fig c) . remarkably, mhv h a displayed a sensitivity to ifn-i treatment that is comparable to that of the highly ifn-i sensitive omt-deficient mutant mhv d a (s fig). however, compared to the omt-deficient mutant mhv d a the phenotype of the endou-deficient mhv h a differs mainly in the lack of restoration of replication under conditions with strongly reduced ifn-i expression (e.g. in mda -/macrophages; fig a) , suggesting that other, most likely ifn-i inducible, antiviral effector mechanisms account for restriction of mhv h a replication. collectively, these results demonstrate that the coronavirus endou activity plays a pivotal role in innate immune evasion in the context of the ifn-i system. endou-deficient coronaviruses induce activation of the oas/rnase l pathway as noted above, coronavirus endou-deficiency results in a pronounced sensitivity to ifn-i treatment that is comparable to that of the highly ifn-i sensitive omt-deficient mutant . virus replication was measured at h.p.i. by plaque assay (mhv) and at h.p.i. by qrt-pcr (hcov- e), respectively. data represent three independent experiments, each performed in two to three replicas. data are displayed as differences to untreated controls and statistical comparisons between wild type and endou-deficient viruses were performed for each concentration. mean and sem are displayed. data points that show significant differences in a two-sided, unpaired student's t-test are depicted. * p < . , ** p < . and *** p < . mhv d a . however, replication of mhv h a was not restored in mda -deficient macrophages. this observation prompted us to consider that replication of endou-deficient coronaviruses may activate additional dsrna-triggered antiviral pathways. we therefore assessed the integrity of ribosomal rna (rrna), a marker for the activation of the oas/rnase l pathway [ ] , during mhv h a infection in primary murine macrophages. indeed, the breakdown of rrna in mhv h a infected wild-type macrophages was readily detectable as early as - h. p.i., thus coinciding with the induction of ifn-β expression during the early phase of the infection (compare figs a and c). this finding is highly surprising since mhv-a encodes a pde activity that has been shown to degrade ', '-oligoadenylate messenger molecules essential for rnase l activation [ ] . however, the pde activity was apparently not sufficient to prevent rnase l activation in macrophages that had been infected with endou-deficient mhv h a . to exclude that the lack of endou activity may directly impact on viral rna synthesis and lead to reduced levels of subgenomic mrnas, we assessed the level of genomic and subgenomic mrna (encoding the pde activity) by qrt-pcr. as shown in s fig, genomic rna and subgenomic mrna were equally reduced in mhv h a infected wild-type macrophages, suggesting that the lack of endou activity does not result in selective reduction of subgenomic mrnas. importantly, while breakdown of rrna was also readily detectable in mda -and mavs-deficient macrophages, rrna remained intact in rnase l-deficient macrophages, demonstrating that infection of endou-deficient mhv h a indeed results in the activation of the oas-rnase l pathway and subsequent degradation of rrna (fig a; s fig) . notably, a breakdown of rrna was not detected in mhv h a -infected ifnar-deficient macrophages ( fig a) concurring with partial restoration of mhv h a replication in these cells. accordingly, and as previously published [ , ] , the degree of rnase l activation correlates with levels of oas expression and we noted indeed reduced baseline expression of oas a, and in ifnar-deficient compared to wild-type c bl/ macrophages (s fig). likewise, we did not observe breakdown of rrna in l cells (fig b) . we therefore assessed the levels of oas a, , and expression in l with or without ifn-i treatment ( . u). as expected, expression of ifn-β was elevated in mhv h a -, but not in mhv-a -infected l cells, irrespectively of ifn-i pre-treatment (s fig). importantly however, expression of oas a, and in l cells was significantly elevated following ifn-i treatment (s fig), and accordingly, rrna breakdown was readily detectable in ifn-i treated l cells that had been infected with mhv h a (fig b) . this data provide evidence for a functional link between the observed pronounced ifn-i sensitivity of mhv h a and restriction of mhv h a replication by the oas/rnase l pathway. surprisingly however, mhv h a replication was not restored in rnase l-deficient macrophages despite the fact that rrna remained intact during the entire replication cycle (compare fig a and fig a) . this strongly suggests that yet another antiviral pathway, in addition to oas/rnase l, is activated during mhv h a infection. one obvious candidate is pkr, a kinase that can be directly activated by dsrna to phosphorylate the eukaryotic initiation factor α (eif α), resulting in translation inhibition of cellular and viral mrnas. indeed, as shown in fig b (left panel) , we readily detected phosphorylated eif α at h.p.i.. in addition, we assessed the extent of translational inhibition at h.p.i. by using puromycin and subsequent facs analysis. as shown in fig b, wild-type mhv-a infected cells showed active translation comparable to mock infected macrophages, while translational inhibition was observed in mhv h a -infected macrophages. finally, we assessed replication of mhv-a and mhv h a in pkr-deficient macrophages, and as shown in fig c, pkr-deficiency alone was also not sufficient for the restoration of mhv h a replication. importantly, however, we observed elevated replication of mhv h a in primary macrophages that are deficient for both, pkr and rnase l that almost reached that of mhv-a ( fig d) [ ] . in order to more precisely analyse the degree of restoration of mhv h a replication in ifnar-and in rnase l/pkr-deficient macrophages, we performed a statistical analysis and compared the differences of calculated mhv-a and mhv h a peak titers between c bl/ and ifnar -/-(p = . ), between c bl/ and rnase l -/-/pkr -/-(p = . ) and between ifnar -/and rnase l -/-/pkr -/-(p = . ) (fig e) . this result shows that mhv h a replication is restored to a comparable degree in ifnar -/and rnase l -/-/pkr -/macrophages. collectively, these results suggest that mhv h a replication results in the early and simultaneous activation of at least three dsrna-triggered pathways, namely ifn-β expression via mda , and antiviral effectors pkr and oas/rnase l. since activation of mda , pkr and oas/rnase l are triggered by dsrna, we assessed if mhv h a infection results in increased appearance of cytosolic dsrna. by using the dsrna-specific antibody j for intracellular staining and facs analysis we assessed the fig b) , ifnar -/-(data level of dsrna in mhv-a -and mhv h a -infected wild-type and ifnar-deficient macrophages at , , , and h.p.i.. at h.p.i. dsrna was not yet convincingly detectable in both mhv-a -and mhv h a -infected wild-type macrophages (fig a) . at h.p.i. dsrna peaks are clearly separated over mock and we observed a slightly stronger dsrna signal in mhv h a -than in mhv-a -infected cells. this difference became convincingly apparent and statistically significant at and h.p.i. with dsrna peaks of mhv h ainfected cells that clearly separated from dsrna peaks of mhv-a -infected cells (fig a and c, right panel) . importantly, we also controlled for virus infection by staining for the replicase complex (nsp / ), and as shown in fig c (left panel) the peaks for nsp / from in mhv h a -infected wild-type macrophages did not exceed those of mhv-a -infected cells. we obtained essentially the same result when we assessed dsrna and nsp / by facs analysis following infection of ifnar -/macrophages (fig b and d) , suggesting that dsrna is also increased in mhv h a -infection under conditions of reduced host cell responses. collectively, these results demonstrate that cytosolic dsrna is increased in endou-mutant virus infection and suggest that elevated dsrna is the trigger for the activation of mda , pkr, and oas. coronaviruses have long been known to efficiently evade host innate immune responses during the early phase of the infection. however, a defined viral function accounting for the apparent lack of efficient sensing of coronavirus infection has remained elusive. here we show that the highly conserved coronavirus endou activity within the viral rtc plays a major role in providing a first line of innate immune evasion during the early phase of coronavirus infection. we show that at least three dsrna-triggered antiviral pathways are involved in restricting replication of endou-deficient coronaviruses (fig ) . first, infection with endou-deficient mhv and hcov- e results in rapid mda -mediated induction of ifn-β expression. second, we observe breakdown of ribosomal rna indicative of activation of the oas/rnase l pathway that temporally coincides with ifn-β expression. third, we show that efficient restriction of endou-deficient coronaviruses is furthermore dependent on pkr since restoration of endou-deficient mhv h a replication required the absence of both, pkr and rnase l. our data suggest that direct restriction of replication of endou-deficient coronaviruses is mediated by rnase l-mediated rna degradation and inhibition of host cell translation through activation of pkr. in contrast, the effect of ifn-i appears to be indirect through the induction of isg expression, that includes oas/rnase l and pkr. whether other isgs may contribute to the restriction of endou-deficient coronavirus replication remains to be determined. finally, we show that mhv h a replication is associated with increased dsrna levels during the early phase of the infection, providing a likely pamp for the observed simultaneous activation of multiple cytoplasmic dsrna-sensors in cells infected with endou-deficient coronaviruses. the concerted activation of multiple cytoplasmic antiviral pathways strongly suggests sensing of the same pamp during replication of endou-deficient coronaviruses. all three types of sensors, mda , oas - , and pkr, are known to recognize dsrna, suggesting that the pamp (s) relevant for their activation during infection is/are of viral origin. notably, rnase l correspond to fig a) and rnase l -/-/pkr -/-(data correspond to fig c) macrophages are displayed. statistical analysis was performed to compare differences of calculated mhv-a and mhv h a peak titers between c bl/ and ifnar -/-(**, p = . ), between c bl/ and rnase l -/-/pkr -/-(**, p = . ) and between ifnar -/and rnase l -/-/pkr -/-(p = . ; ns) macrophages following mhv-a and mhv h a infection. viral endonuclease and innate immunity activation can be triggered by different oas proteins and may depend on particular cell types and virus infections [ ] . for example overexpression of oas was shown to provide rnase l-dependent activity against dengue virus and chikungunya virus infection [ , ] , while oas and oas have been implicated in antiviral activity against hepatitis c virus [ ] . it recently has been shown that among the human oas proteins , , and , oas seems to be mainly responsible for mediating rnase l activation following either polyi:c transfection or virus infection, suggesting a superior role of human oas over oas and oas in restricting virus replication [ ] . interestingly, structural and biochemical studies revealed that oas is selective for binding of long dsrna (> bp) by involvement of an rna-binding, but noncatalytic domain, and that oas is weakly or not activated by short dsrna or single-stranded rna, respectively [ ] . likewise, pkr preferentially dimerizes upon binding to dsrna of similar length (> bp) [ , ] and mda is actually most efficiently activated by even longer dsrna (> kbp) [ ] and higher-order structured rna containing single-stranded and dsrna [ ] . it is thus tempting to propose that viral dsrna represents the natural substrate of the coronavirus endou. however, it remains to be determined which kind of viral dsrna is cleaved by the endou or triggers mda , oas, and pkr activation. compared to mhv wildtype infection we observed a slight but reproducible increase of dsrna in mhv h a -infected macrophages by facs analysis during the early phase of the infection ( - h.p.i.) that became more prominent at - h.p.i., suggesting that the majority of dsrna is not cleaved by endou. this likely includes dsrna being shielded within double-membrane vesicles and replication intermediates actively involved in viral rna synthesis that are likely protected by the rtc and the nucleocapsid protein. we therefore speculate that coronaviruses may have evolved a viral rna quality control mechanism to evade dsrnas sensing, and that endou substrates may comprise dsrna intermediates within stalled rtcs engaged in genome replication or transcription that are no longer active in viral rna synthesis. within the order nidovirales, the endou domain is highly conserved within the families coronaviridae (comprising two subfamilies coronavirinae and torovirinae) and arteriviridae and has been considered a major genetic marker that discriminates nidoviruses from all other rna viruses [ ] [ ] [ ] . however, the recent discovery of insect nidoviruses (family mesoniviridae) [ , ] and re-analysis of the ronivirus genome (family roniviridae; infecting crustaceans) revealed that these two nidovirus families do not encode an endou domain [ ] . in the light of our results it is tempting to speculate that the endou domain has evolved in vertebrate nidoviruses (corona-and arteriviridae) to counteract vertebrate-specific innate immune sensing of viral rna in the context of the type-i interferon system, while the absence of the endou domain in roni-and mesoniviruses is indicative of fundamentally different mechanisms of rna virus innate immune sensing and antiviral effector pathways in invertebrates (e.g. crustaceans and insects) [ ] . mhv as a natural mouse pathogen has been instrumental to understand the delicate balance between host ifn-i responses to infection and counteracting mechanisms of coronavirus innate immune evasion. while mhv evades innate immune sensing in most target cells, plasmacytoid dendritic cells (pdcs) remain as major ifn-i producer cells during early coronavirus replication to ensure protection of mhv target cells and control of potentially lethal coronavirus infections [ , ] . while pdcs sense coronaviral rna within endosomes through tlr , our data demonstrate that the coronaviral endou delays mda -mediated cytoplasmic sensing in macrophages and likely other cell types. this enables coronaviruses to establish robust replication and spread at the entry port of infection. however, in the case of highly pathogenic strains or newly emerging zoonotic coronaviruses, delayed ifn-i responses can have detrimental consequences as recently demonstrated in a murine model of sars-cov infection [ ] . early and rapid sars-cov replication in the respiratory tract combined with a delayed ifn-i response can result in dysregulated innate immune responses and inflammatory cytokine-driven extensive lung damage. therefore, antiviral intervention aiming at inhibiting the coronavirus endou activity may be a promising approach to restore efficient sensing of coronaviral rna and thereby activating ifn-i expression as well as antiviral effector pathways such as pkr and oas/rnase l. there is a growing number of virus-encoded ribonucleases that have been reported to execute diverse steps in the context of cellular mrna quality control and mrna decay [ , ] . for example, herpesvirus-encoded endonucleases are known to broadly target cellular and also viral mrnas that are subsequently further processed by cellular exonucleases, such as xrn , in order to broadly restrict host cell gene expression [ ] . while this strategy indirectly impacts host cell innate immune responses, a more specific interaction of rna decay and host cell dsrna responses has recently been described for vaccinia virus decapping enzymes d and d [ , ] . they remove '-cap structures of partially overlapping vaccinia virus mrnas that arise during the late phase of infection in order to preclude any accumulation of viral dsrna and subsequent activation of dsrna sensors such as pkr and oas. notably, also in this case xrn is required to further process the de-capped mrnas. our data show that early during coronavirus infection the endou activity conceptually fulfils the same task, namely the removal of dsrna that would otherwise trigger host cell dsrna responses, such as ifn-β expression and activation of pkr and oas/rnase l. it will be interesting to address whether xrn is involved in further degrading the coronavirus endou cleavage products or if this function could be fulfilled by the coronavirus-encoded exonuclease (exon) activity residing in nsp . interestingly, like the endou, the coronavirus exon is an integral component of the coronaviral rtc, and exon has been demonstrated to provide an rna proofreading function that permits coronaviruses to stably maintain their extraordinary large rna genome [ ] [ ] [ ] . a functional link between the two coronaviral ribonucleases, exon and endou, would suggest an unprecedented concept of viral rna quality/decay control that goes beyond rna proofreading and includes the removal of dsrna-based pamps at the site of rna synthesis to efficiently evade innate and intrinsic antiviral host cell responses. recombinant mhv strain a , hcov- e, mhv h a , hcov- e h a , and mhv d a were generated using the vaccinia virus-based reverse genetic system as previously described [ , [ ] [ ] [ ] . viruses were propagated on cl mouse fibroblasts (mhv) and on huh- hepacarcinoma cells (hcov- e) and their identity was confirmed by sequencing. to monitor viral spread in vivo, mice at the age of - weeks were injected intraperitoneally with plaque-forming units (pfu) of mhv-a and mhv h a , diluted in mem % ( % heat-inactivated fetal calf serum, penicillin ( μg/ml) and streptomycin ( μg/ml)). mice were euthanized two days post infection (d.p.i.) and liver and spleen were harvested, weighed and homogenized. viral load (pfu/g organ) was determined by plaque assay. murine l fibroblasts (sigma), cl cells (gift from s.g. sawicki) were cultured in mem %. murine embryonic fibroblasts (c bl/ mefs) were maintained at low passage in dmem % (dulbecco's modified eagle medium-glutamax). huh- cells (gift from v. lohnmann) were cultured in dmem % and . mm sodium pyruvate. mrc- cells (human lung fibroblast-like cells; sigma) were maintained at low passage in mem %, supplemented with % non-essential amino acids (neaa). hek -mx -luc cells (gift from g. kochs) were maintained in dmem %, supplemented with g ( μl/ml) [ ] . murine bone marrow-derived macrophages were obtained from mice at the age of - weeks. progenitor cells were isolated from hind limbs, passed through a cell strainer and red blood cell lysis was carried out in ml lysis buffer/mouse ( . m nh cl, mm khco , . mm edta). cells were washed x with pbs and taken up in macrophage medium (imdm iscove's modified dulbecco's medium, - % m-csf (l -supernatant), . % mm -mercaptoethanol). new medium was added d.p.i. and adherent cells were harvested d.p.i. primary human macrophages were obtained from peripheral blood of healthy human donors as previously described [ ] . peripheral blood mononuclear cells were isolated by centrifugation of buffy coat blood over a leucosep tube (greiner bio one). cells from the enriched interphase were collected, washed twice with pbs and red blood cells were removed. cells were taken up in imdm and plated in -well cell culture plates. non-adherent cells were removed three h.p. seeding and adherent cells were cultured for days in imdm %. medium was changed every second day. all experiments using human blood were in accordance with the swiss federal legislation and the institutional guidelines of the cantonal hospital st. gallen and the blutspendedienst srk bern. total cellular rna was isolated from murine macrophages with trizol (life technologies) and genomic dna was removed with dnase (ambion, dna-free dnase treatment). rna concentration was measured by nanodrop and input for cdna synthesis was standardized to ng. synthesis of cdna was carried out using the m-mlv reverse transcriptase from promega and the μl cdna were diluted with μl dh o. the faststart universal sybr green master (rox) mix (roche) was used for measuring mrna expression of ifn-β, gapdh and tbp (s table) . induction of ifn-β was normalized to levels of the household genes gapdh and tbp (geometric mean) and expressed as ΔΔc t over mock (Δc t values calculated as c t reference-c t target) [ ] . expression of oas a, oas , oas and rnase l mrna in mock infected macrophages and l cells was normalized to levels of gapdh [ ] (s table) . expression levels were displayed as Δc t values (c t reference-c t target). copy number of cell-associated viral rna isolated from mhv infected macrophages was determined using the rt taqman pcr system (taqman fast universal pcr master mix ( x), no amperase ung, applied biosystems) with primers and probe specific to the mhv genome fragment encoding the nucleocapsid (s table) . copy numbers were determined by using a standard curve, consisting of an in vitro transcribed rna of known copy number, obtained from a plasmid comprising the mhv-nucleoprotein sequence. quantitative rt-pcr to determine mhv genomic rna and subgenomic mrna two rna standards encompassing mhv nucleotides (nts) - (genomic standard), and mhv nts - / - (corresponding to the mhv mrna leader-body junction; subgenomic mrna standard) were prepared as follows. one rt-pcr product corresponding to the ' region of mhv-a genomic rna was generated by rt-pcr using viral rna from mhv-a infected cells as template and primers t -mhv-leader -frw and mhv-orf -rev (s table) . the resulting rt-pcr product comprised the t -rna-polymerase promoter, mhv nts - . a second rt-pcr product corresponding to the ' region of mhv-a mrna was generated by rt-pcr using viral rna from mhv-a infected cells as template and primers t -mhv-leader -frw and mhv-ns -rev (s table) . the resulting rt-pcr product comprised the t -rna-polymerase promoter, mhv nts - , and mhv nts - . both rt-pcr products were separated on an agarose gel and dna fragments of the appropriate size were excised and purified. in vitro transcribed (ivt) rna was prepared using the ribomax large scale rna production system-t (promega). rq rnase-free dnase was added to the ivt rna and incubated for minutes at ˚c. the in vitro transcribed rna was purified using the nucleospin rna kit (macherey-nagel), its quantity was determined (absorbance at nm) and eight -fold dilutions were prepared. synthesis of cdna was carried out for each dilution using the m-mlv reverse transcriptase and random primers (promega). the ul of cdna were diluted with μl dh o and used as a standard for the quantitative rt-pcr reaction. copy numbers of genomic and subgenomic viral rna were determined for samples obtained from c bl/ macrophages infected with mhv-a and and mhv h a (moi = ). a multiplex reaction (taqman fast universal pcr master mix ( x), no amperase ung, applied biosystems) and primers and a probe specific to the genomic or subgenomic sequence (s table) , respectively, were used. total type-i ifn in supernatants obtained from mhv infected macrophages was measured by an ifn-β enzyme-linked immunosorbent assay (bpl assay science, verikine mouse ifn beta elisa kit, . - pg/ml). technical replicates were pooled. the level of biologically active human type i ifn in the supernatant of infected human macrophages was measured with hek cells that were stably transfected with a luciferase reporter plasmid under the control of the mx-promoter [ , ] . recombinant ifnα a/d (sigma) was used as a cytokine standard and luciferase activity was detected hours p.i. by a luminometer (luciferase assay system, promega). to assess the sensitivity of mhv and hcov- e towards ifn-i, l cells (mhv) and mrc cells (hcov- e) were treated with recombinant ifnα a/d (sigma, as indicated in the figures) for four hours, and then infected with mhv-a , mhv h a , mhv d a , hcov- e and hcov h a (moi = ). virus supernatant was harvested h.p.i (mhv) and h.p.i (hcov- e). viral replication was measured by plaque assay (mhv) or by using primers and a probe specific to the hcov- e membrane protein (s table) and the quantitect probe one- step rt-pcr kit (qiagen). to assess baseline expression, l cells were pre-treated with . u of ifnα a/d for h and then infected with mhv-a and mhv h a (moi = ). cellular rna was isolated with trizol, cdna was prepared and qrt-pcr was performed (s table) . total rna isolated from mhv-infected macrophages and l cells was analysed with a fragment analyzer (labgene) using the dnf- standard sensitivity rna analysis kit ( nt lower marker, advanced analytical technologies). to assess the amount of dsrna-positive cells, c bl/ and ifnar -/macrophages ( x cells) were infected with mhv-a and mhv h a (moi = ). for facs, cells were detached with pbs at ˚c, centrifuged and fixed with % formalin. cells were permeabilized with . % triton and stained with the mouse monoclonal antibody j directed against dsrna ( : , english & scientific consulting bt) and the anti-mhv nsp / rabbit antiserum [ ] ( : ) at ˚c for h. cells were washed, stained with a secondary antibody goat f(ab') anti-mouse igg a, human ads-pe ( : , southernbiotech) and a donkey anti-rabbit alexa- ( : ) for min. facs was performed using the bd facs canto ii and data were analysed using flowjo v. . cell debris was excluded based on a gate of fsca/ssca, followed by a doublet discrimination fsca and fsw. to assess the extent of translation inhibition, c bl/ macrophages were infected with mhv-a and mhv h a (moi = ). min prior to each time point, puromycin (sigma) was added to the wells at a final concentration of μg/ml to label active translation. at the indicated time points, cells were washed twice with pbs, and subsequently detached with pbs at c. cells were fixed in % pfa for min at rt, and washed once with facs buffer (pbs + % bsa). cells were incubated in ice-cold methanol for min at c. after two wash steps in facs buffer, cells were incubated with a primary mouse antibody directed against puromycin ( : , milipore) and a primary rabbit anti-eif α-p (abcam; : ) in facs buffer for min at rt. cells were washed twice with facs buffer and incubated with the secondary antibody donkey anti-mouse-alexa ( : ), and donkey anti-rabbit-alexa ( : ) in facs buffer for min at rt in the dark. cells were washed once in facs buffer, and kept in % pfa in the dark until cells were analysed with the facs canto (bd) using the bd facs diva software. kinetics of virus growth, viral rna and ifn-β mrna were analyzed using non-linear regression. the regression model is an exponential saturation model (increasing response with constant asymptote) that additionally allows a peak response. it is described by the formula where y is the response value (log virus titer or log expression value), t is the time (in hours), a is the value of the asymptote, m is a "midpoint value"representing the time where the exponential increase has reached half of the asymptotic value and where the peak is located, p is a value describing the additional peak height, and s is a scale parameter specifying the steepness of the exponential increase and the width of the peak. the coefficients a, m and p were determined individually for the groups to be compared (mhv-a and mhv h a ), as symbolized by the index g. the model was chosen on pragmatic grounds because it was able to describe the time courses of all data very well and the coefficients represent biologically relevant aspects of the kinetics that can be addressed directly by statistical tests. the analyses were performed in r, version . . [ ] with the function nls [ ] using the algorithm "port" restricting the coefficients to positive values. p-values and confidence intervals were determined by parametric bootstrapping, resampling the residuals from a normal distribution with mean and variance estimated from the variance of residuals of the fitted model. confidence bands were generated by connecting the point-wise % confidence intervals of the predictions. the significance of the difference between treatments in differences between maxima of the groups (i.e., the group-treatment interaction) was determined by bootstapping the difference-in-difference. the assumption of normally distributed residuals was checked and confirmed with normal-quantile quantile plots. all other data were analysed using r v. all experiments using human blood (buffy coat) were in accordance with the swiss federal legislation and the institutional guidelines (including informed consent) of the cantonal hospital st. gallen and the blutspendedienst srk bern. the evolution of antiviral defense systems interferon-stimulated genes: a complex web of host defenses dysregulated type i interferon and inflammatory monocyte-macrophage responses cause lethal pneumonia in sars-cov-infected mice mouse hepatitis virus does not induce beta interferon synthesis and does not inhibit its induction by double-stranded rna efficient replication of the novel human betacoronavirus emc on primary human epithelium highlights its zoonotic potential inhibition of the alpha/beta interferon response by mouse hepatitis virus at multiple levels -o methylation of the viral mrna cap evades host restriction by ifit family members sars-coronavirus replication is supported by a reticulovesicular network of modified endoplasmic reticulum ribose '-o-methylation provides a molecular signature for the distinction of self and non-self mrna dependent on the rna sensor mda severe acute respiratory syndrome coronavirus nsp protein suppresses host gene expression by promoting host mrna degradation coronavirus nonstructural protein : common and distinct functions in the regulation of host and viral gene expression deubiquitinating and interferon antagonism activities of coronavirus papain-like proteases nidovirus papain-like proteases: multifunctional enzymes with protease, deubiquitinating and deisgylating activities antagonism of the interferon-induced oas-rnase l pathway by murine coronavirus ns protein is required for virus replication and liver pathology middle east respiratory syndrome coronavirus ns b protein inhibits host rnase l activation unique and conserved features of genome and proteome of sars-coronavirus, an early split-off from the coronavirus group lineage major genetic marker of nidoviruses encodes a replicative endoribonuclease nidovirus ribonucleases: structures and functions in viral replication identification and subcellular localization of a kda, polyprotein ab processing product in human coronavirus e-infected cells comparative in vivo analysis of the nsp endoribonuclease of murine, porcine and severe acute respiratory syndrome coronaviruses colocalization and membrane association of murine hepatitis virus gene products and de novo-synthesized viral rna in infected cells rna replication of mouse hepatitis virus takes place at double-membrane vesicles purification, cloning, and characterization of xendou, a novel endoribonuclease involved in processing of intron-encoded small nucleolar rnas in xenopus laevis the severe acute respiratory syndrome coronavirus nsp protein is an endoribonuclease that prefers manganese as a cofactor the coronavirus endoribonuclease nsp interacts with retinoblastoma tumor suppressor protein biochemical and genetic analyses of murine hepatitis virus nsp endoribonuclease crystal structure and mechanistic determinants of sars coronavirus nonstructural protein define an endoribonuclease family new antiviral target revealed by the hexameric structure of mouse hepatitis virus nonstructural protein nsp murine coronavirus mouse hepatitis virus is recognized by mda and induces type i interferon in brain macrophages/microglia autocrine interferon priming in macrophages but not dendritic cells results in enhanced cytokine and chemokine production after coronavirus infection rrna cleavage as an index of ppp(a 'p) na activity in interferon-treated encephalomyocarditis virus-infected cells activation of rnase l by murine coronavirus in myeloid cells is dependent on basal oas gene expression and independent of virus-induced interferon activation of rnase l is dependent on oas expression during infection with diverse human viruses interferon action in triply deficient mice reveals the existence of alternative antiviral pathways oligoadenylate synthase-like (oasl) proteins: dual functions and associations with diseases the large form of human ', '-oligoadenylate synthetase (oas ) exerts antiviral effect against chikungunya virus distinct antiviral roles for human ', '-oligoadenylate synthetase family members against dengue virus infection the ribonuclease l-dependent antiviral roles of human ', '-oligoadenylate synthetase family members against hepatitis c virus structural mechanism of sensing long dsrna via a noncatalytic domain in human oligoadenylate synthetase regulation of innate immunity through rna structure and the protein kinase pkr the regulation of the protein kinase pkr by rna length-dependent recognition of double-stranded ribonucleic acids by retinoic acid-inducible gene-i and melanoma differentiationassociated gene activation of mda requires higher-order rna structures generated during virus infection discovery of the first insect nidovirus, a missing evolutionary link in the emergence of the largest rna virus genomes an insect nidovirus emerging from a primary tropical rainforest type i ifn-mediated protection of macrophages and dendritic cells secures control of murine coronavirus infection control of coronavirus infection through plasmacytoid dendritic-cell-derived type i interferon emerging roles for rna degradation in viral replication and antiviral defense a common strategy for host rna degradation by divergent viruses coordinated destruction of cellular messages in translation complexes by the gammaherpesvirus host shutoff factor and the mammalian exonuclease xrn cellular '- ' mrna exonuclease xrn controls double-stranded rna accumulation and anti-viral responses poxvirus decapping enzymes enhance virulence by preventing the accumulation of dsrna and the induction of innate antiviral responses coronaviruses: an rna proofreading machine regulates replication fidelity and diversity high fidelity of murine hepatitis virus replication is decreased in nsp exoribonuclease mutants rna '-end mismatch excision by the severe acute respiratory syndrome coronavirus nonstructural protein nsp /nsp exoribonuclease complex recombinant mouse hepatitis virus strain a from cloned, full-length cdna replicates to high titers in vitro and is fully pathogenic in vivo generation of recombinant coronaviruses using vaccinia virus as the cloning vector and stable cell lines containing coronaviral replicon rnas infectious rna transcribed in vitro from a cdna copy of the human coronavirus genome cloned in vaccinia virus interferon action and apoptosis are defective in mice devoid of ', '-oligoadenylate-dependent rnase l rapid and simple detection of ifn-neutralizing antibodies in chronic hepatitis c non-responsive to ifn-alpha hepatitis a virus suppresses monocyte-to-macrophage maturation in vitro accurate normalization of real-time quantitative rt-pcr data by geometric averaging of multiple internal control genes research . pubmed central pmcid: pmcpmc cell-type-specific activation of the oligoadenylate synthetase-rnase l pathway by a murine coronavirus human and mouse mx proteins inhibit different steps of the influenza virus multiplication cycle processing of the coronavirus mhv-jhm polymerase polyprotein: identification of precursors and proteolytic products spanning kilodaltons of orf a. virology r development core team: a language and environment for statistical computing.: r foundation for statistical computing nonlinear models we would like to thank muriel fragnière, arnaud baumann, and ronald dijkman for various technical assistance. we are grateful to susan baker for providing the anti-mhv nsp / rabbit antiserum. we are grateful to matthias schweizer and rune hartmann for input and ideas, and discussing the manuscript. conceptualization: ek rz lcb bl vt. key: cord- -jvl authors: juranic lisnic, vanda; babic cac, marina; lisnic, berislav; trsan, tihana; mefferd, adam; das mukhopadhyay, chitrangada; cook, charles h.; jonjic, stipan; trgovcich, joanne title: dual analysis of the murine cytomegalovirus and host cell transcriptomes reveal new aspects of the virus-host cell interface date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: jvl major gaps in our knowledge of pathogen genes and how these gene products interact with host gene products to cause disease represent a major obstacle to progress in vaccine and antiviral drug development for the herpesviruses. to begin to bridge these gaps, we conducted a dual analysis of murine cytomegalovirus (mcmv) and host cell transcriptomes during lytic infection. we analyzed the mcmv transcriptome during lytic infection using both classical cdna cloning and sequencing of viral transcripts and next generation sequencing of transcripts (rna-seq). we also investigated the host transcriptome using rna-seq combined with differential gene expression analysis, biological pathway analysis, and gene ontology analysis. we identify numerous novel spliced and unspliced transcripts of mcmv. unexpectedly, the most abundantly transcribed viral genes are of unknown function. we found that the most abundant viral transcript, recently identified as a noncoding rna regulating cellular micrornas, also codes for a novel protein. to our knowledge, this is the first viral transcript that functions both as a noncoding rna and an mrna. we also report that lytic infection elicits a profound cellular response in fibroblasts. highly upregulated and induced host genes included those involved in inflammation and immunity, but also many unexpected transcription factors and host genes related to development and differentiation. many top downregulated and repressed genes are associated with functions whose roles in infection are obscure, including host long intergenic noncoding rnas, antisense rnas or small nucleolar rnas. correspondingly, many differentially expressed genes cluster in biological pathways that may shed new light on cytomegalovirus pathogenesis. together, these findings provide new insights into the molecular warfare at the virus-host interface and suggest new areas of research to advance the understanding and treatment of cytomegalovirus-associated diseases. the cytomegaloviruses, classified within the betherpesvirinae subfamily, are a group of species-specific herpes viruses that establish life-long infection of their hosts. human cytomegalovirus (hcmv) can cause devastating disease and death in congenitallyinfected infants, and long-term neurological complications in survivors. in adults, hcmv can cause a spectrum of diseases in immune compromised patients involving multiple organs and tissues and is a primary cause of graft loss in transplant patients [ , ] . in recent years, hcmv has been linked to lung injury in trauma patients [ ] and is also postulated to act as a cofactor in atherosclerosis and some cancers [ , ] . for these reasons, there is an urgent need for an effective vaccine and new antiviral intervention strategies that mitigate the toxicity and drug resistance shortcomings of current antiviral drugs [ , ] . there exist a number of challenges to our understanding of cmv pathogenesis as well as progress in vaccine and antiviral drug development. two outstanding challenges are the gaps in our knowledge of viral genes and how these gene products interact with host cellular gene products to cause disease. despite the publication of the first sequence of the hcmv genome in [ , ] , and the first sequence of the murine cytomegalovirus (mcmv) genome in [ ] , there are still important questions regarding the nature and number of genes for these viruses. mcmv is the most widely used model to study hcmv diseases and recapitulates many of clinical and pathological findings found in human diseases. our understanding of mcmv viral genes and genomes has evolved with the technology used to study them. a major milestone in understanding mcmv came with decoding the first mcmv complete genome sequence by rawlinson and colleagues [ ] . the authors identified a kb genome predicted to encode genes. subsequent refinements in the annotation of the mcmv were introduced by classical molecular and biochemical studies that are reflected in the current ncbi reference sequence. the application of new technologies to study the mcmv genome emerged in the last decade and include proteomic [ ] , in silico [ ] , and gene array [ , ] approaches that have led to major revisions in gene annotation. more recently cheng and colleagues [ ] proposed additional changes after sequencing isolates to measure genome stability after in vitro and in vivo passage. also, lacaze and colleagues [ ] extended the microarray approach to include probes specific to both strands of the genome, leading to the discovery of noncoding and bi-directional transcription at late stages of mcmv infection. finally, a recent transcriptomic analysis of newly synthesized rna in mcmv infected fibroblasts [ ] applied rna-seq technology to study regulation of viral gene expression and identified a very early peak of viral gene transcriptional activity at - hours post infection followed by rapid cellular countermeasures but did not attempt to re-annotate mcmv genome. altogether, these new technologies have refined and advanced our knowledge of viral genes and the mcmv genome. nevertheless, we still lack definitive annotation for the standard lab strains of mcmv and specific knowledge of how many of these genes function during natural infection and disease. currently, two annotations of mcmv genomes are used -the original rawlinson's annotation with minor modifications (genbank accession no. gu . ) where open reading frames (orfs) are identified and the ncbi reference sequence annotation (genbank accession no: nc_ . ) with orfs. we previously used a transcriptomic approach to analyze gene products of hcmv [ ] . this was the first report to characterize abundant antisense and noncoding transcription in the hcmv genome showing that there is greater complexity of herpesvirus genomes than previously appreciated. using rna-seq technology, gatherer et al. [ ] showed that most protein coding genes are also transcribed in antisense but are generally expressed at lower levels than their sense counterparts. a more recent analysis of translational products of hcmv [ ] by ribosomal footprinting indentified translated orfs, further underscoring the complexity of herpes virus genomes. we describe mcmv transcriptional products that differ from predicted orfs, novel spliced transcripts, and novel transcripts derived from intergenic regions of the genome. additionally, we found that the most abundant viral transcript (mat) is a spliced transcript recently identified as a noncoding rna that limits accumulation of cellular mirnas [ , ] . here we report that this transcript also specifies a novel protein and to our knowledge, this is the first viral transcript that functions both as a noncoding rna and mrna. analysis of the host transcriptional response to infection revealed many unexpected host genes that are regulated by virus infection, including many noncoding rna genes. correspondingly, many host genes regulated by virus infection cluster in unexpected biological pathways that may shed new light on the pathogenesis of cytomegalovirus-associated diseases. together, these findings suggest important revisions are required for mcmv genome annotation and emphasize numerous aspects of mcmv biology and the host response to this infection that are unknown and require further study. in this study, we set out to complete a transcriptomic analysis of mcmv infection. we analyzed viral transcripts through classical cdna cloning and sequencing and through next generation sequencing of cdna generated from total cellular rna (rna-seq). analysis of cdna libraries is a well-proven approach to analyze viral transcripts based on isolation of long transcripts, molecular cloning of the transcripts, and traditional sanger-based sequencing of the cdna clones. traditional cloning has many advantages, including isolation of novel transcripts that may not be identified by probe-based technologies, as well as precise analysis of transcript splice sites and transcript ends. the introduction of massively parallel sequencing techniques represents a major new technology to study gene expression. basically, rna (total or fractionated) is converted to a library of smaller cdna fragments. adaptors are added to the fragments, and the shorter fragments are sequenced in a high-throughput manner using next generation sequencing technology. this rna-sequencing (rna-seq) approach is free of selection biases associated with traditional cloning or probe-based methods and allows for the entire transcriptome to be analyzed in a quantitative manner (reviewed in [ ] ). first, cdna libraries representing the major temporal classes of viral gene expression were generated by collecting rna from infected mouse embryonic fibroblasts (mefs) at time points after infection. for rna-seq analysis, rnas collected at the same time points were pooled, converted to cdna, and sequenced on the illumina genome analyzer iix. of the , , reads that passed the filter from infected cells, % aligned to mcmv genome indicating a -fold coverage of the viral genome. a total of cdna clones were included in the final analyses [ from the immediate early (ie) library, from the early (e) library, and from the late (l) library]. generally, temporal assignment of cdna clones in this study agrees with previous studies and a detailed comparison, including discrepancies to earlier studies is provided in dataset s . as shown in figures and , transcriptomic data generated using these two experimental approaches were compared to currently available genome annotation (the ncbi reference sequence, genbank accession. no. nc_ . , and a more recent sequence analysis of the smith strain, genbank accession no. gu . ). using this schematic overview, current anno- we have conducted a comprehensive analysis of the murine cytomegalovirus and host cell transcriptomes during lytic infection. we identify numerous novel spliced and unspliced transcripts of mcmv. unexpectedly, the most abundantly transcribed viral genes are of unknown function. we found that the most abundant viral transcript, recently identified as a noncoding rna regulating cellular micrornas, also codes for a novel protein. to our knowledge, this is the first viral transcript that functions both as a noncoding rna and an mrna. infection alters expression of many unexpected host genes, including many noncoding rna genes. correspondingly, many cluster in unexpected biological pathways that may shed new light on cytomegalovirus pathogenesis. together, these findings provide new insights into the molecular warfare at the virus-host interface and suggest new areas of research to advance the understanding and treatment of cytomegalovirus-associated diseases. figure . comparison of cdna cloning and rna-seq data in relation to current genome annotation. comparison of poly(a) cdna library (green arrows) and rna-seq analysis of murine cytomegalovirus (gray histograms). the longest clone from each group of clones in the cdna library is shown. eland alignments of rna-seq reads were loaded in integrative genomics viewer and compared to nc_ . , (red arrows) and gu . (blue arrows). the data range for rna-seq data was set to - . data is shown in kb ranges with kb overlap. data is shown for the first kb of the mcmv genome and the figure legend is shown in figure . doi: . /journal.ppat. .g tations (red and blue arrows) largely agree. the mcmv transcripts identified through our classical cdna cloning and sequencing (green arrows) and the rna-seq expression profiles (gray histograms), showed complementary results to each other but diverged dramatically from current annotations. a summary of the cdna clones relative to genes annotated in the ncbi reference gu . (blue arrows). the data range for rna-seq data was set to - . data is shown in kb ranges with kb overlap. data is shown for genomic region spanning - kb of the mcmv genome. doi: . /journal.ppat. .g table s , and a complete list of the cdna clones isolated in this study and their characteristics are presented in table s . a detailed comparison of transcripts cloned in this study compared to current annotations (gu . and nc_ ) is presented in table s , and includes estimated relative expression measured as reads per kilobase per million mapped reads (rpkm) derived from rna-seq analysis. analysis of the cdna clones revealed many novel transcripts with the following characteristics: ( ) novel antisense transcripts. excluding cdna clones that mapped to intergenic regions, ( %) of cdna clones were in the sense (s) orientation, ( %) were antisense (as), and ( %) overlapped more than one gene in both s and as orientations relative to original annotation provided by rawlinson and colleagues [ ] . these designations were reevaluated according to the current ncbi reference sequence (nc_ ) in which as transcripts to hypothetical or putative proteins were revised as s transcripts due to lack of evidence for the predicted sense transcript. if as transcripts that overlapped hypothetical proteins also overlapped s transcripts in this library, the as designation was preserved. according to these criteria, ( %) transcripts were in s orientation, only ( . %) were in as orientation, and ( %) overlapped more than one gene in both s and as orientations. ( ) transcripts overlapping more than one currently annotated genes. several cdna clones were isolated that overlap more than one currently annotated gene. for example, four cdna clones in our library overlapped both the m and m genes. the longest of these clones specifies a bp transcript, whereas current annotation predicts two genes of bp (m ) and bp (m ). these data suggest the possibility that several orfs are translated from unexpectedly long or polycistronic transcripts. unusually long and polycistronic transcripts have recently been shown to be a feature of hcmv transcriptome [ ] . ( ) the absence of transcripts in currently annotated regions. several gene regions were not well represented in the cdna cloning study. for example, no cdna clones overlapping m or m were found in this study, and these regions had the lowest rpkm reads by rna-seq of and , respectively (gray histograms in figures and and table s ), consistent with earlier gene array-based studies [ ] . for comparison, the well-defined mcmv genes m and m , both represented with multiple clones in our cdna library, have rpkm values of , and , respectively. because some viral transcripts with higher rpkm reads were not represented in the classical cdna library (for example, an rpkm of for the m gene), we conclude the cdna library cloning did not capture all viral transcripts. nevertheless, the failure to identify clones in regions poorly represented by rna-seq data suggests that further attention is required to prove or disprove the existence of genes predicted by in silico (orf) analysis. ( ) novel spliced transcripts. one of the most striking aspects of the cloning study was the abundance of novel spliced transcripts. a total of novel spliced transcripts were cloned in this study as well as spliced transcripts reported by others. a complete list of spliced transcripts identified in this and other studies is provided in table s . one of the most abundant novel spliced transcripts identified in this study maps to the m gene ( cdna clones). current annotation for m predicts a protein of aa, whereas the clones in this study predict a novel truncated protein product. splicing results in a change of open reading frame at amino acid residue of the currently annotated m protein and introduction of a new stop codon at position (dataset s ). by far the most abundant novel spliced transcript is that overlapping m in the s orientation, and m in the as orientation and is discussed in detail below. approximately % of the viral cdna clones and % of all viral reads from the rna-seq analysis mapped to the novel spliced transcript at the right end of the genome. the structure of this transcript relative to current gene annotation is shown in figure a and the spliced nature of this transcript is also apparent in the rna-seq profile (gray histogram). the longest predicted orf extends into the second exon and predicts a protein of aa, of which the first residues matches the predicted m protein sequence ( figure b ). to determine if this transcript is translated, an antibody was prepared to the protein sequence predicted for m . this antibody was used in immunoblot analysis of cell lysates prepared from mock-infected cells and cells infected with wild-type (wt) bac-derived smith strain virus, or various multi-gene and single-gene mutant virus strains. this antibody reacted with a protein of approximately the expected size ( kda) ( figure d ) in cells infected with wt virus and mutant viruses that express most or all or the mat transcript as determined by northern blot analysis ( figure c ). while the mat protein is first detected hrs after infection, maximal amounts are observed at and hrs after infection ( figure e ). mat protein was also detected in fibroblasts exposed to five different strains of mcmv isolated from wild mice, indicating that the coding region of the gene is conserved in laboratory and wild strains of mcmv ( figure g ). most remarkably, these findings demonstrate that the mat gene generates a single transcript with both noncoding [ , ] and protein coding functions. we also investigated the possibility that mat protein accumulation is directly related to control of the mat transcript levels by cellular mir- [ ] . marcinowski and colleagues have shown that when the binding site for mir- is mutated (m -mut virus), mat transcript levels were increased twofold in comparison to levels obtained in cells infected with wt mcmv at hours after infection due to loss of transcript regulation by the microrna. the difference in mat transcript abundance between wt and the m -mut virus was lost by hours after infection. however, we observed that mat protein levels were similar in cells infected with the wt and m -mut viruses at and hrs after infection ( figure h ). we conclude that the noncoding function of the mat transcript (regulation of cellular mir- ) is unrelated to mat protein accumulation. in addition to providing valuable insight into transcript structure, rna-seq analysis revealed several new facets of the viral transcriptome. first, accumulation of individual viral transcripts varies by several orders of magnitude. figure a depicts the number of rna-seq reads mapped against the mcmv genome in which rows , and visualize the data with the maximum number reads set to , , , and , respectively. validating the classical cloning study, the most abundant transcript identified by rna-seq is the mat transcript. enumeration (rpkm) of the most abundant transcripts is presented in figures b and c , and shows that after the mat transcript, the most highly expressed genes are m , m , and m , all genes without assigned functions. also highly expressed are the immune evasion genes m and m , m (glycoprotein b), and additional genes of unknown functions (m and m ). second, as shown by comparing figure b to c, both the overall magnitude of expression and the ranking of the abundance of different transcripts vary according to annotation used for the rpkm analysis. third, analysis of reads mapped at the highest resolution ( figure a , row ) indicates that most of the viral genome is transcribed to some degree. remarkably, or % of the reads mapped to intergenic regions, depending on annotation ( figure d ). this percentage is reduced to % when annotation is modified to reflect the correct mat gene structure identified in this study. rna-seq also detected significant transcription in m -m ( figure s ), m -m and m -m ( figure e ) intergenic regions. in contrast, the transcriptional profile of the annotated m shows less transcriptional activity than the adjacent intergenic regions. similarly, rna-seq identified transcription from genes that were not isolated in the classical cdna library or in previous studies using microarray technology [ , ] . a detailed analysis of the sensitivity of this rna-seq study to previous studies is provided in supplemental datasets s a-c. we also compared our rna-seq data to a recent rna-seq analysis of the mcmv transcriptome using bac-derived wt virus on nih- t fibroblasts [ ] . as shown in supplemental figure s , the profiles obtained from these two different rna-seq experiments are remarkably similar despite using different sequencing platforms and library generation approaches. also, either seven or eight of the most abundant genes were identical in both datasets (supplemental dataset s c). minor differences in abundance of some transcripts can be attributed to differences in the time points analyzed in these two studies as well as the fact that our analysis achieved an order of magnitude greater sequencing depth (compare reads analyzed for each histogram set in figure s ). together these findings demonstrate that rna-seq analysis is a highly sensitive method for detection of viral gene expression during infection. moreover, these findings highlight numerous incongruencies with current annotation for the mcmv genome. finally, rna-seq analysis revealed that many of the most abundantly expressed viral genes are of unknown function. because cdna cloning and rna-seq identified significant differences between the mcmv transcriptome and current annotations, we performed an in depth analysis of several genomic regions by northern analyses ( figure , figures s , s , s , s ) using our cdna clones to generate strand specific riboprobes ( table ) . to investigate genomic regions where transcripts overlapping more than one gene were detected, we analyzed transcription in m - and m - regions. in both regions multiple transcripts were detected with different temporal expression patterns. smaller transcripts tended to accumulate at later time points, a feature previously reported for certain transcripts in both hcmv and mcmv [ ] [ ] [ ] . in the m -m region transcripts were cloned, all of which overlapped the predicted m and m genes, and one transcript was spliced ( figure a ). the rna-seq profile figure . verification of new transcripts by northern blot. balb/c mef cells were infected with bac derived smith virus and harvested at indicated times post infection. total rna was separated by denaturing gel electrophoresis, transferred to nylon membrane and incubated with probes specific for s and as transcripts. rna integrity and loading was evaluated by inspecting s (not shown) and s rrna bands under uv light after transfer to membrane. transcripts in the m - (a), m -m (b), m (c) and m -m (d) gene regions were analyzed (due to smiling effects during gel electrophoresis for the image shown in a and c, the ladder was not accurate for inner lanes of the gel and the position of the ribosomal bands was therefore used to estimate the band sizes). predicted genes (rawlinson's annotation) are depicted as empty arrows, while thin black arrows show longest transcripts cloned in our cdna library as well as clones used to generate probes (marked with *). ends of transcripts are marked with arrowheads. the nucleotide coordinates relative to smith sequence (nc_ . ) of isolated transcripts are given below thin arrows, while the names of the clones are written above. thin gray lines show isolated transcripts that cannot be detected with the probe. gray histograms showrna-seqreads aligned to mcmv genome. maximal possible exposure times were used to ensure even low abundance transcripts are detected and are noted on the blots. doi: . /journal.ppat. .g ( figure a , gray histogram) also strongly indicated transcription that spans both predicted genes. consistent with our cdna library, no antisense transcripts were detected while the sense probe detected transcripts ( figure a , bands - ). the end of all cdna clones end at or close to nucleotide position (supplemental table s ) and rna-seq data alignment to mcmv genome indicates a sudden drop in reads around this nucleotide position. assuming that transcripts in this region are coterminal, band sizes predict transcript initiation sites in m , m , m or m , and m ( figure a , bands - ). similar results were found in this region in cells infected with wild isolates of mcmv (alec redwood, personal communication). while the smallest band observed by northern analysis ( figure a , band ) corresponds in size to the novel spliced transcript, e ( bp), we could not confirm splicing by pcr using intronflanking primers (data not shown). therefore, it is likely this spliced transcript represents an aberrant transcript or a result of intramolecular template switching during reverse transcription [ ] . the m gene region also diverged substantially from current annotation. similar to m - region, in the m region transcripts with differential temporal expression patterns were detected by northern analyses using clone ie as a probe ( figure b ). no transcript was detected using an as probe derived from clone ie or l , indicating that m is not transcribed in the sense orientation ( figure b and figure s ). we therefore propose that m should be removed from mcmv genome annotation. this is consistent with our cloning study where transcripts have been isolated in this region and none overlap m in the sense orientation (supplemental table s ). evidence from the cdna library and rna-seq alignment (table s and figure b ) indicates that bands detected in northern are co-terminal, with the end located close to nucleotide position . the largest band at kb ( figure b , band ) is detectable at and hours pi and we predict that this transcript initiates in m . we failed to detect transcripts between the m and m genes consistent with previous studies [ ] and northern analyses using mutant viruses lacking genes in this region (data not shown). the lack of cdna clones can be explained by low abundance and size of this transcript, as well as by propensity of cdna libraries to enrich ends. the band slightly smaller than kb ( figure b , band ) shows a peak accumulation at hours pi and is consistent with a transcript overlapping m -m (approx locations - , . kb). the band of approximately kb ( figure b , band ) shows peak accumulation hours pi. based on the rna-seq profile this band could represent transcripts that initiate at nucleotide position . finally, the late time points are dominated by smaller transcripts of approximately kb ( figure b , band ; predicted start site at ) which correspond in size to the cdna clones detected in our study. in short, northern analyses support the conclusion that transcripts overlapping multiple genes in the m -m region and m -m region accumulate in infected cells and indicate that additional, larger transcripts are transcribed in these regions which have yet to be characterized. next we analyzed gene regions in which we detected novel spliced transcripts. the m region was chosen as an example of a highly abundant spliced transcript of unknown function in addition to m - transcript. current annotations predict an orf of . kb whereas rna-seq profiles and cdna study both detected a slightly shorter ( . kb) transcript with an bp intron. northern blot analysis ( figure c ) identified a strong band of appropriate size ( . kb) that starts to accumulate at ie times and peaks at e and l times after infection. due to the small intron and high abundance of this transcript, unspliced transcripts could not be definitively resolved by northern analysis but were confirmed by pcr using primers flanking the intron ( figure s ) . additionally, northern analysis detected another less abundant band of approximately kb. leatham and colleagues [ ] have detected a band of similar size in the homologous region in hcmv of . kb that encompasses ul - genes. while we failed to isolate cdna clones overlapping m region, we predict the larger, less abundant kb band observed initiates in m , though additional northern or race studies are needed to confirm the start site of the larger transcript. the m -m region was previously shown to have a very complex transcriptional profile [ , ] . cdna library data, rna-seq data and results of northern analyses with l as a probe all are in agreement with the findings of scalzo et al. [ ] of multiple spliced transcripts that share exon . bands - ( figure d ) correspond to previously reported m , m and m . spliced transcripts. in our cloning study four isolated clones correspond to m . transcripts (represented by the longest clone, l ) and one to m (l ) (supplemental table s ). transcripts corresponding to m were not isolated in the cloning study, however the rna-seq profile in the region corresponding to m exon shows active transcription ( figure s d ). we have also detected a . kb band ( figure d , band ) that corresponds to longer m and m transcripts, and bands corresponding to unspliced transcripts of m and m . (approx. kb, figure d , band ). in addition to these previously published transcripts, we have also cloned one novel spliced transcript from this region, e . in accordance with work in the m region [ ] , we propose m s as a designation for this novel gene. like other spliced transcripts from this region, e shares exon with other transcripts while its splice donor site is located at . the spliced nature of this transcript has been confirmed by pcr ( figure s c) , however more analyses are needed to determine its exact start site. northern analysis revealed a band of . kb ( figure d , band ) that corresponds in size to the e spliced transcript whereas the unspliced version is detected around kb ( figure d, band ) . in addition, a band of similar size ( . kb) transcribed from the plus genomic strand detected by the l probe ( figure figure s b ) could correspond to the unspliced version of e . all probes used in this region detected bands transcribed from negative genomic strand that correspond to those previously reported by rapp et al. [ ] . based on additional northern blots using cdna clones l (as to m ) and l (as to m ) ( figure s a and b) as well as previous reports [ , ] we conclude that the kb transcript starts in m and ends in m and corresponds to transcript encoding gh while the kb transcript starts in m and ends in m and corresponds to transcript encoding dutpase (supplemental figure s ). additional very large transcripts transcribed from plus or minus genomic strands detected by the l and l probes, respectively, have yet to be characterized but underscore the complex transcriptional patterns in this region. last, we set out to confirm novel antisense transcripts detected in the cdna library. analysis of transcription in m -m region confirmed previously published findings. we have detected single m transcript from plus genomic strand as described by scalzo [ ] ( figure s a) . a probe derived from m detected a single transcript from negative genomic strand corresponding to m [ ] , and from the positive genomic strand that correspond to those described by cranmer et al. [ ] and are in line with our cdna and rna-seq analysis ( figure s b) . the presence of sense and antisense transcripts in this gene region corresponds to findings for hcmv [ ] . finally, in the m gene region we detect transcripts from plus genomic strand that correspond to those described by lyons et al [ ] (figure s c ). temporal expression of transcripts detected by northern in this region is in line with our cdna analysis and previously published data [ , ] . based on northern analyses of regions, we conclude that our cdna and rna-seq analyses faithfully represents the mcmv transcriptome in infected primary fibroblasts and confirms the presence of novel transcripts. moreover, the distribution of clones in the ie, e and l cdna libraries accurately reflected the accumulation of transcripts detected by northern analyses. rna-seq analysis also enabled us to investigate changes in the host transcriptome. differentially expressed (de) murine genes in mcmv-infected cells compared to mock-infected cells were identified by calculating rpkm. this analysis identified , statistically significant (p, . ) genes altered by infection ( table s ) . the top induced, upregulated, repressed and downregulated genes are presented in tables - , (genes associated with characterized biological pathways are in bold). interferon b (ifnb ) and the interferon-inducible pyhin (a.k.a. ifi- , ifix) were among the top induced genes, consistent with the expected host response to virus infection. also congruous with expected host responses to infection were two highly induced genes associated with apoptosis induction (hrk and tnfsf [a.k.a. trail]). interestingly, transcription factors (foxa , en , insm , tbx , [a. k.a t-bet], and tp ) were among the most strongly induced by mcmv infection. chemokine ligands dominated the group of the top upregulated genes. genes encoding proteins with roles in intrinsic cellular defense were also highly upregulated, including oas , mx , gpb and rsad (a.k.a. viperin). there were also a surprising number of genes involved in development, differentiation, and stem cell renewal strongly induced or upregulated by infection, including foxa , spint , lin b, en , gabrq, esx , trim , trp , cpne , cdh , cited , pou f , and jag . the relevance of these genes, as well as others including art , ugt , and trank to infection is not clear. we analyzed protein levels of several induced and upregulated transcripts whose relevance to mcmv infection is unknown (figure ) including the notch ligand jagged , the homeobox-containing transcriptional factor engrailed , and the e ubiquitin-protein ligase trim . protein levels of all proteins tested correlated with their transcript levels in infected balb/c fibroblasts. interestingly, the top repressed and downregulated genes are primarily of unknown relevance to infection, though many are receptor or cell surface molecules (npy r, rxfp, mc r, cd r , antxrl, scara , il r , agtr , gpr , the olfactory receptor genes, olfr and olfr , and the lectin or lectin-like genes clec b and reg a). mcmv infection also caused repression or downregulation of noncoding (nc)rnas including the small nucleolar rna gene, snord a and genes of unknown function including long intergenic noncoding rnas (lincrnas), the miscellanous rna, b rik, and antisense transcripts (gm , gm ). to summarize, while many of the top upregulated genes are associated with host responses to infection, the function of many of cxcl chemokine (c-x-c motif) ligand . ccl chemokine (c-c motif) ligand , rantes . trank tetratricopeptide repeat and ankyrin repeat containing . cxcl chemokine (c-x-c motif) ligand ; mig . genes and their products do not work in isolation but rather form pathways and networks. even small perturbations in gene expression in a pathway can exert profound influences on eventual processes or functions. therefore, we analyzed gene lists for shared common pathways. as expected, the top scoring gene networks for all differentially expressed (de) genes included (i) infectious disease, antimicrobial response, inflammatory response ( focus molecules); (ii) inflammatory response, cellular development, cellmediated immune response ( focus molecules) and (iii) cell morphology, hematological system development and function, inflammatory response ( focus molecules) ( table s a) . these were also top networks when the subset of up-and down-regulated genes were evaluated (table s b) . also identified were networks associated with cell morphology and hematological system development and function. when this analysis was conducted with only induced and repressed genes, the top networks included cellular development, cell-mediated immune response, cellular function and maintenance, gene expression and embryonic development ( table s c ). the relationships among the molecules in top networks for differentially regulated and induced/ repressed genes are shown in figures figure s and figure s . thus, an unexpected outcome of this analysis is that mcmv infection influences a subset of networks controlling development.. the biological functions and/or diseases that were most significant to the molecules in the mcmv-regulated networks are shown in figure a . immunological disease, cardiovascular disease, genetic disorders, and skeletal and muscular disorders ranked as the top bio-functions connected with genes altered by mcmv infection. among molecular and cellular functions, cell growth and proliferation were the top ranked perturbed functions, consistent with known effects of lytic mcmv infection of cells. nervous system development and function is at the top of the list of physiological and developmental biofunctions, followed by organismal and tissue development and, surprisingly, behavior with associated genes. de genes were also evaluated for canonical pathways in the ingenuity library ( figure b) . the pathways most affected by mcmv included g-protein coupled receptor signaling followed by pathogenesis of multiple sclerosis and gaba receptor signaling. together, these analyses point to known and expected consequences of infection at the cellular level (i.e., cell growth and proliferation, g-protein coupled receptor signaling) and physiological level (i.e. nervous system development) but also highlight unexpected cell and molecular functions, as well as physiological systems and disorders that may advance the understanding of cmv pathogenesis. gene ontology (go) enrichment using gorilla ranked lists analysis [ , ] was also used to analyze de genes. the full list of enriched go terms long with associated genes is shown in table s . gorilla analysis highlighted processes associated with upregulated genes including cell differentiation, neuron differen- tiation, regulation of ion transport, and the g-protein coupled receptor signaling pathway. genes downregulated during mcmv infection were associated with many processes, including regulation of cell shape, adhesion, motility, and the extracellular matrix. altogether, gorilla analyses support results of the ingenuity pathway analysis and suggest novel processes regulated in infected cells, notably suggesting that infection leads to a restructuring of the extracellular environment of the infected cells. we report a comprehensive analysis of the mcmv transcriptome during lytic infection derived from cloning and sequencing of viral transcripts and next generation sequencing (rna-seq). by combining the approaches of rna-seq and traditional cdna cloning as well as northern and rt-pcr analyses in certain complex regions, we were able to construct a comprehensive profile of viral and host transcription during lytic infection. we also investigated the host transcriptome using rna-seq combined with differential gene expression analysis, pathway analysis, and gene ontology analysis. the major findings are as follows: ) the mcmv transcriptome diverges substantially from that predicted by current annotation; ) the identification of a novel viral protein specified by the mat transcript indicates that this transcript functions as an mrna and a non-coding rna; ) the majority of the most abundantly transcribed viral genes are of unknown function; and ) the host response to infection includes regulation of many host genes and gene networks of unknown relevance to infection. there are four major findings from the analysis of the mcmv transcriptome. first, we demonstrate novel transcripts of mcmv including novel splice variants, transcripts that map to noncoding regions, and transcripts overlapping multiple genes. earlier, we reported similar novel transcripts of hcmv through analysis of a classical cdna library [ ] . this study revealed a dramatic increase in the complexity of viral gene products compared to currently available predictions and its findings were later on confirmed by rna-seq analysis [ ] . a more recent analysis of hcmv translational products [ ] by ribosomal footprinting identified over translated orfs -a strikingly high number compared to annotated genes. this discrepancy is, at least in part, a consequence of the polycistronic nature of hcmv transcripts which appear to code for many more orfs than previously predicted (internal in frame or out-of-frame orfs, uorfs) as well as orfs coming from antisense or dedicated short transcripts. our analysis demonstrated that the mcmv transcriptome is similarly complex: we identified several regions where multiple co-terminal transcripts expressed in different temporal phases are being transcribed. such transcripts have the potential to code for truncated protein forms or even completely new proteins as described for hcmv, suggesting that the size and complexity of the mcmv proteome, like the mcmv transcriptome, is currently underestimated. accumulation of ncrnas is also a prominent feature of the cytomegalovirus transcriptomes. our rna-seq analysis shows intense transcription in previously described stable mcmv introns and in intergenic regions, consistent with abundant ncrnas reported for hcmv and mcmv [ , , ] . these findings have a profound implication for understanding studies of cmv genes functions and underscore the need for transcriptomic maps in addition to genomic maps depicting only orfs. the functions of many mcmv genes have been elucidated by using deletion mutants [ ] . however in a transcriptionally complex region of the genome any deletion will likely impact multiple transcripts and possibly multiple proteins resulting in complex phenotypes. in line with previous studies [ ] , we identified novel as transcripts of mcmv. interestingly, preliminary estimates in our cloning study indicate that as transcripts occur at much lower frequency than reported for hcmv [ ] . there are likely to be additional as transcripts of mcmv. because we did not capture every known sense transcript of mcmv, we may presume that the cdna cloning study did not capture all as transcripts. in addition, the rna-seq analysis performed in this study was limited by the fact that the methods employed did not provide strand-specific information and could not identify novel as transcripts. as transcripts, even those expressed at low levels, may possess noncoding rna functions and contribute to complexity of the proteome as has described for hcmv [ ] . therefore, further studies are needed to determine the number and nature of as transcripts derived from mcmv and will be critical to generating definitive transcriptome and proteome maps of this virus. the cdna library analysis does suggest that the extent of mcmv as transcription is lower than that described for other herpesviruses, including hcmv. these results are consistent with a strand-specific rna-seq experiment performed by dölken group [ ] that also show poor as transcription in comparison to sense counterparts. very little antisense transcription was also noted for the anguillid herpesvirus (anghv ) infecting eels [ ] , though extensive antisense transcription was reported for other herpesviruses, including kshv and mhvc [ , ] . we conclude that different members of the herpesviridae family differ in the extent of antisense transcription during lytic infection. second, we observed similar inconsistencies between transcriptomic data and gene annotation for mcmv as previously reported for hcmv [ ] . these discrepancies can profoundly impact future studies related to the quantitative analyses of gene expression, interpretation of microarray studies, comparisons to newly sequenced virus strains, and studies using deletion mutant virus strains. the results presented here represent an important first step in re-annotation of the mcmv genome and underscore the utility of transcriptome studies in validating and refining genome annotation for microbial pathogens. third, analysis of the mcmv transcriptome revealed the striking abundance of the spliced mat transcript. this gene is also largely conserved in wild isolates of mcmv (alec redwood, personal communication and [ ] ) and the protein is expressed by wild isolates tested in this study. mat abundance may reflect its multiple functions. the untranslated region (utr) of this transcript facilitates degradation of murine mir- , establishing that this transcript functions as a noncoding rna molecule [ , ] . members of the alpha, beta, and gamma herpes virus subfamilies all encode for abundant, largely enigmatic noncoding rnas including the latency associated transcript (lat) of herpes simplex virus (hsv), ebna rnas of esptein-barr virus (ebv), the b . transcript of hcmv, the pan rna of kaposi's sarcoma herpes virus (kshv) and the hsur transcripts of herpesvirus samiri (hvs) which also downregulates the cellular mir (reviewed in [ ] ). in addition to the noncoding function of the mat, we demonstrate that this transcript also encodes for a novel small protein of approximately kda. to our knowledge, this is the first herpes virus transcript we know of that functions both as a noncoding rna, and an mrna that specifies a novel viral protein. fourth, a somewhat startling finding from the quantitative rna-seq analysis was that after mat, the most abundant viral transcripts in infected cells are derived from genes without known functions, including m . we report that m is a novel spliced transcript predicted to specify a much smaller protein compared to current annotation. these results highlight fundamental gaps in our understanding of basic mcmv biology. we found that the cdna library and rna-seq approaches yielded remarkably complementary data including identification of novel transcripts and new insights into transcript abundance, despite different biases in each of these methods. for example, while there may be selection bias for isolating transcripts with long tracts of adenosines during cdna library construction [ ] , gc content, bias in the sites of fragmentation, primer affinity and transcript-end effects may influence rna-seq results [ ] . future rna-seq studies may also facilitate novel gene identification as rna-seq has now been applied to ab initio reconstruction of gene structure [ ] using only rna-seq data and the genome sequence. however, currently available algorithms are unable to cope with highly dense genomes, such as mcmv and other viral genomes. until such tools are developed for very dense genomes, rna-seq data relies upon comparison to existing gene annotation and other experimental methods for gene structure prediction. in this study, we compared rna-seq to currently used annotations but also to the cdna library study, northern analysis, and rt-pcr studies to identify and validate numerous novel transcripts. we also report that lytic infection elicits a profound cellular response in fibroblasts. this study identified , differentially regulated genes. as the number of mouse genes is estimated to be , [ ] we estimate that over % of mouse genes are altered in response to infection. many of the top upregulated and induced genes and gene networks were associated with immune responses to infection, including interferon and interferon-inducible genes such as phyin , a potential activator of p [ ] , the inflammasone regulator gpb [ ] and rsad (a.k.a. viperin), also known to be induced by hcmv [ ] . inflammatory chemokine ligand genes are also highly upregulated during infection. mcmv encodes virally-derived chemokine homologs specified by the m /m genes [ , ] and at least one chemokine receptor homolog, m [ ] . numerous host chemokine receptors are also upregulated by infection, suggesting a remarkably complex interplay between mcmv-derived and host derived chemokine signaling during infection. induction of inflammatory gene networks by mcmv also lends credence to the hypothesis that inflammatory responses link cmv infection to chronic diseases, such as chronic allograft rejection, cardiovascular disease, and cancer [ , , ] . numerous transcription factors are also induced or upregulated by infection including insulinoma-associated (insm ). recently, insm was found to be strongly upregulated by hsv- infection and shown to promote hsv gene expression, probably by binding the hsv infected cell protein (icp) promoter. [ ] . this raises the intriguing possibility that insm plays a similar role in promoting virus gene expression during mcmv infection. another induced transcription factor induced at the transcript and protein level is engrailed- (en ). this transcription factor is key to patterning cerebellar foliation during development [ ] . we previously described a profound dysregulation of cerebellar development in brains of neonatal mice infected with mcmv [ ] , suggesting a possible physiological link to regulation of this gene. another top induced gene was the gaba receptor, gabrq. glutamate receptor signaling was also identified as significantly impacted canonical pathways in our dataset. in the developing brain gaba and glutamate receptors influence neuronal proliferation, migration, differentiation or survival processes [ ] . whether and how these observations relate to our previous findings that mcmv infection of neonates results in decreased granular neuron proliferation and migration [ ] are important areas for future study and may impact our understanding of neurological damage and sequelae associated with hcmv in congenitally-infected infants. perhaps most importantly, many top regulated genes, especially downregulated and repressed genes, are associated with functions whose roles in infection are obscure, including many genes of unknown function. many downregulated or repressed genes are cell surface molecules, host lincrnas, antisense rnas, or small nucleolar rnas. regulation of lincrnas was recently observed during infection with severe acute respiratory syndrome coronavirus (sars-cov) and influenza virus, and have been suggested to impact host defenses and innate immunity [ ] . further studies to identify the functions of these downregulated and repressed genes and noncoding rnas during mcmv infection may well provide novel insights into the virus-host molecular interface as well as possible therapeutic targets. this analysis also revealed immunological disease, cardiovascular disease, genetic disorders and skeletal and muscular disorders as top bio-functions connected with genes altered by mcmv infection. while mcmv involvement in cardiovascular disease is a subject of intensive research, potential involvement in skeletal and muscular disorders is not well documented but may be relevant to the novel observation that mcmv infection of mice with a heterozygous trp mutation develop rhabdomyosarcomas at high frequency [ ] . a primary caveat of rna-seq analysis is determining whether changes in gene transcript levels are also reflected at the protein level. this is particularly important as herpesviruses can control protein accumulation at the post-transcriptional, translational, and post-translational levels [ ] [ ] [ ] . we confirmed that the notch ligand, jagged , is highly upregulated by infection at both the transcript and protein level. notch signaling is a highly conserved signaling pathway that plays important roles in development, including neurogenesis and differentiation of immune cell subsets [ ] . jagged is also upregulated by the alphaherpesviruses, hsv- and psuedorabies viruses [ ] . kshv and ebv also exploit the notch signaling pathway to facilitate aspects of their life cycle [ ] and notch signaling is proposed to influence hsv- -induced interferon responses [ ] . we show for the first time that the betaherpesvirus, mcmv, also influences notch signaling. dysregulation of jagged as a consequence of mcmv infection is highly interesting since it plays a role in important processes affected by cmv including inner ear development [ , ] , generation of motor neurons [ ] and differentiation of immune cell subsets [ , ] . to summarize, this study has refined the understanding of mcmv gene expression and identified new areas of research to advance our understanding of the host response to these ancient viruses. we describe what is to our knowledge, the first herpes virus transcript that functions as both a noncoding rna that limits accumulation of cellular mirnas, and an mrna that specifies a protein. this study also revealed novel features of the host response to infection. perhaps most importantly, this study identified many virus and host genes of unknown function that are regulated during infection. it is highly likely that further study of these genes may lead to breakthroughs in the understanding and treatment of cytomegalovirus-related diseases. primary mouse embryonic fibroblasts (mefs) from balb/c or balb.k mice were prepared and maintained as described [ ] and used between passages - . immortalized murine balb.k mefs, (mef.k) [ ] and svec - (atcc crl- ) were used for immunoblot studies. mcmv smith strain (atcc vr- ) was propagated and titrated on primary balb/c mef by standard plaque assay as described in detail in [ ] . wild type mcmv isolates k (genbank acc no: am . ), c d, k and wp b (genbank acc no: eu . ) [ ] were a kind gift from a. redwood (university of western australia, australia). construction of the d s , dm , dm , dm , dm , and m -mut mutant viruses were previously described and were generated by et-cloning [ ] using the full-length mcmv bac psm fr [ ] . the double deletion mutants (dm dm and dm dm ) were constructed exactly as described previously [ ] . primers for construction of the double deletion mutants are also as described [ ] using the forward primer for the first gene and reverse primer for the second gene. all infections were conducted by exposing cells to . pfu/cell followed by centrifugal enhancement for minutes at g, as described in [ ] . smith mcmv infected cbalb/c mefs were harvested h post infection and viral dna was isolated as described [ ] . rna was extracted from smith mcmv infected balb/c mef at , and hrs after infection (ie library); , and hrs after infection (e library); and , and hrs after infection (l library). no drug was used to select for different temporal classes of transcripts and equal amounts of rna from each time point were pooled prior to library construction. cdna libraries were generated as described previously for hcmv [ ] by following the instruction manual for the superscript plasmid system with gateway technology for cdna synthesis and cloning (invitrogen) with some minor modifications. briefly, total rna was isolated using the trizol reagent (invitrogen, ca, usa). a poly(t)-tailed paci primer-adapter was used for first-strand cdna synthesis ( -gcggccgcttaattaacc(t) - ) . after sec-ond-strand synthesis, an ecori-pmei adapter was added to the end and cdnas were cleaved with ecori and paci. the ecori-pmei adapter was generated by annealing following oligonucleotides: -aattcccgcgggtttaaacg- and -pho-cgtttaaacccgcggg- . cdna fragments were inserted into a modified pcdna . (+) previously digested with ecori and paci and transformed into xl -blue supercompetent e. coli cells (stratagene, ca, usa). positive selection of viral cdna clones was performed as described previously [ ] . mse i-digested genomic mcmv dna was labeled using a dig high prime dna labeling detection starter kit ii (roche applied science) and used to identify virallyderived cdna clones. plasmids harboring cdna clones that reacted with probe were isolated and sequenced from the end using t primer for pcdna . (+) or the ends using primer ( gcaccttccagggtcaaggaag) or standard poly (t) primers at the osu plant-microbe genomics facility. sequences were compared to the mcmv smith strain genome [genbank accession no. nc_ ] using mega blast. total rna was extracted from balb/c mef cells cultured in mm petri dishes and exposed to . pfu/cell of the mw . strain of murine cytomegalovirus or mock-infected. at , , , , , , , and hours after infection, rna was isolated using trizol reagent. rna integrity was assessed on agilent bioanalyzer and only samples with rna index values of at least were used. equal amounts of rna from each time point were pooled ( . mg of rna per time point) and treated with dnasei. libraries were prepared with illumina truseq rna kit according to manufacturer's instructions and sequenced on illumina genome analyzer iix as single-end bp reads. the illumina truseq rna kit employed does not allow for strandspecific information to be derived from the sequence data. datasets are available at the national center for biotechnology information (ncbi) sequence read archive (sra) accession no. srr (sequence reads from mcmv-infected mefs) and accession no. srr (sequence reads from mockinfected mefs). reads were aligned to mouse (ncbi /mm assembly) and mcmv genome (genbank acc.no. nc_ . ) using eland aligner or bowtie aligner (for comparison with data provided by lars dölken). it is important to note that both eland and bowtie aligners do not map splice junctions and thus give concordant results. alignments were visualized using integrative genomics viewer (http://www.broadinstitute.org/igv/) [ ] . differential gene expression was assessed by calculating rpkm (reads per kilobase of million mapped reads (rpkm) using sammate . . . release with edger (http://sammate. sourceforge.net/) [ ] . gene ontology (go) enrichment analysis was performed on filtered lists of differentially expressed genes (p, . ) using gorilla ranked lists analysis [ , ] . ingenuity core analysis (ingenuity systems, www.ingenuity.com) was used for gene interaction network and canonical pathway analysis. gene lists were filtered for statistically significant differentially expressed genes (p, . ) and a fold change cutoff of was set to identify molecules whose expression was significantly differentially regulated. for network generation, these molecules (network eligible molecules), were overlaid onto a global molecular network developed from information in the ingenuity knowledge base based on their connectivity. the functional analysis of a network identified the biological functions and/or diseases that were most significant to the molecules in the network. right-tailed fisher's exact test was used to confirm that biological functions and/or disease assigned to data sets were not due to chance. the nature of individual de genes was also investigated using the mouse genome informatics databases (http://www.informatics.jax.org/) [ ] and entrez gene (http://www.ncbi.nlm.nih.gov/gene) [ ] . rna was isolated using trizol reagent from mock or mcmvinfected balb/c mef at hours (mat) or , and hrs after infection. rna ( mg/lane or mg/lane (mat)) was separated by formaldehyde agarose gel electrophoresis and transferred to positively charged nylon membrane and crosslinked by uv irradiation. membranes were reacted to dig-labeled probes overnight at uc. for mat detection, a dig labeled double-stranded dna probe was made using fragments corresponding to the mat gene sequences derived from cdna library clones e , e and e using roche's dig-high prime dna labeling and detection starter kit i. for all other northern blots, single-stranded dig-labeled rna probes were used generated using roche's dig northern starter kit. antisense probes were generated by in vitro transcription from t promoter present in pcdna . plasmids containing cdna clones that harbor the desired gene fragments (table ) . therefore antisense probes are identical to transcripts cloned in cdna library and can detect transcripts antisense to cdna clones. to generate sense probes, t promoter was added to end of complimentary strand of the gene fragments used for antisense probes by pcr ( table ). the pcr fragments were then in vitro transcribed and dig labeled using t rna polymerase. care was taken to generate sense probes of length comparable to corresponding antisense probes. the m gene sequence was amplified by pcr using viral dna isolated from mcmv bac psm fr using following primers: f: -tttttggatccatgagcaacgcggtcccgttc- and r: -tttttctgcagtcatcacggggggcacc-tacc- , reacted with bamhi and hindiii (new england biolabs), inserted into pqe expression vector and introduced to e.coli bl prep strain (qiagen). the protein was induced according to manufacturers' instructions and purified on a his-tag column. purified protein was used for immunization of balb/c mice and antibody titer in blood serum was measured by elisa. when antibody titer in serum reached adequate levels, animals were sacrificed, their spleens isolated and fused with sp /o cells. supernatants from motherwells were tested by immunoblot blot on purified mat protein and positive wells were rescreened by immunoblot using lysates from mefs infected with wt, d s , d - , d - , dm , dm and dm mutants as described below. rna from mock-or mcmv-infected cells isolated for northern blots was also reverse transcribed using oligo-dt primers (proto-script m-mulv taq rt-pcr kit, new england biolabs). no reverse transcriptase (-rt) controls were run in parallel. splicing was then verified by pcr amplification using primers that flank putative introns (m ; f: cttcatcggattcggaggc; r: tgttgttgtcgacgtctgatgtg; m -m ; f: atc-tcctctgcctccgacctc, r: cgatgtcatcttggaa-tccgacga; m -m ; f: ccggatacgaccgtcagc, r: cgatgtcatcttggaatccgacga) using phusion high fidelity polymerase (new england biolabs). mock-infected or mcmv-infected primary mefs, or murine cell lines (mef.k, svec - ) were lysed in ripa buffer. protein lysates were separated by sds-page and transferred to pvdf. mat protein was detected with anti-m antibody described above, jag with antibody n- (santa cruz), engrailed with en pa - antibody (thermo scientific), trim with pa - (thermo scientific), and actin with antibody c (millipore) followed by peroxidase-labeled secondary antibodies (jackson immunoresearch or abcam). proteins were visualized using amersham ecl prime western blotting reagent (ge healthcare) and quantified using imagej software (http://rsbweb.nih.gov/ij/). dataset s comparison of sensitivity and temporal gene expression data from this study to previous microarray studies of mcmv (s a and s b) and comparison of rpkm values in and this rnaseq experiment (s c). dataset s spliced cdna clone overlapping m and comparison of predicted protein to current annotation. (pdf) figure s rna-seq profiles comparison. rna-seq data from total rna obtained from mcmv infected nih- t fibroblasts from and hrs pi sequenced by dölken group (gse ) was aligned against mcmv genome (gb acc no nc_ . ) using bowtie aligner and visualized in igv in comparison to our rna-seq data. the view of the complete genome is shown at the top with areas magnified below (labeled a-d) and the number of reads displayed are noted on the side. since viral genes display a wide range of expression levels, the whole genome view is shown in wide data range (upper panel) more suitable for displaying highly transcribed regions and a narrowed data range (lower panel) that is more suitable for less transcribed regions. as can be seen, the profiles of the compared alignments are remarkably similar, the only differences being abundance of certain transcripts which are due to different time points analyzed in comparison to the pooled data of our rna-seq and significantly greater depth of at least one order of magnitude of our data in comparison to marcinowski data. (tif) figure s analysis of the m - region. balb/c mef cells were infected with bac derived smith virus and harvested , and hrs post infection. total rna was separated by denaturing gel electrophoresis, transferred to nylon membrane and incubated with probe generated by in vitro transcription from t promoter of l [a; probe should detect predicted m (s) transcripts] or probe generated by in vitro transcription from t promoter of ie transcript [probe should detect m (s)-m (as) transcripts]. rna integrity and loading was evaluated by inspecting s (not shown) and s rrna bands under uv light after transfer to membrane. predicted genes (rawlinson's annotation) are depicted as empty arrows, while thin black arrows show longest transcripts cloned in our cdna library as well as clones used to generate probes (marked with *). ends of transcripts are marked with arrowheads. the nucleotide coordinates relative to smith sequence (nc_ . ) of isolated transcripts are given below thin arrows, while the names of the clones are written above. gray histograms showrna-seqreads aligned to mcmv genome. maximal possible exposure times were used to ensure even low abundance transcripts are detected and are noted on the blots. (tif) figure s verification of m splicing by pcr. schematic of the m gene region and clone l (a) and pcr analysis of the splice site (b). in (a) predicted genes (rawlinson's annotation) are depicted as empty arrows, while the thin black arrow depicts clone l with red arrows depicting the primers used in (b). the ends of transcripts are marked with arrowheads. the nucleotide coordinates relative to smith sequence (nc_ . ). gray histograms showrna-seq reads aligned to mcmv genome. rna isolated from wt infected balb/c mef used in northern blots was reverse transcribed with oligo-dt primers, and then amplified with primers specific for m that flank the putative intron (marked by red arrows). no rt controls were run in parallel. spliced cdna clones and viral dna were used as spliced and unspliced amplification controls, respectively. (tif) figure s analysis of the m -m region spliced transcripts by northern blot and pcr. balb/c mef cells were infected with bac derived smith virus and harvested at indicated times post infection (a and b). total rna was separated on denaturing gel electrophoresis, transferred to nylon membrane and incubated with probes specific for s and as transcripts. rna integrity and loading was evaluated by inspecting s (not shown) and s rrna bands under uv light after transfer to membrane. predicted genes (rawlinson's annotation) are depicted as empty arrows, while thin black arrows show longest transcripts cloned in our cdna library as well as clones used to generate probes (marked with *). images of northern blots are shown using probes derived from the m region in (a) and the m in (b). the ends of transcripts are marked with arrowheads. the nucleotide coordinates relative to smith sequence (nc_ . ) of isolated transcripts are given below thin arrows, while the names of the clones are written above. thin gray lines show isolated transcripts that cannot be detected with the probe. gray histograms showrna-seqreads aligned to mcmv genome. maximal possible exposure times were used to ensure even low abundance transcripts are detected and are noted on the blots (a and b). in (c), the m - spliced transcript and one of two possible m - spliced transcripts were verified by pcr amplification using primers that flank their putative introns. rna isolated from wt infected balb/c mef used in northern blots was reverse transcribed with oligo-dt primers, and then amplified with primers specific to m -m or m -m spliced transcripts (marked by red arrows). no rt controls were run in parallel. spliced cdna clones and viral dna were used as spliced and unspliced amplification controls, respectively. in (d), the position of exon of m reported by scalzo et. al. [ ] , is compared to rna-seq data for this genomic region. (tif) figure s northern blot analysis of the m -m region. balb/c mef cells were infected with bac derived smith virus and harvested at indicated times post infection. total rna was separated on denaturing gel electrophoresis, transferred to nylon membrane and incubated with probes specific for s and as transcripts overlapping m (a), m (b) and m (c) genes. rna integrity and loading was evaluated by inspecting s (not shown) and s rrna bands under uv light after transfer to membrane. predicted genes (rawlinson's annotation) are depicted as empty arrows, while thin black arrows show longest transcripts cloned in our cdna library as well as clones used to generate probes (marked with *). ends of transcripts are marked with arrowheads. the nucleotide coordinates relative to smith sequence (nc_ . ) of isolated transcripts are given below thin arrows, while the names of the clones are written above. thin gray lines show isolated transcripts that cannot be detected with the probe. gray histograms showrna-seqreads aligned to mcmv genome. maximal possible exposure times were used to ensure even low abundance transcripts are detected and are noted on the blots. (tif) figure s graphical representation of top genetic networks for differentially regulated genes. upregulated genes are shown in red, while downregulated are shown in green. level of differential expression is represented by color saturation with most dramatically changed genes being shown in the most saturated color (strong red or green). these overlapping genetic networks are associated with (i) cell-mediated immune response, cellular development, cellular function and maintenance ( focus molecules); (ii) infectious disease, antimicrobial response, inflammatory response ( focus molecules) and (iii) antimicrobial response, inflammatory response, gene expression ( focus molecules) (see supplemental table s ). (tif) figure s graphical representation of top genetic networks for genes induced or repressed by infection. induced genes are shown in red, while repressed are shown in green. level of differential expression is represented by color saturation with most dramatically changed genes being shown in the most saturated color (strong red or green). these overlapping genetic networks are associated with various developmental processes (see supplemental table s ). (tif) cytomegalovirus: pathogen, paradigm, and puzzle manifestations of human cytomegalovirus infection: proposed mechanisms of acute and chronic disease cytomegalovirus reactivation in critically ill immunocompetent hosts: a decade of progress and remaining challenges infection and atherosclerosis. an alternative view on an outdated hypothesis does cytomegalovirus play a causative role in the development of various inflammatory diseases and cancer? the search for new therapies for human cytomegalovirus infections analysis of the protein-coding content of the sequence of human cytomegalovirus strain ad the dna sequence of the human cytomegalovirus genome analysis of the complete dna sequence of murine cytomegalovirus identification of proteins associated with murine cytomegalovirus virions predicting coding potential from genome sequence: application to betaherpesviruses infecting rats and mice experimental confirmation of global murine cytomegalovirus open reading frames by transcriptional detection and partial characterization of newly described gene products temporal profiling of the coding and noncoding murine cytomegalovirus transcriptomes stability of murine cytomegalovirus genome after in vitro and in vivo passage real-time transcriptional profiling of cellular and viral gene expression during lytic cytomegalovirus infection antisense transcription in the human cytomegalovirus transcriptome high-resolution human cytomegalovirus transcriptome decoding human cytomegalovirus posttranscriptional regulation of mir- in murine cytomegalovirus infection degradation of cellular mir- by a novel, highly abundant viral transcript is important for efficient virus replication in vivo uncovering the complexity of transcriptomes with rna-seq characterization of the human cytomegalovirus ul gene the murine cytomegalovirus m . gene, a member of a co-terminal alternatively spliced gene family, encodes the gp virion glycoprotein regulation of cytomegalovirus late-gene expression: differential use of three start sites in the transcriptional activation of icp gene expression reverse transcriptase template switching and false alternative transcripts alternate promoter selection within a human cytomegalovirus immediate-early and early transcription unit (ul - ) defines true late transcripts containing open reading frames for putative viral glycoproteins expression of the murine cytomegalovirus glycoprotein h by recombinant vaccinia virus dna sequence and transcriptional analysis of the glycoprotein m gene of murine cytomegalovirus cloning, characterization, and expression of the murine cytomegalovirus homologue of the human cytomegalovirus -kda matrix phosphoprotein (ul ) mapping and transcriptional analysis of the murine cytomegalovirus homologue of the human cytomegalovirus ul open reading frame gorilla: a tool for discovery and visualization of enriched go terms in ranked gene lists discovering motifs in ranked lists of dna sequences murine cytomegalovirus encodes a stable intron that facilitates persistent replication in the mouse strategies for the identification and analysis of viral immune-evasive genescytomegalovirus as an example genome-wide gene expression analysis of anguillid herpesvirus redefining the genetics of murine gammaherpesvirus via transcriptome-based annotation the lytic transcriptome of kaposi's sarcoma-associated herpesvirus reveals extensive transcription of noncoding regions, including regions antisense to important genes laboratory strains of murine cytomegalovirus are genetically similar to but phenotypically distinct from wild strains of virus noncoding rnps of viral origin local and global factors affecting rna sequencing analysis ab initio reconstruction of cell type-specific transcriptomes in mouse reveals the conserved multi-exonic structure of lincrnas the mouse genome database (mgd): from genes to mice-a community resource for mouse biology stabilization of p in human cytomegalovirus-initiated cells is associated with sequestration of hdm and decreased p ubiquitination gbp promotes nlrp inflammasome assembly and immunity in mammals human cytomegalovirus directly induces the antiviral protein viperin to enhance infectivity cytomegalovirus mck- controls mobilization and recruitment of myeloid progenitor cells to facilitate dissemination spliced mrna encoding the murine cytomegalovirus chemokine homolog predicts a beta chemokine of novel structure functional analysis of the murine cytomegalovirus chemokine receptor homologue m : ablation of constitutive signaling is associated with an attenuated phenotype in vivo herpes simplex virus induces the marked up-regulation of the zinc finger transcriptional factor insm , which modulates the expression and localization of the immediate early protein icp the engrailed homeobox genes determine the different foliation patterns in the vermis and hemispheres of the mammalian cerebellum altered development of the brain after focal herpesvirus infection of the central nervous system glutamate and gaba receptor signalling in the developing brain unique signatures of long noncoding rna expression in response to virus infection and altered innate immune signaling cytomegalovirus infection leads to pleomorphic rhabdomyosarcomas in trp +/ mice the virion-packaged endoribonuclease of herpes simplex virus cleaves mrna in polyribosomes getting the message direct manipulation of host mrna accumulation during gammaherpesvirus lytic infection regulation of translation initiation by herpesviruses notch: control of lymphocyte differentiation in the periphery transcriptional response of a common permissive cell type to infection by two diverse alphaherpesviruses notch and wnt signaling: mimicry and manipulation by gamma herpesviruses inhibition of gammasecretase cleavage in the notch signaling pathway blocks hsv- -induced type i and type ii interferon production notch signaling regulates the pattern of auditory hair cell differentiation in mammals notch signaling and the developing inner ear jagged controls the generation of motor neuron and oligodendrocyte progenitors in the ventral spinal cord the notch ligands jagged , delta , and delta induce differentiation and expansion of functional human nk cells from cd + cord blood hematopoietic progenitor cells expression of notch receptors and ligands on immature and mature t cells a mouse model for cytomegalovirus infection. current protocols in immunology ly p recognition of cytomegalovirus-infected cells expressing h -dk and cmvencoded m correlates with the nk cell antiviral response dissection of the antiviral nk cell response by mcmv mutants mutagenesis of viral bacs with linear pcr fragments (et recombination) systematic excision of vector sequences from the bac-cloned herpesvirus genome during virus reconstitution integrative genomics viewer sammate: a gui tool for processing short read alignments in sam/bam format the mouse genome database (mgd): comprehensive resource for genetics and genomics of the laboratory mouse entrez gene: genecentered information at ncbi we thank corinna benkartek and martin messerle for the mutant viruses and alec redwood for the gift of the wild mcmv viruses and for generously sharing unpublished data. we thank lars dolken for kindly providing their rna-seq data for comparison. we also thank andrea henkel, misel satrak and guojuan zhang for help with the cdna library. key: cord- -o vxqm authors: visser, linda j.; aloise, chiara; swatek, kirby n.; medina, gisselle n.; olek, karin m.; rabouw, huib h.; de groot, raoul j.; langereis, martijn a.; de los santos, teresa; komander, david; skern, tim; van kuppeveld, frank j. m. title: dissecting distinct proteolytic activities of fmdv l(pro) implicates cleavage and degradation of rlr signaling proteins, not its deisgylase/dub activity, in type i interferon suppression date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: o vxqm the type i interferon response is an important innate antiviral pathway. recognition of viral rna by rig-i-like receptors (rlrs) activates a signaling cascade that leads to type i interferon (ifn-α/β) gene transcription. multiple proteins in this signaling pathway (e.g. rig-i, mda , mavs, tbk , irf ) are regulated by (de)ubiquitination events. most viruses have evolved mechanisms to counter this antiviral response. the leader protease (l(pro)) of foot-and-mouth-disease virus (fmdv) has been recognized to reduce ifn-α/β gene transcription; however, the exact mechanism is unknown. the proteolytic activity of l(pro) is vital for releasing itself from the viral polyprotein and for cleaving and degrading specific host cell proteins, such as eif g and nf-κb. in addition, l(pro) has been demonstrated to have deubiquitination/deisgylation activity. l(pro)’s deubiquitination/deisgylation activity and the cleavage/degradation of signaling proteins have both been postulated to be important for reduced ifn-α/β gene transcription. here, we demonstrate that tbk , the kinase that phosphorylates and activates the transcription factor irf , is cleaved by l(pro) in fmdv-infected cells as well as in cells infected with a recombinant emcv expressing l(pro). in vitro cleavage experiments revealed that l(pro) cleaves tbk at residues – . we also observed cleavage of mavs in hela cells infected with emcv-l(pro), but only observed decreasing levels of mavs in fmdv-infected porcine lfpk αvβ cells. we set out to dissect l(pro)’s ability to cleave rlr signaling proteins from its deubiquitination/deisgylation activity to determine their relative contributions to the reduction of ifn-α/β gene transcription. the introduction of specific mutations, of which several were based on the recently published structure of l(pro) in complex with isg , allowed us to identify specific amino acid substitutions that separate the different proteolytic activities of l(pro). characterization of the effects of these mutations revealed that l(pro)’s ability to cleave rlr signaling proteins but not its deubiquitination/deisgylation activity correlates with the reduced ifn-β gene transcription. introduction a virally infected cell activates a plethora of antiviral responses. one of the best-known antiviral responses is the induction of type i interferons (ifn-α/β). replication of the viral genome generates double-stranded rna (dsrna) replication intermediates that can be recognized by cytoplasmic rig-i like receptors (rlrs). for example, picornaviruses, small (~ nm) nonenveloped viruses with a positive-sense rna genome, synthesize replication intermediates that are predominantly recognized by mda [ ] [ ] [ ] [ ] . upon recognition of viral dsrna, mda interacts with mavs, which subsequently activates traf and tbk . tbk phosphorylates the transcription factors irf and irf , resulting in their activation and dimerization. simultaneously, traf interacts with the ikk complex to activate the transcription factor nf-κb. upon activation, irf , irf and nf-κb translocate to the nucleus, where they induce expression of ifn-α/β and other proinflammatory cytokines. subsequent ifn-α/β signaling via the type i ifn receptor (ifnar) and the jak-stat pathway induces the expression of hundreds of interferon stimulated genes (isgs) (reviewed in [ , ] ). ifn-α/β gene transcription is extensively regulated by post-translational modification of rlrs and their downstream signaling proteins, including phosphorylation and ubiquitination. ubiquitin is a . kda protein that can be covalently linked through an ε-amino peptide linkage to lysine residues in target proteins. within the rlr signaling pathway rig-i, mavs, tbk , traf , traf and ikkγ are ubiquitinated and this affects their molecular interactions, localization, stability, or activity (reviewed in [ , ] ). ubiquitination of rlr signaling proteins can both positively and negatively regulate the signaling pathway, which allows for rapid fine-tuning of the innate immune response against viral infection (reviewed in [ ] [ ] [ ] ). consequently, many viruses encode enzymes with deubiquitinating (dub) activity to manipulate the rlr signaling pathway and thereby suppress expression of ifn-α/β (reviewed in [ ] ). in addition to ubiquitin, there are multiple ubiquitin-like modifiers, which can also be attached to target proteins. of special interest is isg , an ifn-induced modifier of . kda comprised of two ubiquitin-like domains in tandem. the exact antiviral properties of isg are not yet fully understood (reviewed in [ , ] ). early work on isg depended on mouse models and showed that expression of isg protected mice from viral infection [ ] [ ] [ ] [ ] . however, important biological and structural differences between isg of murine and human origin have since been reported [ ] [ ] [ ] [ ] . more recently, a picture is emerging that proteins are isgylated co-translationally, explaining why predominantly viral proteins and isgs are isgylated upon infection in humans [ ] . isgylation of rlr signaling proteins has been reported, but the effect of these modifications on the outcome of the signaling pathway is still unclear ( [ ] [ ] [ ] [ ] , reviewed in [ ] ). in addition, isg has been reported to act as a cytokine [ , ] . ifn-α/β signals in autocrine and paracrine ways to induce a tissue-wide antiviral state, thereby limiting viral spread. to establish infection in their host, it is essential for viruses to suppress both the rlr signaling pathway and the downstream signaling of ifn-α/β. affecting protein levels of important signaling molecules, either via cleaving them or inducing their degradation, is a strategy commonly used by viruses to suppress antiviral signaling [ ] [ ] [ ] [ ] . one such example is the picornavirus foot-and-mouth disease virus (fmdv). fmdv is a member of the genus aphthovirus, which also contains bovine rhinitis a and b viruses, and equine rhinitis a virus (erav). the genetic information on the fmdv rna genome is translated as one polyprotein that is autocatalytically processed into the mature proteins, two of which have been shown to possess proteolytic activity and also been implicated in suppressing ifn-α/β induction (reviewed in [ ] ). the c pro , the protease that processes the majority of cleavage sites on the polyprotein, cleaves nf-κb essential modulator (nemo), an adaptor protein that is essential to activate the nfκb and irf signaling pathways [ ] . the second protease on the polyprotein implicated in suppressing ifn-α/β induction is l pro , a papain-like cysteine protease located at the n-terminus of the polyprotein [ ] . once synthesized, l pro immediately frees itself from the growing peptide chain by autocleavage at its own c-terminus. l pro then efficiently cleaves the two isoforms of eif (eukaryotic initiation factor) g to reduce protein synthesis from cellular mrna [ ] and suppresses the induction of ifn-α/β via several mechanisms. l pro has been shown to induce the degradation of nf-κb subunit p /rela [ , ] , and decrease the levels of ifn regulatory factor (irf ) and irf [ ] . further, l pro can also interact with adnp, a negative regulator of transcription [ ] . in addition to cleaving or degrading important signaling molecules, l pro possesses deubiquitinase (dub) activity which has been proposed to modulate rlr signaling [ ] . a subsequent study demonstrated that l pro should be predominantly regarded as a deisgylase rather than a dub as biochemical evidence showed that l pro has a -fold higher affinity for isg than for ubiquitin [ ] . structural studies and biochemical studies have shown separate substrate binding sites on l pro for the viral polyprotein, the isoforms of eif g as well as for ubiquitin and isg [ ] [ ] [ ] , suggesting that it may be possible to uncouple the activities of l pro by the introduction of specific amino acid substitutions. we therefore set out to uncouple the different activities of l pro to discover whether l pro suppresses rlr signaling through its deisgylase/dub activity or through its ability to cleave and degrade multiple rlr signaling proteins. in this work, utilizing encephalomyocarditis virus (emcv) expressing fmdv l pro (emcv-l pro ), we identified mavs and tbk as new l pro substrates and determined the cleavage site in tbk . by introducing specifically designed mutations into l pro , we further identified residues that are important for either the cleavage/ degradation of rlr signaling proteins or for its deisgylase/dub activity, thereby uncoupling the two catalytic activities of l pro . we demonstrate that cleavage/degradation of rlr signaling proteins, but not the deisgylase/dub activity of l pro , correlates with suppressing ifn-α/β gene transcription. to study the effects of l pro on the induction of type i ifn in picornavirus-infected cells, we used two previously generated recombinant viruses; emcv-l zn , which contains inactivating mutations in the zinc-finger domain of the leader (i.e. emcv's rlr signaling antagonist) [ , , ] , and emcv-l pro , which was derived from emcv-l zn and additionally encodes fmdv l pro at the n-terminus of its polyprotein ( fig a) [ ]. we also constructed a similar ). in emcv-l zn , l contains inactivating mutations in its zn-finger domain (c a/c a) which abolishes its ability to suppress antiviral responses. to generate emcv-l pro , the lb pro gene of fmdv o-strain was introduced at the ' end of the emcv-l zn open reading frame. the l pro cleavage site at its own c-terminus was mutated to aaa. instead l pro is released from this viral polyprotein via an emcv c cleavage site. (b) hela r cells were infected at moi with the indicated viruses and cells were lysed at , , , h pi. total rna was isolated and used for rt-qpcr analysis for ifn-β and actin mrna, and emcv vrna. the ifn-β levels are depicted as a fold induction compared to levels in mock-infected cells, after correction for actin mrna levels. the emcv vrna is depicted as a copy number per cell, calculated from a plasmid standard. error bars depict the sd. one-way anova with the dunnet post hoc test was used to determine statistical significance compared to the results for emcv-l zn -infected cells ( � , p< . ; ��� , p< . ; ���� , p< . ; ns, no significant difference). recombinant emcv carrying a catalytically inactive l pro (i.e. emcv-l pro c a) [ ] . to determine whether emcv-l pro can suppress ifn-β induction, we infected hela cells with emcv, emcv-l zn , emcv-l pro or emcv-l pro c a and determined the ifn-β mrna levels over time via rt-qpcr analysis (fig b) . consistent with previous studies [ , , ] , wt l pro , but not l pro c a, reduced the induction of ifn-β mrna approximately -fold, indicating that the catalytic activity of l pro is needed to suppress rlr signaling. in conclusion, the viruses that we generated (emcv-l pro and emcv-l pro c a) accurately mimic the suppression of rlr signaling by l pro as previously reported for fmdv-infected cells [ ] , providing us a model system to determine the mechanism via which l pro suppresses type i ifn induction. l pro has been reported to degrade important signaling proteins such as the p subunit of nf-κb, irf and irf [ , ] . to determine whether l pro targets additional rlr signaling proteins, we subjected cell lysates of hela cells infected with emcv-l zn , emcv-l pro or emcv-l pro c a to western blot analysis for the signaling proteins mavs, tbk and irf , as well as the known l pro -substrates eif g and g bp (fig a) . emcv capsid proteins and tubulin served as infection and loading controls, respectively. infection with emcv-l pro , but not emcv-l pro c a, resulted in the rapid cleavage of eif g (from hpi onwards) and the cleavage of g bp (from hpi onwards). we did not observe cleavage or degradation of irf as was suggested by others [ ] . in addition to these known cleavages, we observed cleavage of mavs and tbk at hpi. for mavs, we observed multiple cleavage products ranging in apparent molecular weight from~ kda to~ kda. tbk cleavage resulted in a single cleavage product with an apparent molecular weight of~ - kda. we also attempted to detect mda and investigate whether this dsrna sensor is targeted by l pro . unfortunately, the low levels of mda prevented us from detecting the endogenous protein. mda expression could be boosted by pretreatment with recombinant ifn-α , but ifn-α pretreatment inhibited efficient emcv infection, thereby interfering with the subsequent analysis. we next focused our attention to identifying the cleavage site in tbk . to this end, we overexpressed n-terminally flag-tagged tbk together with gfp-tagged l pro and performed western blot analysis. as seen in fig b , gfp-l pro was able to cleave flag-tbk . we observed an αflag-reactive cleavage product migrating at~ - kda, the same apparent molecular weight as the cleavage product we observed in emcv-l pro infected cells (fig a) , suggesting that l pro cleaves tbk at its c-terminus. we also co-incubated recombinant his-tbk with increasing amounts of recombinantly expressed l pro and l pro c a ( fig c) . the in vitro incubation of his-tbk with wt l pro also resulted in a~ - kda αhis-reactive cleavage product, confirming that l pro cleaves tbk at its c-terminus and does not rely on other cellular factors. incubation of his-tbk with catalytically inactive l pro did not result in the formation of a cleavage product, confirming that the cleavage is dependent on l pro 's proteolytic activity. subsequently, we showed that l pro also cleaves tbk of murine origin (fig d) , which suggests that l pro cleaves tbk in a conserved region. we identified residues klk -which localize at the very c-terminus of tbk and are well conserved between human, murine and porcine tbk -as a possible cleavage site ( fig e) . indeed, mutation of these residues prevented the cleavage of tbk by l pro (fig d) , confirming these residues are the cleavage site. upon identifying the cleavage site in tbk , we next investigated whether the cleavage of tbk by l pro inhibits its function in the rlr signaling pathway. to this end, we generated cells in which the endogenous tbk gene is replaced with a tbk truncation mutant representative of the~ - kda cleavage product. we first generated hela tbk k.o. cells using crispr/cas technology and characterized the remaining rlr signaling capacity of these cells cleavage of rlr signaling proteins by fmdv l pro correlates with type i ifn suppression (s a and s b fig) . we found that depletion of tbk is not sufficient to fully impair rlr signaling, probably because of functional redundancy with ikkε (s b fig) . tbk k.o. cells have ã -fold lower ifn-β induction upon infection with emcv-l zn , transfection of vrna or upon overexpression of mavs. as expected, ifn-β induction resulting from transfection of irf , which acts downstream of tbk , was not affected in the tbk k.o. cells. subsequently, we expressed full length tbk or tbk Δ aa, which represents the n-terminal cleavage product, in tbk k.o. cells (s c fig) and determined whether expression of tbk and tbk Δ aa restored ifn-β mrna expression upon transfection of poly (i:c). expression of full length tbk in tbk k.o. cells fully restored ifn-β mrna induction (s d fig) and tbk Δ aa was similarly efficient in this (s e fig), indicating that the l pro -generated n-terminal cleavage product is signaling competent. to investigate whether tbk , mavs and irf are cleaved during fmdv infection, we infected porcine lfpk αvβ cells with wt fmdv-a or fmdv-a lacking l pro (leaderless virus, a -llv). western blot analysis revealed cleavage of tbk , but not of mavs or irf , during wt fmdv-a infection ( fig a) . tbk cleavage was observed from hpi onwards upon infection with wt fmdv, but not upon infection with leaderless fmdv. the cleavage product had an apparent molecular weight of~ - kda, consistent with our previous observations of the size of this cleavage product. although a mavs cleavage product was not detected during fmdv infection, densitometry analysis revealed a strong and progressive decrease in the relative ratio of mavs/tubulin from - hpi post infection with wt fmdv compared to mockinfected cells, whereas only a small decrease was detected in leaderless-infected cells (mavs/ tubulin ratio is indicated in fig a) . this suggests that expression of l pro induces degradation of mavs, also in fmdv-infected cells. consistent with our observations in emcv-l pro infected cells, we did not observe a decrease in irf signal in fmdv-infected cells. to investigate whether the cleavage of tbk is conserved amongst aphthoviruses, we infected cells with erav, the closest relative of fmdv. infection with erav, but not emcv, resulted in the cleavage of tbk ( fig b) . however, the cleavage product was less prominent than for emcv-l pro , suggesting that cleavage was inefficient or infection was delayed. notably, in our hela cells, erav displayed a replicative cycle of~ h. this is considerably slower than fmdv, which replicates in - hours. to study tbk cleavage by the two different l pro 's irrespective of variation in viral replication kinetics, we infected cells with emcv-l pro or an emcv expressing erav l pro (emcv-erav l pro ), for which we previously determined the replication kinetics to be similar [ ] . both viral proteases cleaved tbk resulting in a~ - kda cleavage product ( fig c) . fmdv l pro cleaved tbk more efficiently than erav l pro , consistent with the results observed during infection with fmdv or erav (fig a and b ). notwithstanding the differences between erav l pro and fmdv l pro , our data demonstrate that the ability to cleave tbk is conserved amongst these two aphthoviruses. we also investigated the effect of the pan-caspase inhibitor q-vd-ph (q-vd) on tbk cleavage in erav-infected cells ( fig d) . while addition of q-vd decreased the cleavage of known caspase substrate parp, the cleavage of tbk was unaffected. collectively, these results demonstrate that l pro directly cleaves tbk and that this activity is conserved amongst aphthoviruses. construction of l pro mutants to uncouple cleavage/degradation of rlr signaling proteins from its deisgylase/dub activity l pro also possesses deisgylase and-to a lesser extent-dub activity [ , , ] , and this latter activity was previously suggested to be important for l pro 's ability to suppress rlr signaling cleavage of rlr signaling proteins by fmdv l pro correlates with type i ifn suppression plos pathogens | https://doi.org/ . /journal.ppat. july , [ ]. to investigate how l pro reduces the induction of ifn-β gene transcription, we set out to uncouple these two abilities of l pro . to this end, we introduced previously described mutations in the chimeric emcv-l pro and determined whether these mutations affect the deisgylase/ dub activity of l pro and/or its ability to cleave/degrade rlr signaling proteins. a mutation in the sap domain (i a/l a), was previously reported to abolish the ability of l pro to suppress type i ifn expression, to degrade signaling proteins (i.e. nf-κb p , irf and irf ), and to disrupt its dub activity [ , , ], and was therefore included in our screening. analysis of the crystal structure of l pro bound to isg suggested that l pro residues l , p and l are important for isg binding [ ] . fig a shows the structure of l pro (grey) in complex with isg (blue) and indicates the residues of isg (w and r , l , g ) that interact with l pro . mutation of l , p or l in l pro reduced its affinity for isg and impaired its deisgylase activity, without affecting eif g cleavage [ ] . homology modeling showed that isg and ubiquitin interact with the same surfaces of l pro [ ] , suggesting that l pro 's deisgylase activity also reflects its dub activity. two of these mutations (l a and l a) were introduced in emcv-l pro . in addition, we introduced the mutations l a and c s. mutation c s was reported to reduce the affinity of l pro for eif g [ ] whereas mutation of to l to alanine with its shorter side-chain rescued polyprotein processing in the context of the additional mutation l f [ ] . based on the structure of l pro , mutation l a has been predicted to open up the catalytic pocket. fig shows the locations of the residues that are mutated in this study ( fig a) and summarizes the reported effects of these mutations on l pro 's various proteolytic activities ( fig b) . first, we determined the effect of the introduced mutations on l pro 's ability to cleave or degrade rlr signaling proteins. we infected hela cells with emcv, emcv-l zn or the different emcv-l pro carrying the described mutations, lysed the cells at the indicated timepoints and subjected the lysates to western blot analysis for mavs, tbk , nf-κb subunit p , irf , eif g and g bp , as well as l pro and emcv capsid proteins (fig ) . our data show that infection with the different l pro mutant viruses resulted in l pro expression and accumulation of emcv capsid proteins from hpi onwards, which is indicative of efficient infection. interestingly, the introduced mutations in l pro had different effects on the cleavage or degradation of rlr signaling proteins. upon infection with emcv-l pro c s, we observed a~ hr delay in eif g cleavage, consistent with a previous report [ ] . this mutation did not affect the cleavage/degradation of the various rlr signaling proteins. mutation of l or l has been reported to reduce the activity of l pro towards isg , without affecting eif g cleavage [ ] . indeed, infection with emcv-l pro l a, resulted in cleavage of eif g, as well as all other l pro substrates (i.e. mavs, tbk , nf-κb p and g bp ) (fig ) . infection with both emcv-l pro l a and emcv-l pro l a resulted in efficient cleavage of rlr signaling proteins, confirming that these two l pro mutants have the same proteolytic profiles (fig ) . of note, the cleavage cleavage of rlr signaling proteins by fmdv l pro correlates with type i ifn suppression of eif g appears to occur faster by l pro l a and l a compared to wt l pro , although a more in-depth analysis is necessary to confirm a true difference in eif g cleavage kinetics. importantly, both mutations allow us to separate the deisgylase/dub activity of l pro from its ability to cleave rlr signaling proteins. serendipitously, we observed that l pro carrying cleavage of rlr signaling proteins by fmdv l pro correlates with type i ifn suppression mutation l a was strongly impaired in degrading nf-κb p and cleaving mavs and tbk , while cleavage of g bp and eif g was delayed but could be observed clearly at later timepoints (eif g cleavage is~ hr delayed, comparable to the delay observed for l pro c s). mutation of l pro 's sap domain (i a/l a), which was previously shown to abolish cleavage of rlr signaling proteins by fmdv l pro correlates with type i ifn suppression degradation of nf-κb p and dub activity as well as to impair l pro 's ability to reduce ifn-β mrna expression [ , ] , also affected l pro 's ability to cleave mavs and tbk . overall, our data demonstrate that the mutations have differential effects on the cleavage/degradation of rlr signaling proteins. importantly, we demonstrate that l pro residues l and l , which are essential for its deisgylase/dub activity, are not essential for is ability to cleave/degrade rlr signaling proteins, indicating that the two different catalytic activities of l pro can be uncoupled. it has been previously reported that mutations l a, l a and i a/l a affect l pro 's deis-gylase/dub activity [ , ] . to determine the effect of mutations l a or c s on these activities, mutant l pro 's were expressed and purified from e. coli and in vitro catalytic activities towards ubiquitin-tamra and isg -tamra were measured (fig a) . l pro c s and l a displayed wt-like activity towards isg and ubiquitin. we next determined dub activity of wt l pro , l pro c a, c s and l a in cells. to this end, we transfected hek- t cells with a combination of ha-ubiquitin, flag-rig-i and increasing amounts of gfp-l pro encoding plasmids ( . , . and . μg), and visualized ha-tagged ubiquitinated proteins by western blot analysis (fig b) . flag-rig-i, which was included to monitor the effects of l pro -induced translational shut-off on the overexpressed proteins, was clearly detectable even at the highest level of l pro (i.e. transfection of . μg of l pro plasmid), indicating that cleavage of rlr signaling proteins by fmdv l pro correlates with type i ifn suppression l pro -induced translational shut-off did not significantly reduce the protein expression from the transfected plasmids. both wt l pro as well as the c s and l a mutants displayed dub activity upon overexpression in cells, as indicated by the reduction of ha-tagged ubiquitinated proteins upon increasing l pro levels. notably, we observed ubiquitinated proteins upon transfection of low amount of l pro c s plasmid ( . μg), although our in vitro data (fig a) indicated that mutation c s does not reduce the activity for ubiquitin. as the in vitro data are much more quantitative in nature, we consider the relatively decreased dub activity of l pro c s in comparison to l pro wt or mutant l a as observed in fig b the result of variations in expression of the transfected plasmids. thus far, our data showed that mutations c s and l a did not affect l pro 's dub activity towards a mono-ubiquitin fused to a fluorescent tamra molecule. to exclude the possibility that mutations c s and l a affect l pro 's dub activity towards other substrates, we cleavage of rlr signaling proteins by fmdv l pro correlates with type i ifn suppression determined its ability to cleave differently-linked di-ubiquitin molecules. co-incubation of l pro with di-ubiquitin molecules of different linkages indicated that l pro preferentially targets k -and k -linked ubiquitin chains, displayed some activity towards k -, k -, k -, and m -linked chains, but at this enzyme concentration and incubation times has no activity towards k -or k -linked ubiquitin chains (fig ) . neither mutation l a nor c s affected the ability of l pro to cleave the di-ubiquitin molecules. overall, our analysis shows that l pro carrying mutation l a or c s have wt-like deisgylase and dub activity. importantly, as mutation l a impairs l pro 's ability to cleave/degrade rlr signaling proteins ( fig ) , but does not affect its deisgylase/dub activity, introduction of this mutation also allows us to make a distinction between the two proteolytic activities of l pro . l pro 's ability to reduce ifn-β mrna levels correlates with its ability to cleave/degrade rlr signaling proteins, not with its deisgylase/dub activity having characterized how the different mutations in l pro affect its deisgylase/dub activity or the cleavage/degradation of rlr proteins, we next set out to determine which mutants reduce the induction of ifn-β mrna. we infected hela cells with emcv, emcv-l zn or the different emcv-l pro mutants and determined the ifn-β mrna and emcv vrna levels over time via rt-qpcr analysis (fig ) . wildtype l pro as well as l pro c s, l a and l a consistently reduced the induction of ifn-β mrna, while l pro c a and l a were unable to do so (fig a) . the sap domain mutant (emcv-l pro i a/l a) also failed to suppress ifn-β mrna levels (fig b) , which is in agreement with observations in fmdv-infected cells [ , ] . notably, infection with emcv l pro l a, which displayed wt deisgylase/dub activity but is strongly impaired in its ability to cleave/degrade rlr signaling proteins mavs, tbk and nfκb p , failed to suppress the induction of ifn-β mrna. in contrast, l pro l a and l a,which are strongly impaired in their deisgylase/dub activity [ ] but not in their ability to cleave/degrade rlr signaling proteins, reduced ifn-β mrna levels. these combined observations (summarized in fig ) demonstrate that cleavage/degradation of rlr signaling proteins, but not the deisgylase/dub activity of l pro , correlate with suppressing ifnα/β gene transcription. notably, l pro c s reduced the induction of ifn-β mrna despite a h delay in eif g cleavage, indicating that the rapid eif g cleavage and the subsequent translational shut-off is not sufficient to suppress rlr signaling. fmdv suppresses ifn-α/β both at the mrna and at the protein level [ ] , but the molecular mechanism underlying the reduced induction of ifn-α/β gene transcription (rlr signaling) is poorly understood. both the dub activity of l pro as well as its ability to cleave/degrade rlr signaling proteins have been implicated in the suppression of rlr signaling [ , , ] . in this study, we identified mavs and tbk as novel l pro substrates and mapped the cleavage site in tbk . moreover, by introducing specific mutations we were able to separate l pro 's deisgylase/dub activity from its ability to target rlr signaling proteins. using l pro carrying either of these uncoupling mutations, we demonstrated that the cleavage/degradation of rlr signaling proteins, not the deisgylase/dub activity, correlates with the ability to reduce ifn-β gene transcription. collectively, our data strongly suggest that the ability of l pro to cleave/degrade rlr signaling proteins is needed to reduce the ifn-β mrna levels. we identified tbk as a new l pro substrate and identified the cleavage site. we observed cleavage of tbk both in hela cells infected with emcv-l pro and in fmdv-infected lfpk cells, and we demonstrated that the l pro cleavage site is located towards the c terminus of cleavage of rlr signaling proteins by fmdv l pro correlates with type i ifn suppression tbk , more specifically in the coiled-coil (cc ) domain. previous work indicated that tbk Δcc was signaling competent upon overexpression of tbk . however, it was also shown that mutation of residues l and k , which are located in the cc domain, abolishes ifn-β mrna induction upon polyi:c transfection and vsv infection [ ] . l pro cleaves tbk at these residues. yet, we observed that the n-terminal cleavage product restored rlr signaling in tbk k.o. cells upon poly(i:c) stimulation. whether this implies that the l pro -mediated cleavage of tbk does not contribute to the viral strategy to suppress rlr signaling and ifnβ gene transcription remains unknown. unfortunately, it is very difficult to investigate the effect of tbk cleavage by l pro on ifn-β gene transcription in infected cells, as l pro also cleaves other rlr signaling proteins, i.e. mavs (this study) and lgp ( [ ] , see below), and the transcription factor nf-κb. dissection of the effect of tbk cleavage on ifn-β gene transcription from the effect of mavs, lgp and nf-κb cleavage would require the identification of all l pro cleavage sites in these known targets followed by the generation of cells with cleavage-resistant versions of these proteins. it remains a question whether such an approach will yield conclusive answers as other rlr signaling proteins may be targeted by l pro , which have cleavage of rlr signaling proteins by fmdv l pro correlates with type i ifn suppression not yet been identified. hence, the relative importance of the l pro -mediated cleavage of tbk for the viral suppression of ifn-β gene transcription remains unknown. cleavage of rlr signaling proteins by fmdv l pro correlates with type i ifn suppression apart from its role in rlr signaling, tbk has been suggested to be involved in autophagosome maturation. tbk was identified as a factor in the autophagosomal clearance of herpes simplex virus and mycobacteria [ , ] . a recent report showed that tbk phosphorylates lipidated lc- to prevent premature removal of lc from autophagosomal membranes by atg , thereby facilitating a unidirectional flow from the autophagosome to the lysosome [ ] . many picornaviruses hijack autophagic pathways to generate sites for viral rna replication and to facilitate non-lytic release of virions [ ] [ ] [ ] [ ] [ ] [ ] [ ] possibly, cleavage of tbk by l pro facilitates the use of autophagy to aid viral infection and propagation. l pro also impacts mavs integrity. a distinct mavs cleavage product was observed upon infection with emcv-l pro . remarkably, no mavs cleavage product was observed in fmdvinfected cells, although mavs levels progressively declined over time. the reason for this difference is unknown, but may be related to differences in human and porcine cells. nevertheless, our data indicate that the integrity of mavs is affected by l pro in both cell types, suggesting this rlr signaling protein is targeted by fmdv to affect ifn induction. of note, mavs is known to localize to the peroxisomes and mitochondrion associated membranes of the er [ , ] , in addition to its default localization on mitochondria. it remains to be established whether l pro targets all forms of mavs or specifically targets mavs at one of these locations. tbk and mavs are not the only rlr signaling proteins that are targeted by l pro . it was previously reported that overexpression of l pro induces the degradation of irf and irf [ ] , and that the p subunit of nf-κb is degraded in fmdv-infected cells [ ] . we also observed degradation of nf-κb p in emcv-l pro infected-cells but we did not observe degradation of irf . notably, degradation of irf was also not observed in fmdv-infected cells. whether irf degradation is restricted to certain cell types or conditions, or merely is an artefact due to overexpression remains unknown. it is remarkable that l pro , comprising just amino acids, can carry out several specific proteolytic activities on both the viral and cellular substrates as summarized in figs and . previous work documented areas of the protease that are required for polyprotein processing [ , ] . these residues included l which is part of the p pocket that can interact with leucine residues in the substrate at the p position. in this work, l was identified as being involved in tbk and mavs cleavage; however, its replacement by alanine affected neither the activity on eif g nor the deisgylase or dub activities. surprisingly, mutation of two residues of the sap domain, (i and l ) also affected tbk and mavs cleavage, even though they are separated by Å (measured between the respective c α atoms) with helix α lying between them. nevertheless, it cannot be excluded that the sap mutations cause some destabilization of l pro , thereby explaining this mutant's defect in proteolytic activities. further structural work will be required to understand how l pro interacts with tbk and mavs. while this work was in progress, it was reported that lgp , a factor that is essential for mda activation, is cleaved by l pro [ ] . the mutations in l pro that impaired the reduction of ifn-β mrna levels (l a and mutations in the sap domain) displayed an overall defect in the cleavage and/or degradation of each of the rlr signaling proteins we studied (i.e. mavs, tbk and nf-κb p ). we anticipate that the cleavage of lgp is also likely impaired by introduction of these mutations. our data suggest that expression of l pro results in cleavage and/or degradation of multiple rlr signaling proteins (mavs, tbk , nf-κb p , and most likely lgp ). the relative contribution of each cleavage event to the reduction in ifn-β gene transcription remains unknown. a search for other substrates of l pro is of importance to further our understanding of the role and mechanism of how l pro reduces ifn-α/β induction. possibly, such a search may identify rlr signaling proteins that are cleaved earlier than the ones identified so far and may thereby have an influence on the early induction of type i ifn in infected cells. the l pro mutants that are defective in either the deisgylase/dub activity or the cleavage/ degradation of rlr signaling proteins allowed us to study which ability is needed to suppress rlr signaling. mutation l a, which rendered l pro unable to reduce ifn-β mrna levels, impaired the cleavage of rlr signaling molecules, but had no effect on the deisgylase or dub activity of l pro . meanwhile, mutations l a and l a resulted in the opposite phenotypic effect; rlr signaling proteins were cleaved with similar efficiency as wt l pro , but the deisgylase activity was significantly reduced by these mutations [ ] . yet, these l pro mutants still reduced ifn-β mrna levels. collectively, these data indicate that the activity of l pro to cleave/degrade rlr signaling proteins, not its deisgylase/dub activity, is important for reduction of ifn-β induction. medina et. al. have just reported that impairment of the deisgylation activity of l pro causes viral attenuation in vitro and in vivo [ ] . in support of our hypothesis, the mutations introduced by medina et. al. did not affect ifn or isg mrna expression levels [ ] . it was previously suggested that the dub activity of l pro is important for the suppression of rlr signaling [ ], which contrasts our findings. importantly, most experiments by wang et. al. relied on overexpression of l pro , ubiquitin, and several targets proteins (i.e. rig-i, tbk , traf and traf ). a recent study showed that l pro should be predominantly regarded as a deisgylase rather than a dub, as biochemical evidence showed that l pro has a -fold higher activity towards isg than ubiquitin [ ] . given the weak dub activity of l pro in vitro, it remains to be established whether l pro genuinely acts as a dub in fmdv-infected cells under physiological conditions (i.e. without overexpression of components of the ubiquitination system or known ubiquitination target proteins). previously, we found no differences in the levels of ubiquitinated proteins in cells infected with emcv expressing wt l pro or l pro c a, whereas the levels of isgylated protein were decreased in cells infected with emcv-l pro [ ] , suggesting that l pro predominantly acts as a deisgylase in infected cells. it is well established that certain viruses (i.a. adenoviruses, herpesviruses and nidoviruses) rely on viral proteases with dub and deisgylase activity to suppress the induction of ifn-α/β [ ]. fmdv l pro is a papain-like protease and thus l pro is best compared to other virally encoded papain-like cysteine proteases that suppress ifn-α/β gene transcription. members of the order nidovirales (i.e. coronaviruses and arteriviruses) encode one or two papain-like cysteine protease (plp), referred to as pl pro , or plp and plp when the virus encodes two plps. in addition to cleaving the viral polyprotein, pl pro and the equivalent plp have acquired dub and deisgylase activity [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . structure-guided mutagenesis of pl pro of mers-cov and plp of equine arterivirus (eav) allowed the dub activity to be separated from the proteolytic activity portrayed towards the viral polyprotein [ , ] . this uncoupling of these two different proteolytic activities indicated that the dub activity of pl pro /plp contributes to the suppression of ifn-α/β transcription [ , ] . unfortunately, it has not been determined whether pl pro /plp cleaves rlr signaling proteins and thus it is unclear what other proteolytic activities could be affected by the mutations that were introduced. notably, the cleavage site of nidovirus pl pro /plp and fmdv l pro in ubiquitin and isg is different. while sars-cov pl pro breaks the iso-peptide bond between ubiquitin or isg and the target protein [ , ] , fmdv l pro is a non-canonical deisgylase that targets a peptide bond in isg itself, resulting in a diglycylated-lysine in the target protein [ ]. in conclusion, nidovirus pl pro /plp and fdmv l pro are both papain-like proteases, but they likely have evolved different strategies to suppress ifn-α/β gene transcription. fmdv l pro and enterovirus a pro are structurally different enzymes that share many functions; both cleave translation initiation factor eif g [ , , ] , both reduce ifn-α/β gene transcription [ , , , ] , both have been implicated in the suppression of sg formation [ ] [ ] [ ] and both have been suggested to rescue viral translation from the inhibitory effects of p-eif [ , ] . importantly, l pro and a pro both cleave several rlr signaling proteins, but the only overlapping rlr protein is mavs [ , ] . although a causal relationship between cleavage of rlr proteins and suppression of ifn-α/β transcription remains to be established for both proteases, the convergence on the cleavage of mavs is noteworthy. in the absence of sequence homology, no evolutionary basis for the functional similarities between the two picornavirus proteases can be determined. possibly, the extensive similarities between fmdv l pro and enterovirus a pro , both picornavirus proteases, is illustrative of the urgency for picornaviruses to suppress these particular antiviral host responses. our data suggests that the deisgylase activity of l pro is not critically needed to suppress rlr signaling, but rather its role should be sought in the broader antiviral activities of isg (reviewed in [ , ] ). it should be noted that our experiments were performed in naïve cells at high multiplicity of infection. expression of the isgylation machinery as well as isg itself are boosted by ifn-α/β and therefore we cannot formally exclude a role for the dub and/or deisgylase activity of l pro in suppressing rlr signaling under different conditions (e.g. in ifn-primed cells). isg has many functions, both intracellular and extracellular. intracellular isg can act both pro-inflammatory and immunomodulatory, either via isgylation of target proteins or as free isg . moreover, isg can be secreted to act as a cytokine [ , ] . isg also plays a role in damage repair after clearing viral infection [ ] and can regulate cellular processes such as autophagy and metabolism [ , [ ] [ ] [ ] . how the deisgylase activity of l pro contributes to efficient in vivo infection, remains to be established. hela r , hela r tbk k.o. and hek t cells were maintained in dulbecco's modified eagle's medium (dmem) supplemented with % fcs (v/v). hela ohio cells were maintained in dmem supplemented with % fcs (v/v) and % penicillin/streptomycin. lfpk αvβ cells [ ] were obtained from the foreign animal disease diagnostic laboratory (faddl) at the piadc. these cells were maintained in minimal essential medium (mem) supplemented with % fcs (v/v) and % antibiotics and non-essential amino acids. bhk- cells used for fmdv propagation were maintained in mem supplemented with % fcs (v/v), % tryptose phosphate broth, % antibiotics and non-essential amino acids. hela r tbk k.o. cells were generated via crispr/cas methodology using a pcrispr plasmid, as described previously [ ] . the used grna sequences are '-gctactgcaaatgtctttcg- ' and '-gaggaaaacagattggtt- '. fmdv a -wt (wild type) was generated from the full-length serotype a infectious clone, prmc [ ] and a -llv (leaderless virus) was derived from the infectious clone lacking the lb coding region, prm-llv [ ] . viruses were propagated in bhk- and concentrated by polyethylene glycol precipitation, titrated on bhk- cells, and stored at - ˚c. erav (nm- / strain) (gift from d. rowlands and t. tuthill [ ] ) was obtained after passage on hela r cells and subsequently concentrated by ultracentrifugation through a % sucrose cushion at , xg for hours in a sw ti rotor and stored at - ˚c. recombinant emcvs were generated by cloning the genes of interest into the xhoi/noti restriction sites from the pm . -vfetqg-zn infectious clone that was described previously [ ] . emcv-l pro viruses were recovered by transfection of run-off rna transcripts into bhk- cells. upon total cpe, viruses were concentrated by ultracentrifugation (as described for erav) and stored at - ˚c. the following antibodies were used for western blot staining procedures: αfmdv vp (rabbit polyclonal abmade at piadc), αemcv capsid (gift from ann palmenberg), αl pro (gift from cleavage of rlr signaling proteins by fmdv l pro correlates with type i ifn suppression ewald beck and tim skern), αmavs (enzo life science alx- - ), αtbk (cell signaling ), αirf (santa cruz sc- ), αnf-κb-p /rela (santa cruz biotechnology sc- ), αeif g (bethyl laboratories a - a), αg bp (bd biosciences clone /g bp), αparp (roche diagnostics # ), αflag (sigma m ), αgfp (invitrogen ose g), αhis (ge healthcare, - - ), αmyc (clone a , millipore), αha (abcam ab ) and αtubulin (sigma dm a). respective irdye or irdye conjugated secondary antibodies (licor) or hrp-conjugated secondary antibodies were used for detection. hela r cells were seeded in -wells plates and the next day infected with the indicated viruses at moi or transfected with the indicated plasmids or vrna. plasmids were transfected using fugene (promega) and vrna was transfected using lipofectamine (invitrogen), both according to the manufacturer's instructions. preparation of viral dsrna and the pcdna-gfp-mavs construct have been described previously [ , ] . pegfp-irf [d ] construct was a kind gift from john hiscott [ ] . at the indicated time points cells were lysed and cellular rna was isolated using total rna isolation kit (machery-nagel) according to manufacturer's instructions. reverse transcription was set up using taqman reverse transcription reagents (applied biosystems) before performing qpcr analysis with sybr green (roche) as described previously [ ] . the pires-egfp-fmdv l pro plasmid was described previously [ ] . the pcdna-flag-tbk plasmid was a gift from john hiscott [ ] and the pef-flag-rig-i was a gift from takashi fujita [ ] . ha-ubiquitin was expressed from a pcmv plasmid. hek t cells were seeded in -well plates and the next day transfected with . μg of total plasmid using fugene (promega) according to manufacturer's instructions. h posttransfection cells were lysed μl lysisbuffer ( mm tris ph . , mm edta, mm nacl, % np , protease inhibitor mix (roche)). post nuclear lysate was obtained by centrifugation at xg at ˚c for min. the amount of total protein in the lysates was determined using bca assay (thermo-fisher) and μg of protein was resolved using reducing sodiumdodecyl sulfate-polyacrylamide gel electrophoresis (sds-page) and transferred to . μm nitrocellulose membranes by wet electrophoretic transfer. membranes were incubated h in blocking buffer (pbs + . % tween + % bsa) and successively incubated overnight with primary antibodies diluted in blocking buffer and then for min with respective secondary antibodies diluted in blocking buffer. between and after the incubations, the membranes were washed three times with pbs + . % tween- . finally, membranes were washed once with pbs and scanned using an odyssey imager (li-cor). hela r cells were seeded in cm dishes and infected the next day with the indicated viruses at moi . at the indicated time points cells were released using trypsin, washed once in pbs and lysed in μl lysis buffer ( mm tris ph . , mm edta, mm nacl, % np , protease inhibitor mix (roche)). subsequent steps are identical as described for transfected cells. for the analysis of fmdv-infected lfbk αvβ cultures, cells were lysed in lysis buffer ( . % np- substitute, mm tris ph . , mm nacl, mm edta). lysates were incubated at ˚c for min and cellular debris was collected by centrifugation at , xg for min at ˚c. ng of protein was resolved by sds-page, transferred by western blot and secondary antibodies conjugated with horseradish peroxidase (pierce) were used for detection cleavage of rlr signaling proteins by fmdv l pro correlates with type i ifn suppression of proteins. following incubation with appropriate primary and secondary antibodies, protein bands were visualized using supersignal west dura extended duration substrate (thermo-scientific, rockford, il, usa) according to the manufacturer's directions. in vitro tbk cleavage sl pro was expressed and purified as reported previously [ ] . ng of his-htbk (millipore) was incubated with - μg sl pro for h at ˚c in a hepes buffer ( mm hepes ph . , mm kcl, mm edta) before the reaction mixture was dissolved on sds-page. proteins were transferred to nitrocellulose and western blot staining for the his-tag was performed. myc-mtbk and myc-mtbk aaa were transiently expressed in hela ohio cells from plasmid pcs - myc-mtbk , a gift from t. decker. μg myc-tagged mtbk containing cell lysate was incubated with μg sl pro for h at ˚c in a hepes buffer before resolving the reaction mixture on sds-page, transferring the protein to nitrocellulose membrane and performing western blot staining for myc. ubiquitin/isg -tamra assays were performed according to [ ] . di-ubiquitin in vitro cleavage assays were performed as described previously [ ] . total rna was isolated and used for rt-qpcr analysis for ifn-β and actin mrna. the ifn-β levels are depicted as a fold induction compared to levels in mock-treated cells, after correction for actin mrna levels. error bars depict the sd. (c) hela r tbk k.o. cells were transfected with μg plasmid expressing full-length or truncated tbk (tbk Δ aa). tbk Δ aa is representative for the l pro -generated n-terminal cleavage product. cells were lysed and lysates subjected to western blot analysis for tbk and tubulin. (d) tbk k.o. cells were reconstituted with full-length tbk as described for (c) and subsequently transfected with ng poly(i:c). cells were lysed at h post transfection of poly(i:c). total rna was isolated and used for rt-qpcr analysis for ifn-β and actin mrna. the ifn-β levels are depicted as a fold induction compared to levels in mock-treated cells, after correction for actin mrna levels. error bars depict the sd. (e) tbk k.o. cells were reconstituted with full-length or truncated tbk (tbk Δ aa) as described for (c). subsequent steps as described for (d). error bars depict the sd. (tif) cleavage of rlr signaling proteins by fmdv l pro correlates with type i ifn suppression mda detects the double-stranded rna replicative form in picornavirus-infected cells mda and mavs mediate type i interferon responses to coxsackie b virus mda plays a crucial role in enterovirus rna-mediated irf activation identification of the role of rig-i, mda- and tlr in sensing rna viruses in porcine epithelial cells using lentivirus-driven rna interference regulation of type i interferon responses cytosolic sensing of viruses post-translational control of intracellular pathogen sensing pathways ubiquitin in the activation and attenuation of innate antiviral immunity isg in antiviral immunity and beyond isg : it's complicated mice lacking the isg e enzyme ube l demonstrate increased susceptibility to both mouse-adapted and non-mouse-adapted influenza b virus infection selective inactivation of usp isopeptidase activity in vivo enhances isg conjugation and viral resistance ubiquitin-like protein isg (interferon-stimulated gene of kda) in host defense against heart failure in a mouse model of virusinduced cardiomyopathy ifn-stimulated gene functions as a critical antiviral molecule against influenza, herpes, and sindbis viruses structural insights into the interaction of coronavirus papain-like proteases and interferon-stimulated gene product from different species mycobacterial disease and impaired ifn-γ immunity in humans with inherited isg deficiency. science ( -) human intracellular isg prevents interferon-α/β over-amplification and auto-inflammation isg deficiency and increased viral resistance in humans but not mice the isg conjugation system broadly targets newly synthesized proteins: implications for the antiviral function of isg positive regulation of interferon regulatory factor activation by herc via isg modification acetaldehyde disrupts interferon alpha signaling in hepatitis c virus-infected liver cells by up-regulating usp negative feedback regulation of rig-i-mediated antiviral signaling by interferon-induced isg conjugation lrrc inhibits type i ifn signaling by targeting isg -associated rig-i for autophagic degradation isg : leading a double life as a secreted molecule. experimental and molecular medicine extracellular isg signals cytokine secretion through the lfa- integrin receptor enterovirus apro targets mda and mavs in infected cells the leader proteinase of foot-and-mouth disease virus inhibits the induction of beta interferon mrna and blocks the host innate immune response nmr analysis of the interaction of picornaviral proteinases lb and a with their substrate eukaryotic initiation factor gii residue l of the foot-and-mouth disease virus leader proteinase is a determinant of cleavage specificity functional dissection of the tbk molecular network innate immune sensor lgp is cleaved by the leader protease of foot-and-mouth disease virus tbk- promotes autophagy-mediated antimicrobial defense by controlling autophagosome maturation human tank-binding kinase is required for early autophagy induction upon herpes simplex virus infection tbk -mediated phosphorylation of lc c and gabarap-l controls autophagosome shedding by atg protease subversion of cellular autophagosomal machinery by rna viruses cellular origin and ultrastructure of membranes induced during poliovirus infection viral reorganization of the secretory pathway generates distinct organelles for rna replication picornavirus infection induces temporal release of multiple extracellular vesicle subsets that differ in molecular composition and infectious potential nonlytic viral spread enhanced by autophagy components foot-and-mouth disease virus utilizes an autophagic pathway during viral replication foot-and-mouth disease virus nonstructural protein c interacts with beclin , modulating virus replication peroxisomes are signaling platforms for antiviral innate immunity mitochondrial-associated endoplasmic reticulum membranes (mam) form innate immune synapses and are targeted by hepatitis c virus impairment of the deisgylation activity of fmdv lpro causes attenuation in vitro and in vivo mé nard r. the papain-like protease from the severe acute respiratory syndrome coronavirus is a deubiquitinating enzyme severe acute respiratory syndrome coronavirus papain-like-protease: structure of a viral deubiquitinating enzyme mé nard r. selectivity in isg and ubiquitin recognition by the sars coronavirus papain-like protease papain-like protease from transmissible gastroenteritis virus: crystal structure and enzymatic activity toward viral and cellular substrates proteolytic processing and deubiquitinating activity of papain-like proteases of human coronavirus nl mers-cov papain-like protease has deisgylating and deubiquitinating activities deubiquitinase function of arterivirus papain-like protease suppresses the innate immune response in infected host cells in vivo assessment of equine arteritis virus vaccine improvement by disabling the deubiquitinase activity of papain-like protease crystal structure of the middle east respiratory syndrome coronavirus (mers-cov) papain-like protease bound to ubiquitin facilitates targeted disruption of deubiquitinating activity to demonstrate its role in innate immune suppression the eif g-eif e complex is the target for direct cleavage by the rhinovirus a proteinase poliovirus a proteinase cleaves directly the eif- g subunit of eif- f complex enterovirus protease apro targets mavs to inhibit anti-viral type i interferon responses essential role of enterovirus a protease in counteracting stress granule formation and the induction of type i interferon picornavirus a protease regulates stress granule formation to facilitate viral translation fmdv leader protease cleaves g bp and g bp and inhibits stress granule formation translation without eif promoted by l protease from foot and mouth disease virus confers eif -independent translation for mrnas bearing picornavirus ires novel mode of isg -mediated protection against influenza a virus and sendai virus in mice interferon-stimulated gene (isg ) and isg -linked proteins can associate with members of the selective autophagic process, histone deacetylase (hdac ) and sqstm /p modification of becn by isg plays a crucial role in autophagy regulation by type i ifn/interferon isg governs mitochondrial function in macrophages following vaccinia virus infection a continuous bovine kidney cell line constitutively expressing bovine αvβ integrin has increased susceptibility to foot-and-mouth disease virus knockout of cgas and sting rescues virus infection of plasmid dna-transfected cells genetically engineered foot-and-mouth disease viruses with poly(c) tracts of two nucleotides are virulent in mice the foot-and-mouth disease virus leader proteinase gene is not required for viral replication a novel, broad-spectrum inhibitor of enterovirus replication that targets host cell factor phosphatidylinositol -kinase iii-beta virus-dependent phosphorylation of the irf- transcription factor regulates nuclear translocation, transactivation potential, and proteasome-mediated degradation middle east respiratory coronavirus accessory protein a inhibits pkr-mediated antiviral stress responses triggering the interferon antiviral response through an ikk-related pathway. science ( -) shared and unique functions of the dexd/h-box helicases rig-i, mda , and lgp in antiviral innate immunity foot-andmouth disease virus leader proteinase: purification of the lb form and determination of its cleavage site on eif- γ a general chemical ligation approach towards isopeptide-linked ubiquitin and ubiquitin-like assay reagents molecular basis of lys -polyubiquitin specificity in the deubiquitinase cezanne we are grateful to john hiscott and takashi fujita for supplying plasmids, ann palmenberg and ewald beck for providing antibodies, and david rowlands and toby tuthill for supplying erav. key: cord- -u ggw authors: gao, peng; chai, yue; song, jiangwei; liu, teng; chen, peng; zhou, lei; ge, xinna; guo, xin; han, jun; yang, hanchun title: reprogramming the unfolded protein response for replication by porcine reproductive and respiratory syndrome virus date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: u ggw the unfolded protein response (upr) in the endoplasmic reticulum (er) constitutes a critical component of host innate immunity against microbial infections. in this report, we show that porcine reproductive and respiratory syndrome virus (prrsv) utilizes the upr machinery for its own benefit. we provide evidence that the virus targets the upr central regulator grp for proteasomal degradation via a mechanism that requires viral glycoprotein gp a, while both ire -xbp s and perk-eif α-atf signaling branches of the upr are turned on at early stage of infection. the activated effector xbp s was found to enter the nucleus, but atf was unexpectedly diverted to cytoplasmic viral replication complexes by means of nonstructural proteins nsp / to promote viral rna synthesis. rnai knockdown of either atf or xbp s dramatically attenuated virus titers, while overexpression caused increases. these observations reveal attractive host targets (e.g., atf and xbp s) for antiviral drugs and have implications in vaccine development. the unfolded protein response (upr) in the endoplasmic reticulum (er) represents an ancient but critical cellular mechanism in response to various deleterious stresses within the er lumen. it maintains er homeostasis by increasing protein-folding capacity, reducing global protein synthesis, and clearing misfolded proteins [ ] [ ] [ ] . protein loads and quality within the er lumen are monitored by three transmembrane proteins: pkr-like er kinase (perk), activating transcriptional factor (atf ) and inositol requiring enzyme (ire ) [ , ] . these sensors are normally kept in an inactive state by grp (also known as bip or hspa ), which is an er-resident chaperone and the master regulator of upr [ , ] . under er stress conditions, grp detaches from the sensors to allow their activation as it proceeds to binds to exposed hydrophobic regions of nascent polypeptides [ , ] . the three sensors regulated by grp control distinct pathways. activated perk phosphorylates eif α. although this leads to global translational attenuation, it does allow the preferential translation of a subset of stress-related mrnas, including the activating transcription factor (atf ) [ ] [ ] [ ] , which is a pro-survival protein that regulates the expression of stressrelated genes involved in anti-oxidation, amino acid biosynthesis and transport, apoptosis, etc [ ] [ ] [ ] [ ] . sensor atf can detect folding perturbations such as disruptions in disulfide bonding and protein under-glycosylation, and its activation leads to its translocation and cleavage within golgi by host proteases to release a transcriptionally-active fragment that induces expression of genes involved in protein folding, such as grp , pdi and grp , etc [ , [ ] [ ] [ ] [ ] . lastly, the ire -xbp branch is evolutionarily conserved from yeast to humans and responds to all types of er stress [ , ] . activated ire has the endoribonuclease activity and splices many mrna targets destined for translation at the er, which causes ire -dependent mrna decay [ , ] . although most of the target mrnas are degraded, the unconventional splicing of xbp mrna produces a functional transcript that encodes the transcription factor xbp s, which controls the expression of many target genes involved in lipid synthesis, erassociated degradation (erad), and chaperones [ ] [ ] [ ] . the cellular upr has been increasingly recognized as an intrinsic line among innate defenses that can result in either degradation of microbial components, translational inhibition, or apoptosis [ ] [ ] [ ] . moreover, the upr can regulate autophagy, inflammation, and type i interferon responses [ ] [ ] [ ] . consequently, investigations of how pathogens interface with this cellular response is required for a thorough understanding of how they replicate and cause disease [ ] . the experiments described in this report address the interplay of upr signaling with porcine reproductive and respiratory syndrome virus (prrsv), a positive-strand rna virus within the arteriviridae family in the order nidovirales [ , ] . prrsv mainly causes reproductive failure of sows and respiratory diseases of young pigs, and it has remained a major threat to global pork production since the first outbreak in late s [ ] . currently, there are no effective vaccines or anti-viral drugs available for this virus [ ] . persistent infections, dysregulation of host immunity, and rapid genetic mutation/recombination all contribute to the difficulty of disease control and the emergence of many highly virulent strains, including the deadly chinese highly pathogenic prrsv in [ ] [ ] [ ] [ ] . clearly, a better understanding of the interactions between this pathogen and its host is needed for developing effective prophylactic and therapeutic measures. prrsv has recently been shown to induce the activation of the perk and ire branches of the upr in porcine alveolar macrophage cell line zmac [ ] , but the significance of these events for virus replication is unknown. here, we show that the virus encodes a protein, gp a, which targets grp for degradation. moreover, we show that the upr does not inhibit but instead stimulates efficient replication of prrsv. in particular, the virus appears to hijack atf to viral replication and transcription complexes (rtcs) to facilitate viral rna synthesis. thus, our results reveal that prrsv exploits upr signaling for its own advantage. to investigate the mechanism of upr induction by prrsv, we initially focused on grp because it is the master regulator that binds to and negatively controls sensors perk, atf , and ire [ , ] . when levels of misfolded proteins rise, grp is released, resulting in activation of the upr [ ] . for many dna and rna viruses, their infections cause increased levels of grp , which is also a chaperone, so that the protein folding capacity of the cell can be enhanced [ ] [ ] [ ] . in the case of prrsv, we observed an elevation of grp at the mrna level in both prrsv-infected marc- cells (used for in vitro propagation of the virus; fig a) and primary pams (porcine alveolar macrophages, the major in vivo cell target; fig c) . but in spite of these increases, we found that grp protein levels actually declined as the infection proceeded (fig b and d ). in contrast, treatment with the potent er-stress inducer thapsigargin (tg) resulted in a dramatic increase of grp (fig b) , indicating that uninfected cells have the capacity to produce large amounts of this protein. the mechanism of prrsv-induced grp decay was probed by treating mock or infected marc- cells with either mg- (a proteasome inhibitor), bafilomycin a (a lysosomal inhibitor), z-vad-fmk (an apoptosis inhibitor), or dmso before collecting samples for analysis. additions of dmso, bafilomycin a , or z-vad-fmk did not have a significant effect, but treatment with mg- restored grp to a level similar to that of uninfected cells ( fig e) . thus, the proteasomal degradation pathway contributes to the reduced levels of grp seen in prrsv-infected cells. to identify the viral protein(s) needed for grp decay, we used transfection assays in marc- cells and hek- ft cells. expression of nonstructural proteins (s fig) and the major envelope membrane proteins (s fig) did not decrease grp expression but seemed to activate the upr, as indicated by the apparently increased levels of this protein. in contrast, the minor envelope protein gp a did not increase grp expression (s a fig) but instead caused a decrease of this protein in about % of the cells (s b fig). this activity was confirmed by western blot analyses in a dose-response experiment, which showed that increasing levels of gp a caused decreasing levels of grp (fig f) , and this effect could be reversed by treatment of the cells with mg- ( fig g) . because subgenomic mrna contains the open reading frames for both gp a and the e protein, we made constructs that eliminate the start codon for e (construct gp aΔe-ha) or express only the e protein (e-ha). the results show that gp a but not e protein decreased levels of grp (fig h) . to verify that marc- cells will exhibit the expected responses upon induction of the upr, we transfected them with an sirna that knockdowns expression of grp . in contrast to the mock-transfected control, rnai silencing of grp (fig a) resulted in an increased amount of xbp s (fig b) , elevated phosphorylation of eif α (fig c) , and increased expression of (fig c and d ). as expected, the scrambled-sequence control (sinc) did not induce these effects. while it seemed likely that the reduced levels of grp in prrsv infections (fig b and d ) would result in elevated expression of downstream components of the upr, it was possible that these would be targeted for degradation, too. to investigate this, the expression levels of the various upr components were examined. in marc- cells, we found that both the perk-eif α-atf and ire -xbp s branches of the upr were activated (fig a, c and e ). in particular, there was a gradual increase in the phosphorylation of perk and eif α, a concurrent increased in expression of atf , and an elevation of the spliced form (xbp s) of xbp at both the protein and mrna levels. in contrast, atf was not activated, and the cleaved fragment of atf was not detectable during infection. similar results were observed in infected, primary pams (fig b, d and f); however, the antibodies to atf did not work on pams extracts, and pdi was used as a surrogate since its expression is subject to regulation by atf [ ] . the degradation of grp and strong induction of downstream components raised the possibility that prrsv might utilize some of the upr machinery for its own benefit. to begin exploring this, we measured the temporal relationship between upr induction and infectious virion production. upregulation of atf and xbp s was detectable around hpi in marc- cells ( fig e) and hpi in pams ( fig f) . this coincided with detectable viral protein expression (here assayed for n; fig a and b ), but it occurred earlier than peak virus replication, which was at hpi in marc- cells ( fig g) and hpi in pams (fig h) . the observed correlation of the timing of upr induction and virus production in both cell types is consistent with the possibility of upr machinery being needed for prrsv replication. the importance of both atf and xbp s for prrsv replication was subsequently investigated with an rnai knockdown approach. all sirnas could efficiently silence the expression of the respective protein at h post transfection ( fig a) without affecting cell viability ( fig b) . infection of these cells with prrsv revealed a reduction in virus titers when either atf or xbp s expression was reduced, but depletion of atf did not have this effect ( fig c) . also, overexpression of atf or xbp s in marc- cells by means of lentivirus transduction significantly increased the virus yields ( fig d) . thus, both atf and xbp s, two downstream effectors of the upr, are critical for prrsv replication. to explore the roles of atf and xbp s in prrsv replication, we began by using a cytoplasmic-nuclear fractionation assay to measure their cellular distribution. marc- cells were either infected with prrsv or mock infected, and duplicates of these cultures were then treated with either thapsigargin (tg) or dmso at hpi for . h. the cytoplasmic and nuclear fractions were isolated and probed for the presence of atf and xbp s (fig a) . the quality of the fractionation was checked by assaying for cytoplasmic gapdh and nuclear histone subunit h . as expected, tg-treatment and prrsv infection upregulated the expression of both atf and xbp s (fig a, compare lanes and with lane ). in the case of xbp s, the protein was enriched in the nuclear fraction whether the cells were treated with tg (compare lanes and ) or infected (compare lanes and ). to our surprise, atf behaved differently. in tg-treated cells, this protein was almost exclusively found in the nuclear fraction (compare lanes and ), but in infected cells, it was found almost exclusively in the cytoplasmic fraction (compare lanes and ). in addition, prrsv infection blocked the tg-induced atf (fig a, compare lanes and ) . thus, prrsv appears to have a mechanism for retaining atf in the cytoplasm. to verify these observations in situ, an immunofluorescence assay (ifa) was used to detect the subcellular location of atf . (unfortunately, the antibodies against xbp s did not work in this assay). we found that atf was not within nuclei but instead was present at perinuclear regions in prrsv-infected cells (fig b, right panels) , and the tg-induced atf nuclear translocation was blocked by prrsv infection (s a fig). moreover, atf was found to colocalize very well with prrsv nsp and nsp ( fig b, right panels) , which are key components of viral replication and transcription complexes (rtcs) [ ] , suggesting that atf is hijacked to viral rtcs during infection. similar results were obtained in prrsv-infected primary pams (s fig) . consistent with cytoplasmic retention, the canonical atf -target genes gadd and asns [ ] were not upregulated during infection as compared with the mock or tg-treated cells (s b and s c fig) . also, we found that this phenomenon is independent of prrsv strain used (s fig to gain insight to how atf is diverted to viral rtcs, individual nonstructural proteins (nsps) were screened for their ability to cause cytoplasmic retention of atf in marc- cells that were treated with tg for . h at h post-transfection (s a fig) . we found that prrsv nsp was able to partially retain atf in the cytoplasm ( fig c and s b fig) , but coexpression with nsp , which is known to form heterodimer with nsp , dramatically increased the retention efficiency, even though nsp by itself-like nsp -did not have any activity ( fig c) . the interaction between nsp and endogenous atf was also confirmed with a co-ip assay using infected cell lysates ( fig d) . these results indicate that nsp / contributes to the recruitment of atf , which is also consistent with the proposed role of nsp / as membrane scaffolding proteins for viral rtcs assembly [ , ] . to learn when atf is redirected to viral rtcs, we used immunofluorescence staining at different times after infection. the results showed that atf became detectable around hpi in marc- cells, concurrent with the expression of the nsp replicase protein, and both colocalized well at the perinuclear region ( fig a) . similar finding was observed in primary pams ( fig b) . the detection of atf was earlier than observed with western blots (fig ) , and this is likely because a low moi was used ( . ). because most cells are not infected, the ifa would be expected to have greater sensitivity because it enables the detection of protein expression at the single cell level. in any case, it is clear that recruitment of atf to viral rtcs is an early event in prrsv infection. infection, the cells were collected and subject to western blot analysis with antibodies against the indicated proteins. nucleocapsid protein n and β-actin served as the indicator for infection and the loading control, respectively. (c and d) detection of xbp mrna splicing. prrsv-infected cells were harvested at the indicated times post infection. the xbp mrna sequence was amplified by rt-pcr followed by digestion with pst i and the products were analyzed by electrophoresis on a . % agarose gel. (e and f) quantification of the proteins or mrnas during prrsv infections. the abundance of pperk, peif a, atf , xbp s, atf , and pdi were expressed as fold changes compared to mock-infected control after being normalized against β-actin. having found that atf is critical for high virus titers and co-localizes with viral rtcs in prrsv infections, we hypothesized that this component of the upr facilitates viral rna synthesis. consistent with this, rnai knockdown of atf greatly reduced the level of total viral rnas, as measured by rt-qpcr analysis (fig a) , and this correlated well with the impaired virus production (fig c) . to investigate which step of viral rna synthesis was affected, we measured the relative abundance of negative-and positive-stranded rnas in marc- cells that had been transfected with either sirna-atf or the scrambled sinc h prior to infection. rna levels were analyzed during a single viral replication cycle (i.e., within hours) by rt-qpcr amplifying orf rna and calculating the amount present relative to the rna from gapdh, a house-keeping gene. the relative abundance of each viral rna was then calculated as the ratio of the sirna-atf -treated group compared to the sinc-treated control. as expected, no negative-stranded viral rnas could be detected in either the control or siatf -treated cells at hpi (fig b) . at - hpi, this rna became detectable, but its accumulation was greatly reduced when atf was depleted. positive-stranded rna could be detected at hpi due to the input of virions used to start the infection, but a gradual, obvious decrease was observed over time ( fig c) . we also measured the abundance of all subgenomic mrnas with primers that specifically amplify the leader-body junctions. the results show that reduced atf expression had an effect on all rna species regardless of the strands (s a and s b fig) , suggesting that this protein is a host factor required for viral rna synthesis. in support of our hypothesis, antibodies to atf , but not the isotype control (igg), could immunoprecipitate viral rna from infected marc- cells, as detected by rt-qpcr of the prrsv 'utr region ( fig d) . also, a purified gst-atf chimera, but not gst alone, could pull down viral rna from purified infected-cell rna (fig f) , which suggests that an atf -rna interaction can occur in the absence of other viral factors. as negative controls, neither the atf antibodies nor the gst-atf chimera pulled down gapdh rna (fig e and g ). lastly, confocal microscopy of infected cells showed that atf exhibited remarkable co-localization with negative-stranded viral rna (detected by rnascope in situ hybridization), as well as the viral polymerase nsp in infected cells (fig h) . together, the above results provide strong evidence for a critical role of atf in prrsv rna synthesis via an interaction with viral rna. the endoplasmic reticulum has complex roles in autophagy, apoptosis, and innate immunity, and thus, is an important organelle in viral pathogenesis [ ] . this is also the location where the upr originates, and previous studies have revealed an association of this response with prrsv-induced apoptosis and dysregulation of tnf-α production in cell culture [ , ] . in this study, two unexpected findings were made that together reveal a novel way in which prrsv reprograms the cellular upr to its own advantage. in particular, the virus targets grp for degradation to induce or potentiate the upr, and it then hijacks atf to viral strain jxwn at an moi of . . at the indicated times, the total titer of infectious virus present in each culture was measured by the endpoint dilution assay. (d) marc- cells were transduced with lentivirus expressing atf -myc, xbp s-myc, or the empty vector. at h post transduction, the cells were infected with prrsv at an moi of . . at hpi, the total viral titer in each culture was measured by the end-point dilution assay (right panel), and the expression of the transduced proteins was analyzed by western blot (left). data information: statistical analysis was performed by two-tailed student's t-test, and asterisks ( � ) indicate the statistical significance: ns, no significance; � , p < . ; �� , p < . ; ��� , p < . . data were presented as means ± standard deviations (sd) of three independent experiments. https://doi.org/ . /journal.ppat. .g repurposing cellular upr by prrsv the first unexpected finding from this study was the downregulation of grp in a manner that is proteasome dependent and requires a viral membrane protein gp a. it is likely that the erad pathway is utilized to retrotranslocate gpr into the cytosol, where proteasomes reside [ ] , but the details of how gp a might cause this remain to be elucidated. in any case, downregulation of grp was surprising because many viruses, especially enveloped viruses and er-tropic viruses (e.g., the flaviviruses and coronaviruses), quickly produce large amounts of membrane proteins and thereby place a large burden on the folding machinery in the er [ ] [ ] [ ] . in response to this stress, most viruses tend to upregulate chaperon grp and other factors to meet the increased demand for folding and post-translational modifications [ ] . this is not the case for prrsv. because grp is the master regulator, its reduced accumulation likely prevents any modulation of the upr, keeping the er stress pathways induced for the abundance of viral rna was assessed by rt-pcr of orf , normalized against the house-keeping gene gapdh, and then compared the benefit of the virus. in addition to providing downstream factors such as atf that this virus utilizes for replication, the reduced levels of grp will likely result in increased levels of misfolded cellular proteins, potentially aiding in immune evasion [ , , ] . what remains unclear is how prrsv promotes the folding of its own proteins in this situation. the second unexpected, and perhaps most exciting, finding from this study is the dependence of prrsv on two downstream upr effectors, atf and xbp s, which were induced in the reduction of gpr . with regard to the perk-eif α-atf signaling branch of the upr, previous studies of how viruses utilize atf have only revealed effects that depend upon its activity as a dna transcription factor. for example, in the cases of west nile virus (wnv) and infectious bronchitis virus (ibv) [ , ] , atf upregulates transcription factor chop to induce apoptosis and virus release. in these cases, activation takes place during the late stages of infection, after high titers of viruses have been made. other viruses (e.g., hsv- and dengue virus) use atf to upregulate gadd to antagonize eif α phosphorylation [ , ] . in the case of retroviruses (e.g., hiv), atf has been found to be diverted and bound to viral promoters to mediate transcriptional activation, and thus virus replication [ ] . in our studies, atf was found to be sequestered in the cytoplasm during prrsv infections, and consistent with this observation, the canonical atf target genes such as gadd and asns [ ] were not upregulated and eif α remained phosphorylated. this property of prrsv appears to be similar to that of mouse hepatitis virus (mhv) [ ] , where high levels of phosphorylated eif α and activation of atf were found without subsequent expression of gadd ; however, in that work, the authors did not examine possible roles for, or the intracellular location of, atf . mechanistically, we found that atf is recruited by nsp / to viral rtcs to promote viral replication. when this protein was depleted with sirnas, the accumulation of viral rna was severely attenuated for both the positive and negative strands. indeed, the effect seems to be equal on the accumulation of all viral rna species at the transcriptional level, suggesting that atf serves as a general facilitator for viral rna biogenesis. this is unlike host rna helicase ddx , which is relocated to the cytoplasm in coronavirus infections [ ] and is needed for the synthesis of much longer viral rna species [ ] . further evidence supporting the association of atf with the replication machinery of prrsv includes its interaction with viral rna in pulldown assays and co-localization with negative-strand rnas during infection. whatever its exact role, depletion of this component of the upr led to a dramatic attenuation of prrsv titers, whereas overexpression increased the viral yield. future studies are needed to dissect how exactly atf promotes rna biogenesis and to identify the atf -interacting domain or motif within prrsv so that a mutant virus can be tested for vaccine development. to sinc control at to hpi. (b and c) the same as (a) except the abundance of positive-and negative stranded rnas were analyzed by rt-qpcr at , , and hpi. (d) control, rabbit igg or antibodies specific for atf were used for immunoprecipitations from lysates of infected marc- cells (moi = . , hpi). the presence of viral rna was assayed by rt-qpcr targeting '-utr sequence or (e) gapdh as a negative control, and the fold enrichment was calculated against the normal rabbit igg group. (f) gst-atf or gst were purified from e. coli (left panel) and added to rna extracted from prrsv-infected marc- cells in an in vitro binding assay. rt-qpcr with primers specific for a 'utr sequence was used to detect viral rna (middle panel) and the fold of enrichment of viral rna was expressed against the gst control. (g) in parallel, the pull down of cellular gapdh mrna was used as a negative control. (h) to look for atf within viral replication compartments in infected marc- cells (moi = . , hpi), the negative-strand rna was detected by the rnascope in situ hybridization method, and atf and nsp were detected by immunostaining. data information: statistical analysis was performed by two-tailed student's t-test, and error bars indicate standard deviations (sd) of means. asterisks ( � ) indicate the statistical significance: ��� , p < . ; ���� , p< . ; ns, no significance (n = experiments in each condition). confocal images were acquired with nikon a confocal microscope. oil objective: x; zoom in x. https://doi.org/ . /journal.ppat. .g interestingly, atf has also been implicated in cancer progression [ ] . in tumors, atf expression is detected in hypoxic-and nutrient-deprived regions where it promotes metabolic homeostasis and cancer cell survival by transcriptionally regulating amino acid uptake and biosynthesis, autophagy, redox balance and angiogenesis [ , [ ] [ ] [ ] . as atf is normally expressed at low levels and is not essential for normal cells, this molecule serves as a potential target for drug development in both human and veterinary medicine. currently, there are no approved drugs for this target, and a search for one is warranted. we also found that prrsv is dependent on xbp s. the ire branch of the upr is linked to many functions, such as apoptosis through the ire -mediated ridd and jnk pathways, inflammation through ire -ask pathway, autophagy, erad, and lipid synthesis through activation of xbp s [ , , , , ] . our study showed that ire is activated in infected cells, consistent with two previous reports [ , ] . while it is clear that the downstream effector xbp s is important for virus replication the mechanism was not further explored here. there are several ways that xbp s might be important. one is for fulfilling the increased demand for lipid synthesis, as the replication of some viruses, including prrsv, often involves extensive the activated effector xbp s enters nucleus, but atf is diverted to viral replication complexes by nonstructural proteins nsp / to promote viral rna synthesis. to maintain a favorable environment, prrsv targets grp for partial proteasomal degradation by viral glycoprotein gp a, which creates a positive feedback loop to sensitize and potentiate the upr signaling. https://doi.org/ . /journal.ppat. .g modulation, rearrangement and capture of intracellular membranes into virions [ , , ] . second, xbp s may be important for regulation of autophagy [ ] . indeed, xbp s has been shown to be an important regulator of genes involved in autophagy biogenesis, such as lc , ulk , atg , beclin , and bcl [ , , ] . third, xbp s may facilitate the degradation of misfolded proteins via the erad pathway and thereby increase the folding capability via protein chaperones [ , ] . prrsv appears to be different from closely-related coronaviruses in its requirement for xbp s signaling. for example, mhv induces splicing of xbp but suppresses the activation of its downstream target genes [ ] . also, transmissible gastroenteritis virus (tgev) triggers the activation of ire -xbp s signaling, but this pathway is not required for viral replication [ ] . in any case, since xbp s is a stress-inducing gene product and normally expressed at low level, it represents a potential antiviral target for prrsv. in summary, the findings reported here provide further insight into the mechanisms by which prrsv interacts with cellular pathways and reveal a novel paradigm for how pathogens can repurpose components upr for their own use. our results also identify potentially druggable targets (atf and xbp s) and provide new information for making attenuated vaccines to control prrsv. the sampling of primary porcine pulmonary alveolar macrophages (pams) derived from onemonth-old spf pigs was performed according to the chinese regulations of laboratory animals-the guidelines for the care of laboratory animals (ministry of science and technology of people's republic of china) and laboratory animal-requirements of environment and housing facilities (gb ± , national laboratory animal standardization technical committee). the license number associated with this research protocol was cau , which was approved by the laboratory animal ethical committee of china agricultural university. the chinese highly pathogenic prrsv strain jxwn (genbank accession no: ef ), low pathogenic prrsv strain hb / . (eu ), the nadc -like prrsv chsx (kp ) [ , ] , porcine epidemic diarrhea virus (pedv) bj c strain [ ] , and encephalomyocarditis virus (emcv) strain (bjc ) [ ] have been documented previously. antibodies used in this study were obtained from a variety of sources. mouse anti-ha monoclonal antibody (mab) (#h ), rabbit anti-myc polyclonal antibody (pab) (#c ) and mouse anti-β-actin mab (#a ) were all purchased from sigma-aldrich (mo, usa). rabbit anti-grp mab (#cs- ), rabbit anti-pdi pab (# ), rabbit anti-perk mab (# ), rabbit anti-eif α pab (#cs- ), and rabbit anti-phospho-eif α (ser ) mab (#cs- ) were obtained from cell signaling technology (beverly, ma). rabbit anti-atf pab (#nbp - ) and rabbit anti-xbp pab (#nbp - ) were obtained from novus biologicals (littleton, usa). rabbit anti-atf pab (# - -ap), anti-gadd pab (# - -ap), anti-gapdh pab (# - -ap) and histone-h pab (# - -ap) were purchased from proteintech (chicago, il). normal mouse igg used as negative control was purchased from beyotime (#a ). mouse anti-n (prrsv), anti-nsp β (prrsv), anti-nsp (prrsv), anti-nsp (prrsv), mouse anti-n (pedv) and anti-vp (emcv) mab were produced by our lab at china agricultural university and used as previously described [ , ] . the plasmids expressing nsp α, nsp β, nsp , nsp , nsp , nsp , nsp , nsp , nsp , nsp , nsp , nsp , gp a, gp , gp , gp , m and n were constructed by cloning the corresponding coding region from prrsv strain jxwn genome into the vector pcmv-ha (clontech, # ). gp aΔe-ha was created by introducing the corresponding mutations into the plasmid pgp a-ha with the quikchange site-directed mutagenesis kit (agilent technologies, # ). the atf gene from marc- cells was cloned into the vector pgex- p- (ge healthcare # - - ) for expression of the fusion protein gst-atf in e. coli bl cells (takara, # ). transfection of plasmid was performed with lipofectamine™ ltx reagent (thermo fisher, #a ) according to the manufacturer's instructions. marc- cells were infected with prrsv strain jxwn at an moi of . . at hpi, the cells were washed twice with cold phosphate-buffered saline (pbs) ( mm nacl, . mm kcl, mm na hpo , . mm kh po ), harvested with trypsin-edta digestion, and then centrifuged at , rpm for minutes. the cell pellets were resuspended with pbs, and the cytoplasmic and nuclear fractions were prepared by using ne-per nuclear and cytoplasmic extraction reagents (thermo fisher, # ), following the manufacturer's instruction. gapdh and histone-h were used as the respective cytoplasmic and nuclear makers for monitoring fraction quality by western blot. total rnas from cultured cells were extracted with trizol reagent (thermo fisher, # ) according to the manufacturer's instruction. the concentration of the extracted rna was measured using a nanodrop spectrophotometer (thermo fisher). reverse transcription was performed using a fastking rt kit (with gdnase) (tiangen, #kr - ) following the user guide. the xbp gene was amplified by pcr with the forward primer (f ) '-aaacagagtagcagcgcagactgc- ' and the reverse primer (r ) '-ggatctctaa gactagaggcttggtg- '. to resolve the spliced forms (xbp s) and unspliced (xbp u) of xbp , the pcr products were digested with the restriction enzyme pst i-hf (neb, #r l) and separated on the . % agarose gel. the cdna was synthesized from the total cellular rna by reverse transcription using the indicated primers. the relative quantitative pcr (qpcr) was performed with the applied biosys-tems™ sybr™ select master mix (thermo fisher, # ) according to the manufacturer's recommendations. the pcr was assembled in a μl reaction containing . μm gene specific primer, μl sybr™ select master mix, and μl cdna template. the pcr parameter was set up as follows: ˚c for min, ˚c for min; cycles of ˚c for s, ˚c for s, and ˚c for s. to measure viral total rna, a pair of internal primers in the orf gene were used to amplify all sgmrnas and grna. to measure grna and each viral sgmrna, a pair primers in the corresponding orf were used for the qpcr. the cellular gapdh was quantified as the internal control to normalize the cdna amounts. the -ΔΔct method was used to calculate the relative abundance of target genes compared to gapdh. the primers used for qpcr are listed in s and s tables. the lentiviral packaging system containing plasmids pwpxl (# ), pmd .g (# ) and pspax (# ) was purchased from addgene. the genes coding for xbp s and atf were cloned from marc- cells into the plasmid pwpxl and expressed as c-myc epitope fusion protein (xbp s-myc and atf -myc). the recombinant viruses were rescued by co-transfecting the three plasmids into hek- ft cells. at h post-transfection, the supernatants containing lentiviruses were harvested, filtered and concentrated. titers of the lentiviruses were determined using a quicktiter™ lentivirus titer kit according to the manufacturer's instruction (cell biolabs, #vpk- ). marc- cells in -well plates were transduced with × lentiviral particle (lp). at h post-transduction, the cells were infected with prrsv strain jxwn at an moi of . . the expression level of xbp s and atf was assessed by western blotting with rabbit anti-myc polyclonal antibodies to measure the effect of overexpression of atf and xbp s on viral replication, and the total virus titer was assessed by end point dilution assay. for rna interference, small interfering rnas (sirnas) was designed to target two different coding regions for each given gene. the sequences of sirnas are listed in s table. sirnas were transfected with lipofectamine rnaimax (thermo fisher, # ) according to the manufacturer's instruction. cell viability was assessed at h post transfection with celltiter aqueous one solution reagent (promega, #g ), and the knockdown effect was examined by western blot analyses of the whole cell lysate with antibodies to atf , atf , grp or xbp s, and β-actin was used as a loading control. for transfection/infection assay, marc- cells were infected with prrsv strain jxwn at h post transfection of sirnas, and total viruses were collected at the indicated time points for titration, and total rnas were extracted for analyzing the viral rna abundance by relative qpcr. the amount of total cellular proteins was quantified by using pierce™ bca protein assay kit (thermo fisher, # ), and - μg of the whole lysate per sample was subject to western blot analysis. briefly, the protein samples were separated by sds-page, transferred onto pvdf membranes, blocked with pbst (pbs with . % tween- ) containing % milk for . h, and then probed with appropriate primary antibodies at room temperature. for detection of phosphorylated proteins, bovine serum albumin (bsa) was used instead of milk. the membranes were then washed with pbst and incubated with appropriate hrp-conjugated secondary antibodies with dilution ratio : , . the membranes were again washed and then developed with the pierce ecl western blot substrate (thermo fisher, # ). marc- cells in six-well plates were infected with prrsv strain jxwn at an moi of . . at hpi, the cells were harvested and lysed in np- lysis buffer ( . % np- , mm nacl, mm tris-hcl ph . ) containing protease inhibitor (sigma, #p ). after clarifying by centrifugation at , rpm for min, the supernatants were precleared with protein a/g sepharose beads (santa cruz, #sc- ) and then incubated with μl mouse anti-nsp mab (mouse isotype antibody as a negative controls) and μl protein a&g sepharose beads overnight at ˚c with gentle rotation. the beads were washed five times with the np- lysis buffer, and the proteins bounded to the beads were separated by sds-page, followed by western blot analysis. marc- cells grown to - % confluence on coverslips in six-well plates were infected with strain jxwn at an moi of . . at indicated time points post infection, the cells were fixed with . % paraformaldehyde for min at room temperature, permeabilized with pbs containing . % triton x- and % bovine serum albumin (bsa) for min, and then blocked with % bsa-pbs ( % bovine serum albumin in pbs) for min. the cells were then incubated with primary antibodies as indicated in a humid chamber for h at room temperature or overnight at ˚c. after being washed times with pbs for min each, the cells were incubated with appropriate second antibody (alexa , or -conjugated) for additional h. nuclear dna was stained with ', -diamidino- -phenylindole (dapi) (thermo fisher, # ) for min and then washed with pbs three times for min each. the cells were imaged using a nikon a confocal microscope. to detect negative-stranded viral rnas, in situ hybridization was employed by using the rnascope multiplex fluorescent detection reagents v kit (acd, # ). a total of double-z branched pairs targeting the regions of orf , orf and 'utr of the prrsv genome (nt position , - , ) as rna probe (acd, # ) to detect negative-strand prrsv rnas. the hybridization procedure was performed according to the manufacturer's instruction. briefly, marc- cells on lab-tek ii chamber slides (thermo fisher, # ) were infected with prrsv at an moi of . . at the indicated time points post infection, the cells were fixed with % neutral buffered formalin (nbf), followed by dehydration and rehydration steps with suggested concentrations of ethanol and subsequent treatment by hydrogen peroxide (acd) and protease iii (acd). the cells were then incubated with rna probes for h at ˚c in hybez™ oven (acd), followed by a cascade of signal amplification and a series of washing procedures. hybridization signals were detected by tsa plus cyanine (perkinelmer, #nel e kt). for triple staining, the cells were fixed again with . % paraformaldehyde and then processed following the normal immunofluorescence assay procedure as described above. rna immunoprecipitation was carried out using the ez-magna rip™ rna-binding protein immunoprecipitation kit by following the manufacture's protocol (merck millipore). briefly, marc- cells in mm petri dish were infected with prrsv strain jxwn at an moi of . for h and then washed twice with ice-cold pbs. the cells were then scraped off the plates, collected by centrifugation, and lysed by complete rip lysis buffer with protease inhibitor cocktail (sigma, #p ) and rnase inhibitor (thermo fisher, #am ), followed by a step of centrifugation at , rpm for min at ˚c to remove the cell debris. to immunoprecipitate atf -rna complexes, magnetic beads were mixed with rabbit anti-atf polyclonal antibodies or normal rabbit igg (isotype control), and then added to the clarified cell supernatants for incubation overnight at ˚c. after washing with cold rip wash buffer six times, the beads-protein-rna complexes were digested with proteinase k at ˚c for min. afterwards, the supernatants were collected on a magnetic separator and transferred to a new tube for rna exaction with trizol reagent (thermo fisher, # ). the abundance of viral rnas was analyzed by relative rt-qpcr targeting prrsv 'utr or gapdh (as a control). e. coli bl cells containing the plasmid pgst-atf or pgex- p- vector were grown in ml x yt (yeast extract and tryptone) media at ˚c. protein expression was induced for h with isopropyl β-d- -thiogalactopyranoside (iptg) at a concentration of . mm when the optical density at nm reached . . the cultures were pelleted at , rpm for min, resuspended in pbs supplemented with bacterial protease inhibitors, sonicated, and lysed for min on ice with % triton x- . the lysates were cleared twice at , rpm for min at ˚c, and the supernatants were incubated with glutathione-sepharose b beads at room temperature for h, washed times with pbs, and resuspended in μl pbs. to test the interaction with viral rna in vitro, total rna was extracted from prrsvinfected marc- cells using trizol reagent, and μg were used for incubation with gst-atf or gst beads in μl np buffer ( . % np- , mm nacl, mm tris-hcl, ph . ) with protease inhibitor cocktail (sigma, #p ) and rnase inhibitors (thermo fisher, #am ) overnight at ˚c. after washing six times with np- buffer, the protein-rna complexes were eluted from beads with μl elution buffer ( mm tris-hcl, ph . , mm edta, ph . , . % sodium dodecyl sulfate), and the proteins were digested at ˚c for . h using proteinase k at a concentration of . mg/ml. afterwards, the supernatants were collected by centrifugation at , rpm for min and transferred to a new tube where the abundance of viral rna was analyzed by relative rt-qpcr targeting prrsv 'utr or gapdh as a control. following treatment with sirnas or lentivirus transduction, marc- cells were infected with prrsv strain jxwn . after incubation at ˚c for h, unbound viruses were washed off with serum-free dmem three times, and the cell cultures were supplemented with maintenance medium containing % fbs. at indicated time points post infection, the whole cell culture was collected, and the virus was titrated by the end point dilution assay. all the graphs and relevant statistical tests used in the work were created by graphpad prism version . (la jolla, ca, usa). statistical significance between two groups was analyzed by two-tailed unpaired student's t-test or one-way anova, and asterisks indicate the statistical significance: ns, no significance; � , p < . ; �� , p < . ; ��� , p < . . error bars indicate means ± standard deviations (sd). the unfolded protein response: from stress pathway to homeostatic regulation the unfolded protein response intracellular signaling by the unfolded protein response signal integration in the endoplasmic reticulum unfolded protein response the unfolded protein response and cell fate control dynamic interaction of bip and er stress transducers in the unfolded-protein response master regulator of the unfolded protein response and crucial factor in flavivirus biology dissociation of kar p/bip from an er sensory molecule, ire p, triggers the unfolded protein response in yeast coping with stress: eif kinases and translational control reinitiation involving upstream orfs regulates atf mrna translation in mammalian cells regulated translation initiation controls stress-induced gene expression in mammalian cells an integrated stress response regulates amino acid metabolism and resistance to oxidative stress transcription factor atf directs basal and stress-induced gene expression in the unfolded protein response and cholesterol metabolism in the liver trb , a novel er stress-inducible gene, is induced via atf -chop pathway and is involved in cell death activating transcription factor in vitro reconstitution of er-stress induced atf transport in copii vesicles the luminal domain of atf senses endoplasmic reticulum (er) stress and causes translocation of atf from the er to the golgi underglycosylation of atf as a novel sensing mechanism for activation of the unfolded protein response atf is a transcription factor specializing in the regulation of quality control proteins in the endoplasmic reticulum er stress signaling by regulated splicing: ire / hac /xbp intracellular signaling from the endoplasmic reticulum to the nucleus: the unfolded protein response in yeast and mammals decay of endoplasmic reticulum-localized mrnas during the unfolded protein response rna surveillance is required for endoplasmic reticulum homeostasis xbp : a link between the unfolded protein response, lipid biosynthesis, and biogenesis of the endoplasmic reticulum the multiple roles of xbp in regulation of glucose and lipid metabolism a review of the mammalian unfolded protein response innate sensing of influenza a virus hemagglutinin glycoproteins by the host endoplasmic reticulum (er) stress pathway triggers a potent antiviral response via er-associated protein degradation antiviral activity of an isatin derivative via induction of perk-nrf -mediated suppression of cap-independent translation roles of endoplasmic reticulum stress in immune responses cross talk between er stress, oxidative stress, and inflammation in health and disease crosstalk of er stress-mediated autophagy and er-phagy: involvement of upr and the core autophagy machinery fxr inhibits endoplasmic reticulum stress-induced nlrp inflammasome in hepatocytes and ameliorates liver injury the expanding roles of endoplasmic reticulum stress in virus replication and pathogenesis nidovirales: a new order comprising coronaviridae and arteriviridae reorganization and expansion of the nidoviral family arteriviridae mystery swine disease in the netherlands: the isolation of lelystad virus pathogenesis and control of the chinese highly pathogenic porcine reproductive and respiratory syndrome virus emergence of fatal prrsv variants: unparalleled outbreaks of atypical prrs in china and molecular dissection of the unique hallmark importation and recombination are responsible for the latest emergence of highly pathogenic porcine reproductive and respiratory syndrome virus in china porcine reproductive and respiratory syndrome virus: a persistent infection genotype strains of porcine reproductive and respiratory syndrome virus dysregulate alveolar macrophage cytokine production via the unfolded protein response the er chaperone and signaling regulator grp /bip as a monitor of endoplasmic reticulum stress japanese encephalitis virus infection initiates endoplasmic reticulum stress and an unfolded protein response endoplasmic reticulum stress is induced and modulated by enterovirus induction of the unfolded protein response (upr) during marek's disease virus (mdv) infection. virology protein disulfide isomerase-associated is an atf -inducible er stress response protein that protects cardiac myocytes from ischemia/reperfusionmediated cell death the prrsv replicase: exploring the multifunctionality of an intriguing set of nonstructural proteins atf -dependent transcription mediates signaling of amino acid limitation orf a-encoded replicase subunits are involved in the membrane association of the arterivirus replication complex non-structural proteins and interact to modify host cell membranes during the formation of the arterivirus replication complex involvement of unfolded protein response, p and akt in modulation of porcine reproductive and respiratory syndrome virus-mediated jnk activation new insights into the physiological role of endoplasmic reticulum-associated degradation er stress, autophagy, and rna viruses how does the stressed out er find relief during virus infection? coronavirus infection, er stress, apoptosis and innate immunity viruses, endoplasmic reticulum stress, and interferon responses the molecular chaperone grp contributes to toll-like receptor -mediated innate immune response to hepatitis c virus in hepatocytes grp plays an integral role in tumor cell inflammationrelated migration induced by m macrophages west nile virus infection activates the unfolded protein response, leading to chop induction and apoptosis upregulation of chop/gadd during coronavirus infectious bronchitis virus infection modulates apoptosis by restricting activation of the extracellular signal-regulated kinase pathway herpes simplex virus infection activates the endoplasmic reticulum resident kinase perk and mediates eif- alpha dephosphorylation by the gamma( ) . protein the role of the unfolded protein response in dengue virus pathogenesis hiv exploits antiviral host innate gcn -atf signaling for establishing viral replication early in infection coronavirus infection modulates the unfolded protein response and mediates sustained translational repression the cellular rna helicase ddx interacts with coronavirus nonstructural protein and enhances viral replication nucleocapsid phosphorylation and rna helicase ddx recruitment enables coronavirus transition from discontinuous to continuous transcription targeting the atf pathway in cancer therapy psat is regulated by atf and enhances cell proliferation via the gsk beta/beta-catenin/cyclin d signaling pathway in er-negative breast cancer role of atf in regulation of autophagy and resistance to drugs and hypoxia the endoplasmic reticulum stress sensor ire alpha protects cells from apoptosis induced by the coronavirus infectious bronchitis virus xbp mrna splicing triggers an autophagic response in endothelial cells through beclin- transcriptional activation mouse hepatitis virus infection activates the ire /xbp pathway of the unfolded protein response japanese encephalitis virus activates autophagy through xbp and atf er stress sensors in neuronal cells a regulatory link between er-associated protein degradation and the unfolded-protein response the perk arm of the unfolded protein response negatively regulates transmissible gastroenteritis virus replication by suppressing protein translation and promoting type i interferon production nadc -like strain of porcine reproductive and respiratory syndrome virus the -amino-acid deletion in the nsp of highly pathogenic porcine reproductive and respiratory syndrome virus emerging in china is not related to its virulence development of the full-length cdna clones of two porcine epidemic diarrhea disease virus isolates with different virulence genomic analysis of two porcine encephalomyocarditis virus strains isolated in china an infectious arterivirus cdna clone: identification of a replicase point mutation that abolishes discontinuous mrna transcription mapping the nonstructural protein interaction network of porcine reproductive and respiratory syndrome virus targeting swine leukocyte antigen class i molecules for proteasomal degradation by the nsp alpha replicase protein of the chinese highly pathogenic porcine reproductive and respiratory syndrome virus strain jxwn we sincerely thank dr. eric j. snijder (leiden medical center, netherlands) for providing eav infectious cdna clone and dr. qin wang for providing csfv and the antibodies to e protein. we also sincerely thank dr. john w. wills (pennsylvania state university college of medicine) for language editing of the manuscript. key: cord- -pa p dif authors: rozen-gagnon, kathryn; stapleford, kenneth a.; mongelli, vanesa; blanc, hervé; failloux, anna-bella; saleh, maria-carla; vignuzzi, marco title: alphavirus mutator variants present host-specific defects and attenuation in mammalian and insect models date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: pa p dif arboviruses cycle through both vertebrates and invertebrates, which requires them to adapt to disparate hosts while maintaining genetic integrity during genome replication. to study the genetic mechanisms and determinants of these processes, we use chikungunya virus (chikv), a re-emerging human pathogen transmitted by the aedes mosquito. we previously isolated a high fidelity (or antimutator) polymerase variant, c y, which had decreased fitness in both mammalian and mosquito hosts, suggesting this residue may be a key molecular determinant. to further investigate effects of position on rna-dependent rna-polymerase (rdrp) fidelity, we substituted every amino acid at this position. we isolated novel mutators with decreased replication fidelity and higher mutation frequencies, allowing us to examine the fitness of error-prone arbovirus variants. although chikv mutators displayed no major replication defects in mammalian cell culture, they had reduced specific infectivity and were attenuated in vivo. unexpectedly, mutator phenotypes were suppressed in mosquito cells and the variants exhibited significant defects in rna synthesis. consequently, these replication defects resulted in strong selection for reversion during infection of mosquitoes. since residue is conserved among alphaviruses, we examined the analogous mutations in sindbis virus (sinv), which also reduced polymerase fidelity and generated replication defects in mosquito cells. however, replication defects were mosquito cell-specific and were not observed in drosophila s cells, allowing us to evaluate the potential attenuation of mutators in insect models where pressure for reversion was absent. indeed, the sinv mutator variant was attenuated in fruit flies. these findings confirm that residue is a determinant regulating alphavirus polymerase fidelity and demonstrate proof of principle that arboviruses can be attenuated in mammalian and insect hosts by reducing fidelity. during replication, rna viruses generate approximately error per nucleotides copied, giving rise to an immense population of genetically distinct but closely related variants [ , , , ] . the genetic diversity of these ''mutant swarms'' is not detected by consensus sequencing, which to-date has been the basis for most studies of viral infection. however, this lack of information on genetic diversity has obscured crucial aspects of virus biology. although rna-dependent rna polymerases (rdrp) have a high intrinsic error rate, their mutation rates can be altered to generate both higher and lower fidelity variants (antimutators and mutators, respectively) [ , , , , , , , , , ] . thus far, antimutator variants are thought to replicate more slowly, making fewer genomes with greater accuracy; in contrast, mutator variants have been shown to replicate more quickly, synthesizing more viral genomes but introducing many errors during the replication process [ , , , , ] . despite this, overall growth and titers of polymerase fidelity variants are not significantly different when grown in isolation in cell culture; for mutators the negative effects of accumulating deleterious mutations are only noticeable after several rounds of replication [ , ] . in recent works, these variants have been useful in exploring how the course of viral infection is affected by either restricted or expanded population diversity [ , ] . current evidence indicates that mutation frequencies of rna viruses have been optimized over time to be neither too accurate nor too erroneous [ , , , , , , ] . it is thought that errorprone replication allows the virus to explore sequence space to gain adaptability and accumulate potentially advantageous mutations. for several rna viruses, limiting viral population diversity has fitness costs in vivo. despite similar in vitro growth phenotypes, variants that make fewer errors have reduced titers and exhibit restricted tropism in animal models [ , , ] . this restriction in tropism may be due to cooperative inter-variant interactions or beneficial minority variants that are missing in a situation with restricted population diversity [ ] . it is also proposed that high mutation rates of influenza a may contribute to altered tropism, allowing infection of new hosts [ ] . therefore, it seems that the relatively high error rates of rna viruses generate a level of diversity that facilitates adaptive fitness advantages. in contrast, there is also an upper threshold to mutation frequencies; if crossed, extreme error rates lead to the accumulation of deleterious mutations and loss of genetic integrity. evidence for this is demonstrated by treatment of numerous rna viruses with nucleoside analog mutagens, which increase mutation frequencies and result in extinction by lethal mutagenesis [ , , , , ] . although thus far rdrp mutators have not exhibited growth defects in isolation in vitro, a recent paper showed that hiv mutator and antimutator strains were less fit than wildtype in competition assays [ ] . in addition, several studies recently report in vivo attenuation of mutator strains: coxsackie virus b mutator strains present reduced viral titers in key organs and fail to establish persistent infections in mice [ ] , and a severe acute respiratory syndrome (sars) coronavirus mutator strain exhibits reduced pathogenesis in several mouse models [ ] . antimutator and mutator variants are valuable tools to study where the threshold of advantageous polymerase error exists for viruses facing different selective pressures. in this respect, arboviruses represent a special evolutionary position due to their need to replicate in disparate hosts, which is accompanied by distinct selective pressures. arbovirus fitness is not necessarily reduced due to obligate host-cycling (alternating passages of chikv did not limit viral fitness), yet it has been shown that evolvability may be reduced due to these evolutionary constraints [ , , , , , ] . for alphaviruses, evidence suggests that viral diversity is most restricted in the insect host, due to more stringent population bottlenecks and selective pressures [ , , , , , ] . since minority variants are thought to play important roles in arbovirus pathogenesis, transmission, and emergence [ , , , , , ] , the implications of altered polymerase fidelity and mutation rates merit further study. recently, this question was partially addressed using a chikungunya virus antimutator variant [ ] . chikungunya virus (chikv) is a re-emerging arbovirus, transmitted by aedes species mosquitoes. this positive-stranded rna virus (family togaviridae, genus alphavirus) has an . kb genome, of which the first . kb encode four nonstructural proteins (nsp - ) involved in diverse processes including rna synthesis, immune evasion, and host tropism [ , , , , , , ] . in most cases, functions of these proteins are putative in chikv and have only been shown in related model viruses, such as semliki forest virus and sindbis virus [ , , , ] . nsp is the rdrp, responsible for nucleotide incorporation during replication [ ] . previously, we isolated an antimutator strain of chikv by passaging virus in ribavirin, an rna nucleoside analog. ribavirin causes nucleotide misincorporation by the rdrp, adding selective pressure for an intrinsically more faithful polymerase [ , , ] . this antimutator strain harbored a single amino acid change ( y) in nsp . although y showed no growth defects in vitro, the variant was moderately attenuated in vivo in both mammalian and mosquito hosts [ ] . however, no arbovirus mutators have been isolated thus far. to this end, we mutated the conserved cysteine residue at position to obtain several mutators in the arboviruses chikv and sinv, confirming this position's importance in determining alphavirus fidelity. we used these novel mutator strains to examine how increased polymerase error affects arbovirus fitness in vitro and in vivo; interestingly, mutator strains presented distinct cell-and hostspecific phenotypes. mammalian cell lines vero, hela, and bhk- were maintained in dmem (gibco) supplemented with % newborn calf serum (ncs, gibco) and % penicillin-streptomycin (p/s, sigma), at uc with % co . mosquito cell lines c / and u . (aedes albopictus) and aag (aedes aegypti) were grown in l- media, supplemented with % fetal bovine serum (fbs, gibco), % p/s, % tryptose phosphate, and % non-essential amino acids (neaa), at uc with % co . drosophila melanogaster s cells were grown in schneider's drosophila media (gibco), supplemented with % fbs, % l-glutamine, and % p/s at uc. wildtype chikv was generated from the la reunion strain - infectious clone, previously described [ ] . nsp position mutants were generated by site-directed mutagenesis of the infectious clone using the quikchange ii xl site-directed mutagenesis kit (stratagene). all newly generated dna plasmids were sanger sequenced in full (gatc biotech) to confirm mutagenesis of position and to ensure no second-site mutations were introduced. select sinv mutants were constructed in the same fashion from the ptr wildtype infectious clone [ ] . chikv and sinv expression plasmids were linearized with noti or xhoi respectively, purified by phenol-chloroform extraction and ethanol precipitation, and subsequently used for in vitro transcription of viral rnas using the sp mmessage mmachine kit (ambion). rnas were then purified by phenol:chloroform extraction and ethanol precipitation, quantified, diluted to mg/ml and stored at uc. for rna transfections, bhk- cells were trypsinized, washed twice with ice-cold pbs, and resuspended at a concentration of cells/ml in ice-cold pbs. cells ( . ml) were mixed with mg of in vitro transcribed viral rna, placed in mm cuvette author summary chikungunya (chikv) is a re-emerging mosquito-borne virus that constitutes a major and growing human health burden. like all rna viruses, during viral replication chikv copies its genome using a polymerase that makes an average of one mistake per replication cycle. therefore, a single virus generates millions of viral progeny that carry a multitude of distinct mutations in their genomes. in this study, we isolated chikv mutators (strains that make more errors than the wildtype virus), to study how higher mutation rates affect fitness in arthropod-borne viruses (arboviruses). chikv mutators have reduced virulence in mice and severe replication defects in aedes mosquito cells. however, these replication defects result in selective pressure for reversion of mutators to a wildtype polymerase in mosquito hosts. to examine how mutators would behave in an insect model in absence of this genetic instability, we isolated mutators of a related virus, sindbis virus (sinv). sinv mutators had no replication defect in fruit fly (drosophila) cells, and a sinv mutator strain was stable and attenuated in fruit flies. this work shows proof of principle that arbovirus mutators can exhibit attenuation in both mammalian and insect hosts, and may remain a viable vaccine strategy. and electroporated at . kv, mf with infinite v in a xcell gene pulser (biorad). cells were allowed to recover for minutes at room temperature then mixed with ml of prewarmed media and placed into a t- flask. after hours incubation at uc, viral titers were determined by standard plaque assay. in brief, -fold serial dilutions of each virus in dmem were incubated on a confluent monolayer of vero cells for hour at uc. following incubation, cells were overlaid with . % agarose dissolved in dmem and % ncs and incubated at uc for hours. the cells were then fixed with % formalin for hour, the agarose plugs were removed, and plaques were visualized by the addition of crystal violet. plaque size was quantified by scanning the crystal violet-stained cell monolayer, then quantifying the size of each individual plaque in square millimeters using imagej (http://rsbweb.nih.gov/ij). each virus was then passaged once over a - % confluent monolayer of bhk- cells, titered as described above, aliquoted, and stored at uc until use. to analyze each virus for reversion at position , viral rna was extracted for each electroporation and bhk- passage using trizol reagent (invitrogen). for chikv, this rna was used to amplify a bp region corresponding to nucleotides ( - ), which included position , using the forward primer ( -gatgagcacatctc-catag- ) and the reverse primer ( -gtttgggttgggat-gaact- ) and the titan one tube rt-pcr kit (roche). for sinv, a bp region (nucleotides - ) was amplified in the same fashion using forward primer ( -accaggcacgaaa-cacacagaa- ) and reverse primer ( -actgggcgga-agtctgtatgcg - ). each pcr product was cleaned using the nucleospin pcr and gel extraction kit (macherey-nagel) and sanger sequenced at position / to confirm genetic stability. at passage , all viruses used were fully sequenced to ensure no second site mutations. hela cells ( , cells/well in -well tissue culture plates) were pre-treated for two hours with either media containing no mutagen, or media containing mm or mm ribavirin (sigma). post-treatment, media was removed and the cells were inoculated with virus in dmem at an moi . for one hour at uc. following incubation, mutagen-containing media was replaced and cells were incubated for hours at uc. virus was harvested at hours and mean titers were obtained by tcid . in brief, a -well tissue culture plates was plated for each virus with vero cells/well. viruses were serially diluted in ten-fold dilutions in dmem. each dilution was distributed in a row of the -well plate, with each well receiving ml of diluted virus. viruses and cells were incubated - days at uc with % co . following incubation, cells were fixed with ml of % formalin for minutes. all media were removed, and ml of crystal violet was added to each well. viruses that exhibited significant sensitivity or resistance compared to wildtype at p, . or greater at either mm or mm ribavirin were considered potential fidelity variants, and mutation frequencies were estimated (table ) . to determine mutation frequencies, all mutants were electroporated in tandem into bhk- cells. supernatants were collected hours later and viral rna was extracted. for chikv, an approximately bp region corresponding to nucleotides - was amplified of the e region of the genome using forward primer -tacgaacacgtaacagtgatcc- and reverse primer -cgctcttaccgggtttgttg- . for sinv, the analogous region was amplified using forward primer -tacgaacatgcgaccactgttc- and reverse primer -cgctcggagcggatttactg- , and approximately bases of this fragment was included in the analysis. amplified fragments were purified as described above, and ml of each product was modified by a a-overhang addition reaction ( ml amplitaq gold buffer, ml mm datp). modified products were cloned using the topota cloning kit (invitrogen), and single colonies were picked for sequencing. mutation frequencies were determined as previously described [ ] . mutation frequencies in mosquito cells were obtained in the same fashion using the samples obtained from c / growth curves (we determined mutation frequency for a wildtype sample electroporated into c / cells, and there was no difference between samples generated by infection or electroporation; the nonviability of mutators transfected into mosquito cells made it impossible to estimate mutation frequencies in c / by electroporation). we sequenced approximately clones per viral population in c / cells. mutation frequencies from mouse muscle were determined using rna extracted from homogenized muscle samples from mice that most closely represented the median titer for that variants. for estimating in vivo mutation frequencies, a minimum of clones were sampled per population. to confirm that the presence of rna or aberrant viral particles in supernatants/ homogenates did not affect mutation frequencies, we purified virus on % sucrose cushion and re-estimated mutation frequencies; no differences were observed. to estimate the population diversity of variants by deep sequencing, cdna libraries were prepared by superscript iii from rna extracted from virus generated in bhk- or c / cells, and the viral genome was amplified using a high fidelity polymerase (phusion) to generate overlapping amplicons - kb in length. pcrs were fragmented (fragmentase), multiplexed, clustered, sequenced in the same lane with illumina cbot and gaiix technology and analyzed with established deep sequencing data analysis tools and in house scripts. briefly, per-base phred quality scores were utilized to trim bases with error probabilities higher than . , and sequences with less than bases after trimming were discarded. for this purpose we used the fastq-mcf tool from the ea-utils toolkit at http://code.google. com/p/ea-utils [ ] . the alignment step is performed using burrows wheeler aligner [ ] and pileup is performed using samtools [ ] . once the pileup is done, an in-house script collects the data per-position and calculates the variance at each nucleotide position by root mean square deviation (rmsd) and determines the mean variance and standard error across the whole genome [ ] . to estimate population diversity in a phenotypic assay, we performed neutralization assays using viruses which had been passaged times on bhk- cells, using the n neutralizing antibody chk- (a kind gift from dr. m.s. diamond [ ] ). pfu of wildtype and mutator chikv strains were incubated for hour at uc with serial dilutions of antibody, ranging from mg/ml to . mg/ml, or left untreated. virus-antibody complexes were added to pre-seeded confluent monolayers of vero cells, and allowed to bind at uc for hour. assays were then overlayed with agarose and developed as described above for a plaque assay. plaques were counted and normalized to the untreated control for each virus. virus growth was evaluated for wt and all mutant viruses in bhk- , c / , u . , aag , and s cells and titers were determined by tcid on vero cells as described above. using the hour time point from the c / growth curve, we also performed a cytopathic effect (cpe) assay on c / cells on all viruses (celltiter aqueuos one solution cell proliferation assay (mts) kit; promega). we obtained similar titers by standard tcid and cpe assay, indicating that viruses amplified on mosquito cells were still equally infectious when titered on vero cells. for chikv, genome copy number was determined by extracting viral rna from the supernatant at each time point using the trizol reagent and performing quantitative rt-pcr (qrt-pcr) using the taqman rna-to-ct kit (applied biosystems). ct values were determined in duplicate based on amplification of nsp transcripts using forward ( -tcactccctgctggacttgataga- ) and reverse ( -tgacgaacagagttaggaacatacc- ) primers and probe -[ -fam] aggtacgcgcttcaagtt-cggcg- as previously published [ , ] . to determine genome copy number for sinv, viral rna was extracted in the same manner and quantitative pcr was performed based on amplification of nsp transcripts using forward ( -aaaacgcc-taccatgcagtg- ) and reverse ( -ttttccggctg-cgtaaatgc- ) primers and the sybr green pcr master mix (applied biosystems). standard curves were performed in each run using samples of in vitro transcribed chikv or sinv rna. in vitro transcribed rna was transfected in bhk- cells in duplicate, as described above or at uc, including rna from a construct in which the polymerase active site (gdd) was replaced with gnn by site-directed mutagenesis to abrogate replication and alongside a mock transfection where no rna was added. transfections in c / and u . cells were modified by pulsing with v, mf, and v. forty-eight hours post-transfection, supernatant containing progeny virus was collected. cells were washed twice in pbs and rna was trizol (invitrogen) extracted, quantified and diluted to the same concentration. samples were prepared in northernmax formaldehyde loading dye (ambion) with ml of ethidium bromide, heated to uc for minutes, then separated on a . % le agarose (lonza) gel containing morpholinepropanesulfonic acid (mops) running buffer (ambion) and . % formaldehyde. rna was transferred onto nitrocellulose membrane, cross-linked by ultraviolet irradiation (uvp), and prehybridized at uc for hour in ultrahyb ultrasensitive hybridization buffer (ambion). a plasmid used for the expression of chikv rna probes corresponding to the portion of the e glycoprotein was generated by first amplifying the region of the chikv genome from ( -gaagcgacagacgggacg- ) to ( -gttacatt-tgccagcggaa- ) by pcr and subsequently topo-ta cloning the pcr product into the pcrtopo-ii vector. rna probes complementary to positive strand rna were labeled with p using the maxiscript sp in vitro transcription kit (ambion), unincorporated nucleotides were removed using c bl/ mice (janvier) or cd- mice (charles river) were housed according to institut pasteur guidelines in biosafety level isolators, with the approved experimental protocol # . , reviewed by the institut pasteur ethics committee under dossier #cetea - . at -days old, litters of c bl/ were inoculated with pfu of wildtype or mutant chikv viruses subcutaneously (n = /variant). eight-day old cd- litters were inoculated with pfu of wildtype or mutant sinv strains in the same fashion, and monitored for symptoms of hind limb paralysis and survival. in addition, seven days post-infection, chikv and sinv-infected mice were sacrificed and brains, thigh muscles, livers and blood were harvested and homogenized in ml of pbs at shakes/second for min (mm retsch). rna was extracted and viral genome copies were determined by qrt-pcr as described. principal chikv vectors ae. albopictus providence (alprov, f generation) from la reunion and ae. aegypti paea (pae, a lab colony at institut pasteur since ) from tahiti, in french polynesia were fed on artificial bloodmeals containing pfu/ml of virus in pbs-washed rabbit blood [ ] . chikv wildtype and mutators were fed to both ae. albopictus and ae. aegypti, and sinv wildtype and mutator g were fed to ae. aegypti. the blood meals were warmed to uc and presented to day-old females in membrane feeders, and engorged mosquitoes were incubated for days. seven days post infection, mosquitoes were dissected to obtain legs and wings, and saliva was obtained by in vitro transmission assay; in brief, mosquitoes were salivated for - min by placing the proboscis in a pipette tip containing fbs. following salivation, bodies were frozen. to confirm ingestion, a sample of engorged mosquitoes was immediately homogenized at time . samples were homogenized as described for mouse tissues, rna was extracted, and qrt-pcr was performed. a standard curve was generated using serial dilutions of a chikv bloodmeal of known titer. drosophila melanogaster flies (strain w ) were reared on standard medium at uc. three-to four-day-old female flies were injected with nl of a virus dilution containing pfu in mm tris-hcl (ph . ) using a drummond nanoject injector as previously described [ ] . fly mortality at day was attributed to damage produced by the injection, and these flies were excluded from further analyses. mortality was monitored daily for days, and every - days flies were transferred to fresh vials. in all experiments, - flies per genotype group were injected. homogenates of individual flies were titrated on by plaque assay on vero cells, as described above. all experiments were performed in triplicate unless noted otherwise. statistics, noted where applied, were performed in microsoft excel and graphpad prism. p-values. . were considered non-significant (ns). we previously described a chikv antimutator variant that possessed a single amino acid change from a cysteine to a tyrosine at position (c y) of the rna-dependent rna polymerase nsp [ ] . since coxsackie virus b mutator strains are situated in a structurally analogous area, we hypothesized that this position plays important roles in modulating intrinsic chikv rdrp fidelity [ ] . to address this, we substituted each amino acid at position of the chikv full-length infectious clone (table ) . after three passages in bhk- cells, viruses were sanger sequenced to determine genetic stability. of the substitutions, were viable and genetically stable (table ). this high number of viable variants indicates that position has structural plasticity and can tolerate a wider range of substitutions than in previous attempts at generating fidelity variants of other rna viruses [ , ] . interestingly, unstable viruses did not readily revert to wildtype, but mutated to other variants, including the antimutator form of the protein, y ( table ). the only strict biochemical requirement we observed was a necessity for uncharged residues, as all variants with charged residues ( d, e, h, k, or r) were unstable or not recoverable. in addition, we observed a general correlation between hydrophobicity of the substituted amino acid and stability or viability of the variant, where hydrophobic amino acids were preferred. finally, as a first characterization of virus fitness, we measured the mean size of plaques. variants a, g, l, n, q, t, and w had significantly smaller plaques than wildtype ( table ) . because polymerase fidelity variants have altered intrinsic rates of (in)correct nucleotide incorporation, they have often been identified by their relative resistance or sensitivity to nucleoside analog rna mutagens [ , , ] . therefore, we addressed the sensitivity of all genetically stable variants to ribavirin (table and figure a ). viruses were grown in the presence of either mm or mm ribavirin, or left untreated. we expect antimutator variants (such as y) to demonstrate resistance, and mutator variants to demonstrate sensitivity when compared to wildtype. as previously described, the antimutator y demonstrated significantly higher survival than wildtype (p, . , twoway anova) as did m and n (p, . for both, two-way anova). additionally, we identified several mutator candidates that were significantly more sensitive to ribavirin ( a, g, w, t, q; p, . for all, two-way anova). all ribavirin-sensitive variants presented small-plaque phentoypes, as well as variant n (p, . for all, student's t-test). though these variants presented small plaque phenotypes, virus stocks reached wildtypelike titers, with the exception of n and q ( table ) . as observed previously for picornaviruses [ , , ] , the ribavirinresistant and -sensitive phenotypes of these chikv variants suggested altered polymerase fidelity. to address this further in a genetic assay, we estimated the mutation frequencies of each variant that demonstrated significantly altered ribavirin sensitivity at either concentration of ribavirin. viral rna from the supernatants of bhk- cells was extracted, and an approximately nucleotide fragment of the e genome was amplified by rt-pcr and topo cloned as previously described [ ] . we sequenced approximately individual clones per viral population (corresponding to an average of , nucleotides) to calculate the mutation frequencies ( figure b and table ). since previous studies with y required . clones per population to distinguish more subtle differences in mutation frequencies [ ] , we could only statistically confirm the altered fidelities of three mutator strains ( a, g and w; p, . , p, . , p, . , respectively, x test) ( figure b) . we excluded variants that did not exhibit significant fidelity differences compared to wildtype ( m, n, and q). as a complementary approach, we performed deep sequencing on these same virus populations to characterize the relative diversity in these virus populations. in accordance with the mutation frequency data, the mean variance across the whole genome was significantly lower for the antimutator y variant (p = . , mann-whitney u test) and significantly higher for the a, g and w mutator variants, compared to wildtype virus (p, . for all, mann-whitney u test; figure c ). next, we examined growth of these variants in mammalian cells. as seen previously, the antimutator y presented no significant difference in amount of progeny virus (figure a ) or number of genome copies ( figure b ). as observed with coxsackie virus mutators, chikv mutator strains ( a, g, and w) generated the same or more genomes than wildtype virus ( figure b ), but slightly fewer infectious progeny (figure a) . consequently, these mutator variants have a lower specific infectivity than wildtype in mammalian cells ( figure c ). this is consistent with previously published results showing that mutator variants make more lethally mutagenized rna [ , , , ] . recently, low fidelity polymerase mutators of coxsackie virus and exonuclease activity deficient mutators of coronaviruses were shown to be attenuated in mice [ , ] . to determine whether this holds true for alphaviruses, we administered a sublethal infection of either wildtype or a, g and w viruses to -day old c bl/ mice. at days of infection, when titers peak and virus is rapidly cleared thereafter, viral loads were determined in different compartments (muscle, blood, brain, liver). viral loads were significantly lower for all three mutator strains in each tissue ( figure a ). since the in vivo mutation frequencies of mutator strains had not been previously reported, we examined the virus populations in the muscle of the wildtype-or the mutator-infected mouse that presented the median viral load. although we cannot predict whether selection will act differently on these variants in mice to potentially skew the mutation frequencies, they remained elevated to varying degrees for the mutator strains. interestingly, higher mutation frequencies in vivo correlated with increased attenuation ( figure b ). because arboviruses must cycle through both vertebrate and arthropod hosts, and since mutator strains of other rna viruses were only examined in mammalian systems [ , , , ] , we addressed viral replication in three mosquito cell lines: ae. albopictus c / cells, ae. aegypti aag cells and ae. albopictus u . cells. the replication profile for the antimutator y was indistinguishable from wildtype in all conditions. on the other hand, the mutator strains a, g, and w presented significantly lower infectious progeny in c / (p, . for all, two-way anova; figure a ), aag (p, . for a and g, two-way anova; figure b ) and u . (p, . for a and w, p, . for g, two-way anova; figure c ) cells. unexpectedly, we observed unprecedented reduction in genomic rna released into the supernatant in all three mosquito cell types ( figure d -f). these results are discordant with the existing literature that found mutator polymerases synthesize rna at faster rates than wildtype [ ] , in which case decreases in virus titer resulted directly from the increased mutational burden. here, the reduced viral titers obtained in mosquito cells seem to result from a host-specific replication defect, rather than the effect of lethal mutation. to further distinguish between these two effects, we examined whether mutation frequencies differed in mosquito versus mammalian cells, comparing wildtype chikv to the mutator strains. it is important to note that because mutator strains replicate so poorly in mosquito cells, these strains may present artificially low mutation frequencies. unfortunately, it is not possible to uncouple replication from mutation frequency in this model. nevertheless, the mutation frequencies of all viruses, including wildtype), were lower in c / cells ( figure ) than in bhk- cells ( figure b) . furthermore, the significant differences that existed between mutators and wildtype in mammalian cells were negated in mosquito cells, as evidenced by molecular clone sequencing ( figure a ) and whole-genome deep sequencing ( figure b ). we thus hypothesized that the negative fitness cost of mutator polymerases in mosquito cells is more closely linked to replication defects. to further confirm this, we generated genetically homogenous in vitro transcribed rna corresponding to each variant, which do not present the differences in mutation frequencies of virus stocks generated in cell culture. following transfection of mammalian bhk- cells, there were no significant differences in rna synthesis ( figure a ) or production of infectious virus ( figure c) ; however, in mosquito c / cells, there was a very marked defect in replication for the mutator variants, compared to wildtype virus or the antimutator y strain ( figure b ), that correlated with the significant reduction in progeny (p, . for all mutators, one-way anova; figure d ). similarly, no detectable infectious progeny was produced following transfection of u . cells with the mutator variants ( figure e ), further confirming the replication defect observed during infection of cells with virus stocks. to exclude the possibility that this replication defect is the result of temperature-sensitivity rather than host-specificity, we performed infections in mammalian bhk- cells at uc (mosquito cell temperature). we observed no difference in the growth of any variant compared to wildtype ( figure f ). in addition, we transfected mammalian cells grown at uc, and saw no difference in subgenomic rna synthesis, indicating that the reduced polymerase processivity of mutators in mosquito cells is not due to reduced temperature ( figure b and g) . finally, we determined whether lower temperature could be responsible for the reduced mutation frequencies we observed in mosquito cells. in mammalian cells at uc, mutator g makes significantly more mutations than in mosquito cells at uc (p, . , x test; figure h ). in contrast to what we observed in mosquito cells, mutator g also made significantly more mutations than wt (p, . , x test; figure h ). these data indicate that lower temperature is responsible for neither the replication defects nor the reductions in mutation frequencies we observed in mosquito cells. in the mosquito host, selective pressure against the replication defects of mutator strains causes reversion since host-specific replication defects were observed in mosquito cell culture, we hypothesized that these variants would be even more attenuated in mosquitoes than in mice. we orally infected both aedes species chikv hosts (ae. albopictus and ae. aegypti) with a blood meal containing either wildtype or the a, g and w mutators. seven days after infection, when chikv has reached peak titers, we quantified viral loads in bodies (infection), legs and wings (dissemination) and saliva (transmission) of individual mosquitoes (figure ) . surprisingly, no significant defect was observed in either aedes species for any of the variants. to address the possibility that the fitness cost of defective replication, observed in mosquito cell culture, would favor the reversion of these mutant polymerases to wildtype, we deep sequenced virus from the body of an individual mosquito that presented the median titer from each group. indeed, reversion to wildtype (or other replication competent variants, such as t or v) occurred in a ( %), g ( %) and w ( %). whether position changed to wildtype depended on the genetic distance of the mutated codon from wildtype: for example, w (tgg) reverted completely to wt (tgt), while a (gct) reverted predominantly to a combination of v ( %; gtt) and t (act; %). interestingly, when we examined higher passages (passage ) of mutators in c / cell culture, we also observed varying levels of reversion (ranging from less than % to as much as %), highlighting the strong selective pressure acting against this replication defect. after confirming that polymerase position plays an important role in modulating fidelity in chikv, we examined if this residue is a universal fidelity determinant among the alphaviruses. indeed, this region of the nsp gene containing a cysteine is conserved across the alphavirus family ( figure a ). thus, we generated the analogous fidelity variants ( a, g, w) in the well-studied, distantly related alphavirus sindbis virus (sinv). genetically stable mutants ( a and g) were screened for changes in ribavirin sensitivity. both showed significantly higher sensitivity than wildtype sinv (for a, at least p, . , for g, p, . , two-way anova; figure b ). moreover, the mutation frequencies determined by molecular clone sequencing confirmed the mutator phenotypes suggested by ribavirin screening ( figure c ): in comparison to wildtype that presented . mutations per , nucleotides, a presented . , and g presented . (p, . , x test). this confirms that this conserved residue is a general fidelity determinant for the alphaviruses. we next addressed whether replication defects also existed for these sinv mutators. in mammalian bhk- cells, mutator variants produce near wildtype-like titers of infectious particles ( figure d ), and the same amounts of extracellular rna genomes ( figure e ). importantly, as was observed for chikv strains, the sinv mutators presented more significant drops in virus titers in mosquito c / cells (p, . , two-way anova; figure f ), that correlated with a significant decrease in extracellular rna genomes (p, . , two-way anova; figure g ). given the similarity of in vitro, host-specific phenotypes of chikv and sinv mutators, we hypothesized that sinv mutators would behave as chikv mutators in vivo (exhibiting attenuation in a mouse model and reversion in mosquitoes). we inoculated -day old mice with wildtype and g sinv strains, and observed significantly higher survival in mice infected with the mutator ( % compared to % for the wildtype, p = . ; figure a ). in addition, only % of mice inoculated with g exhibited complete hind limb paralysis, compared to % of mice infected with wildtype sinv (p, . , x test; figure a ). interestingly, this reduced paralysis correlated with significantly lower titers in the brain at day postinfection (p, . , student's t-test), confirming the attenuation of mutator strains in mammalian models in yet another virus ( figure b ). we next examined the in vivo phenotype of sinv mutator g in ae. aegypti mosquitoes. as expected, we observed reversion of position g to wildtype, and therefore, no differences in titers in the mosquito host ( figure c-e) . since sinv has a broader host range than chikv, we examined whether the replication defect was mosquito cellspecific, or more general to insects, by infecting drosophila s cells. interestingly, the mutator strains were replication competent, generating virus titers ( figure a ) and rna genome copies ( figure b ) at levels comparable to wildtype virus. finally, we injected drosophila with sinv wildtype and g mutator and followed the kinetics of infection by titering virus in flies for seven days post-infection. in contrast to chikv and sinv mutators in mosquitoes, when drosophila flies were infected with wildtype and mutator strains of sinv, mutator g presented significantly lower titers than wildtype on day and (p, . , student's t-test; figure c ). sequencing of virus from g-infected flies at day and confirmed that no reversion had occurred. these results indicate that in principle, mutators can be attenuated in insects. previous work on antimutator chikv y suggested this residue could be important for determining intrinsic rdrp fidelity [ ] . although there is no crystal structure available for an alphavirus rdrp, structural models predict that position is located in the same area of the rdrp that generated coxsackie virus rdrp fidelity variants ( figure s ) [ ] . by substituting all other amino acids at this position, more mutator variants were isolated than antimutators, consistent with variants obtained for coxsackie virus and with variants identified by characterizing the mutation frequencies of previously published reverse transcriptase variants for hiv [ , ] . to date, all viable rdrp fidelity variants present error rates that remain within the same order of magnitude as their wildtype counterpart [ , , , , , ] . interestingly, although biochemical assays using purified rdrp of picornaviruses indicate that altering fidelity beyond an order of magnitude is enzymatically possible, these viruses are not viable [ ] . together, these studies suggest that within this viable range, wildtype fidelity sits closer to higher fidelity than lower fidelity. this may be further reflection of how rna viruses are considered to exist close to a maximum threshold of error [ ] . in support of this, in conditions where this reversion does not occur, mutator chikv variants present more significant fitness defects in vivo ( figure ) than antimutator virus [ ] . a review of the antimutator and mutator rdrp variant literature in virology reveals the following trends: antimutator strains tend to generate less rna in vitro, but have higher specific infectivity, and have only been reported to lose fitness in vivo or in competition assays (figures and , and [ , , ] ); while mutators generate more rna in vitro, but of lower specific infectivity, with more prominent fitness defects in mice [ ] . accordingly, chikv mutators showed congruous trends, exhibiting no significant replication defects in bhk- cells, but showing marked attenuation in the mouse model. importantly, we showed that the mutator status of these variants (higher mutation frequencies) was maintained in the mouse model at the primary site of chikv replication and was likely responsible for the observed attenuation ( figure ) . however, when we examined chikv mutators in the invertebrate host, the previous trends for how mutators behave was reversed. first, the differences in mutation frequencies between wildtype and mutator strains became virtually indistinguishable in mosquito cells ( figure ) , although it is difficult to draw clear-cut conclusions given the reduced replication rate of mutators. for all viruses, mutation frequency was lower in mosquito cells compared to mammalian cells (figure ). the role that these differences may play in arbovirus evolvability and fitness remain contradictory. our observations corroborate previous observations in alphaviruses that inter-host cycling slows adaptation [ , , ] ; while flavivirus studies report that diversity is maintained in the mosquito host [ , , , , , , ] . second, and contrary to expectations, we observed a severe replication defect in three different mosquito cell cultures (figure ) , which had never been observed for rdrp fidelity variants in mammalian cell culture. the lower titers of infectious progeny were not the result of accumulation of detrimental mutations as was observed for mutators in mammalian hosts; rather, there was a direct defect in genomic rna synthesis in mosquito cells (figure and ). interestingly, similar host-specific replication defects were observed for rdrp mutants of west nile virus (although it is unclear if these variants have altered fidelity). while differences in host temperature do not seem to be the cause, the cellular host factors implicated or missing in these host cell lines remain to be elucidated. finally, we could not address whether mutators were attenuated in vivo in mosquitoes; sequencing of virus populations from mosquitoes revealed partial or total reversion of the fidelityaltering residues at position . although one could expect a variant with severe replication defects to be highly attenuated, it is possible that when coupled to a mutator phenotype, reversion would more quickly and favorably occur when the pressure to increase replication remains, as is the case in mosquitoes that are persistently infected. whether this defect is general to all mutators in mosquitoes, or whether only amino acids a, g, and w at position bear this curiously coupled mutator/ replication effect remains to be seen (since not all variants at this position were defective, as y has no replication defects in mosquito cells [ ] ). isolation of additional arbovirus mutators mapping to other residues in the polymerase should resolve this issue. since the cysteine at position is conserved in the alphavirus genus, we obtained additional arbovirus mutators in sindbis virus. sinv mutators also showed severe replication defects in mosquito cells, and sinv mutator g exhibited the same phenotypes we previously observed in chikv mutators in both mice and mosquitoes. however, the wider host range of sinv allowed us to test whether these replication defects occur across all insects or if they were mosquito-specific [ , ] . in s cells, mutators did not present replication defects, allowing us to test, in principle, whether mutators could be attenuated in an insect model (drosophila flies). indeed, in the absence of any in vitro replication defect and resulting pressure to revert, the mutator strain was attenuated in fruit flies. thus, our results confirm that arbovirus mutators can, in principle, be attenuated in insects. since the isolation of the first antimutator variant of a rna virus, the growing body of literature shows that either increasing or decreasing replication fidelity has detrimental effects to virus fitness [ , , , , , ] . however, how mutation rates and replication capacity are coupled will require more study, and the degree of attenuation resulting from altering these biochemical properties needs to be more carefully evaluated. a future challenge will be to quantitatively link the measurements of mutation frequencies (average mutations per nucleotide sequenced) performed in this work to actual mutation rates (average mutations per nucleotide site per replication) [ , ] and to in vitro biochemical fidelity (rates of incorporation of correct and incorrect nucleotides in absence of selection) [ , , , , , , ] . it is possible that the higher mutation frequencies measured for these alphavirus mutator strains are partly skewed by their producing more rna genomes in shorter replication cycles and thus accumulating mutations more rapidly, rather than incorporating more errors per genome during each replication cycle. indeed, biochemical studies of single-nucleotide incorporation by other mutator polymerases confirm that mutators are both faster enzymes and have higher frequency of mis-incorporation events per replication. in absence of a biochemical assay for alphaviruses, new technologies using microfluidic single-cell analysis of virus strains during single replication cycles should help correlate mutation frequencies, mutation rates, and enzyme fidelity with more confidence. recent studies have proposed both antimutator and mutator strains as candidates for rationally designed live attenuated vaccines [ , , , , ] . overall, fidelity variants present attenuated titers in vivo that range from one to several orders of magnitude lower than wildtype virus. whether this degree of attenuation is sufficient to elicit protective immunity without causing disease will require more careful evaluation in more relevant animal models, as virtually all work has been performed in mice using viruses that are often not natural mouse pathogens. in vitro systems and artificial hosts may alter many of the selective pressures to which a virus would be subjected in a natural host [ , , , ] . the present study and other work highlight that intrinsic fidelity and the mutant spectrum are labile and subject to stringent and disparate selective pressures in different hosts [ , , , , , , , ] . a more comprehensive understanding of the selective pressures in natural hosts is crucial to predicting how viruses will behave in vivo, and essential to evaluating the feasibility of using fidelity variants as vaccines, whether standalone or coupled with other, conventional attenuating mutations. despite the necessity for further research, from a vaccine development perspective these data support that in principle, mutators can be attenuated in a wider range of hosts and may be viable candidates for live-attenuated vaccines. figure s structural homology model of the chik nsp core polymerase. the model shows the predicted locations of c (green sphere) and two nearby residues (l and t , shown as gold spheres) that are the structural equivalents of known fidelity-altering sites in coxsackie virus polymerase (positions i and f , respectively) ( ). the model was obtained using the i-tasser threading platform (roy, a., et al., ) and is color coded according to polymerase domains. the polymerase palm domain (grey), where our fidelity altering mutations are located, is modeled with fairly high confidence because of the large number of conserved polymerase sequence motifs (motifs a-d) whose structure is also well conserved among the solved rdrp structures. the thumb domain (purple) modeling is less reliable, but secondary structure prediction of the nsp sequences is wholly consistent with the alpha-helix based structure of this domain in known rdrp structures. finally, modeling of the fingers (red) domain is the least reliable as a result of significant sequence and length divergence in this region of rdrps. domains where the modeling is weak are shown as semi-transparent. (tif) author contributions viral quasispecies mutation rates among rna viruses rna virus populations as quasispecies quasispecies theory and the behavior of rna viruses a single mutation in poliovirus rnadependent rna polymerase confers resistance to mutagenic nucleotide analogs via increased fidelity coxsackievirus b mutator strains are attenuated in vivo a live, impaired-fidelity coronavirus vaccine protects in an aged, immunocompromised mouse model of lethal disease remote site control of an active site fidelity checkpoint in a viral rna-dependent rna polymerase structure of foot-and-mouth disease virus mutant polymerases with reduced sensitivity to ribavirin virus mutators and antimutators: roles in evolution, pathogenesis and emergence mutagenic dna polymerase genetic control of mutation rates in bacteriophaget heterogeneity of the mutation rates of influenza a viruses: isolation of mutator mutants ribavirin-resistant mutants of human enterovirus express a high replication fidelity phenotype during growth in cell culture the cost of replication fidelity in human immunodeficiency virus type the cost of replication fidelity in an rna virus a polymerase mechanism-based strategy for viral attenuation and vaccine development structural basis for active site closure by the poliovirus rna-dependent rna polymerase quasispecies diversity determines pathogenesis through cooperative interactions in a viral population error thresholds and the constraints to rna virus evolution mutation rates and rapid evolution of rna viruses mutation frequencies at defined single codon sites in vesicular stomatitis virus and poliovirus can be increased only slightly by chemical mutagenesis lethal mutagenesis of hiv with mutagenic nucleoside analogs increased fidelity reduces poliovirus fitness and virulence under selective pressure in mice arbovirus high fidelity variant loses fitness in mosquitoes and mice evolution of pig influenza viruses ribavirin and lethal mutagenesis of poliovirus: molecular mechanisms, resistance and biological implications viral error catastrophe by mutagenic nucleosides theory of lethal mutagenesis for viruses therapeutically targeting rna viruses via lethal mutagenesis back to the future: revisiting hiv- lethal mutagenesis interrelationship between hiv- fitness and mutation rate host alternation of chikungunya virus increases fitness while restricting population diversity and adaptability to novel selective pressures factors shaping the adaptive landscape for arboviruses: implications for the emergence of disease arbovirus evolution in vivo is constrained by host alternation characterization of mosquito-adapted west nile virus effect of alternating passage on adaptation of sindbis virus to vertebrate and invertebrate cells west nile virus experimental evolution in vivo and the trade-off hypothesis vectorborne transmission imposes a severe bottleneck on an rna virus population genetic diversity and slow rates of evolution in new world alphaviruses role of the mutant spectrum in adaptation and replication of west nile virus study of sequence variation of dengue type virus in naturally infected mosquitoes and human hosts: implications for transmission and evolution genetic variation in west nile virus from naturally infected mosquitoes and birds suggests quasispecies structure and strong purifying selection quasispecies of dengue virus cooperative interactions in the west nile virus mutant swarm ntpase and -rna triphosphatase activities of chikungunya virus nsp protein differential unfolded protein response during chikungunya and sindbis virus infection: chikv nsp suppresses eif alpha phosphorylation the crystal structures of chikungunya and venezuelan equine encephalitis virus nsp macro domains define a conserved adenosine binding pocket chikungunya virus nsp blocks stress granule assembly by recruitment of g bp into cytoplasmic foci bst- / tetherin-mediated restriction of chikungunya (chikv) vlp budding is counteracted by chikv non-structural protein (nsp ) biology and pathogenesis of chikungunya virus replication cycle of chikungunya: a re-emerging arbovirus role for conserved residues of sindbis virus nonstructural protein methyltransferase-like domain in regulation of minus-strand synthesis and development of cytopathic infection roles of nonstructural polyproteins and cleavage products in regulating sindbis virus rna replication and transcription o'nyong nyong virus molecular determinants of unique vector specificity reside in non-structural protein conservation of a packaging signal and the viral genome rna packaging mechanism in alphavirus evolution catalytic core of alphavirus nonstructural protein nsp possesses terminal adenylyltransferase activity the broadspectrum antiviral ribonucleoside ribavirin is an rna virus mutagen the mechanism of action of ribavirin: lethal mutagenesis of rna virus genomes mediated by the viral rna-dependent rna polymerase deduced consensus sequence of sindbis virus strain ar : mutations contained in laboratory strains which affect cell culture and in vivo phenotypes isolation of fidelity variants of rna viruses and characterization of virus mutation frequency command-line tools for processing biological sequencing data fast and accurate short read alignment with burrows-wheeler transform the sequence alignment/map format and samtools andes: statistical tools for the analyses of deep sequencing development of a highly protective combination monoclonal antibody therapy against chikungunya virus chikungunya virus in us travelers returning from india changing patterns of chikungunya virus: reemergence of a zoonotic arbovirus entry is a rate-limiting step for viral infection in a drosophila melanogaster model of pathogenesis coronaviruses: an rna proofreading machine regulates replication fidelity and diversity engineering attenuated virus vaccines by controlling replication fidelity determinants of rna-dependent rna polymerase (in)fidelity revealed by kinetic analysis of the polymerase encoded by a foot-and-mouth disease virus mutant with reduced sensitivity to ribavirin structure-function relationships of the viral rna-dependent rna polymerase: fidelity, replication speed, and initiation mechanism determined by a residue in the ribose-binding pocket rna virus error catastrophe: direct molecular test by using ribavirin mosquitoes put the brake on arbovirus evolution: experimental evolution reveals slower mutation accumulation in mosquito than vertebrate cells the west nile virus mutant spectrum is host-dependant and a determinant of mortality in mice genetic diversity and purifying selection in west nile virus populations are maintained during host switching finescale genetic variation and evolution of west nile virus in a transmission ''hot spot adaptation of two flaviviruses results in differences in genetic heterogeneity and virus adaptability west nile virus genetic diversity is maintained during transmission by culex pipiens quinquefasciatus mosquitoes natural resistance-associated macrophage protein is a cellular receptor for sindbis virus in both insect and mammalian hosts beyond rnai: antiviral defense strategies in drosophila and mosquito belshaw r ( ) viral mutation rates incorporation fidelity of the viral rna-dependent rna polymerase: a kinetic, thermodynamic and structural perspective poliovirus rna-dependent rna polymerase ( d(pol)). divalent cation modulation of primer, template, and nucleotide selection biochemical characterization of the fidelity of poliovirus rna-dependent rna polymerase fidelity variants of rna dependent rna polymerases uncover an indirect, mutagenic activity of amiloride compounds adaptation of sindbis virus to bhk cells selects for use of heparan sulfate as an attachment receptor mutations in the e glycoprotein of venezuelan equine encephalitis virus confer heparan sulfate interaction, low morbidity, and rapid clearance from blood of mice rnai targeting of west nile virus in mosquito midguts promotes virus diversification mouse models for chikungunya virus: deciphering immune mechanisms responsible for disease and pathology experimental passage of st. louis encephalitis virus in vivo in mosquitoes and chickens reveals evolutionarily significant virus characteristics west nile virus population genetics and evolution hydrophobic moments and protein-structure key: cord- - tszqh authors: xu, kai; rockx, barry; xie, yihu; debuysscher, blair l.; fusco, deborah l.; zhu, zhongyu; chan, yee-peng; xu, yan; luu, truong; cer, regina z.; feldmann, heinz; mokashi, vishwesh; dimitrov, dimiter s.; bishop-lilly, kimberly a.; broder, christopher c.; nikolov, dimitar b. title: crystal structure of the hendra virus attachment g glycoprotein bound to a potent cross-reactive neutralizing human monoclonal antibody date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: tszqh the henipaviruses, represented by hendra (hev) and nipah (niv) viruses are highly pathogenic zoonotic paramyxoviruses with uniquely broad host tropisms responsible for repeated outbreaks in australia, southeast asia, india and bangladesh. the high morbidity and mortality rates associated with infection and lack of licensed antiviral therapies make the henipaviruses a potential biological threat to humans and livestock. henipavirus entry is initiated by the attachment of the g envelope glycoprotein to host cell membrane receptors. previously, henipavirus-neutralizing human monoclonal antibodies (hmab) have been isolated using the hev-g glycoprotein and a human naïve antibody library. one cross-reactive and receptor-blocking hmab (m . ) was recently demonstrated to be an effective post-exposure therapy in two animal models of niv and hev infection, has been used in several people on a compassionate use basis, and is currently in development for use in humans. here, we report the crystal structure of the complex of hev-g with m . , an m . derivative, and describe niv and hev escape mutants. this structure provides detailed insight into the mechanism of hev and niv neutralization by m . , and serves as a blueprint for further optimization of m . as a therapeutic agent and for the development of entry inhibitors and vaccines. henipaviruses, hendra virus (hev) and nipah virus (niv) [ ] , are recently emerged, highly pathogenic paramyxovirus zoonoses whose major reservoirs in nature are several species of pteropid fruit bats [ , ] . hev causes lethal respiratory disease and encephalitis in horses and severe respiratory disease or late onset encephalitis in humans. in total, there have now been hev spillover events in australia including cases of human infection with fatalities since [ ] [ ] [ ] [ ] [ ] [ ] . niv subsequently emerged in peninsular malaysia in - , causing a large outbreak of respiratory disease in pigs and encephalitis among pig farmers, and was later shown to be closely related to hev [ ] . similar to hev, nearly annual outbreaks of niv infection have been observed. these niv outbreaks have been associated with significantly higher case fatality rates in people, up to %, and several outbreaks have also been linked to the consumption of raw date palm sap contaminated with virus as well as humanto-human transmission [ ] [ ] [ ] . to date, there have been reported cases of niv infection in people with fatalities [ , , ] . the unusual broad species tropism, high morbidity and mortality rates, as well as the lack of any licensed therapeutics, have rendered the henipaviruses biological safety level- (bsl- ) pathogens and potential biological threats to humans and livestock. an often utilized approach to antivirus drug design is to block viral entry via small molecules, peptides and neutralizing monoclonal antibodies (mabs) that bind to the viral surface glycoproteins. a unique feature of the majority of paramyxoviruses is that they require two surface glycoproteins for host cell entry: a class i fusion (f) glycoprotein and an attachment glycoprotein, which can be a hemagglutinin-neuraminidase (hn), hemagglutinin (h), or as in the case for henipaviruses a g glycoprotein that has neither hemagglutinating nor neuraminidase activities [ ] . the henipavirus g glycoprotein engages the host cell membrane protein receptors ephrin-b and -b , and this initial interaction is believed to be sufficient to trigger the f-mediated fusion event between the viral envelope and the host cell membrane leading to virus entry [ , , ] . in the absence of available vaccines or antiviral drugs, neutralizing hmabs offer the possibility for effective pre-and/or post-exposure treatment for many important human viral infections. previously, several hmabs, m -m , were isolated using a recombinant soluble hendra virus g (hev-g) glycoprotein as the antigen for panning of a large naïve antibody library [ ] . among the hmabs, m and its derivatives (m . - ) generated by heavy chain random mutations and light chain shuffling, showed improved binding to hev-g; clone m . had equal or higher binding affinity than the other clones and was selected for further characterization and converted to an igg format [ ] . the m . hmab was able to cross-react with both niv-g and hev-g in vitro with % inhibitory concentrations (ic )of less than ng/ml and ng/ml for nipah virus and hendra virus, respectively, and is capable of neutralizing all available isolates of hev and niv [ , ] . in animal disease models, m . has been shown capable of protecting ferrets against a lethal niv challenge [ ] , as well as african green monkeys (agm) against a lethal hev challenge [ , ] , in time frames of to even hours post viral exposure, respectively. in light of the experimental success of this post-exposure treatment of both niv and hev infection, m . has since been administered on a compassionate use basis to two individuals in australia with a high risk of hev exposure during the hev spillover, and again in in another person exposed to hev. in , m . was used again by compassionate use protocol in an individual with a laboratory exposure to niv in united states. in all these cases, none of these individuals showed symptoms of hev or niv infection at the time of m . administration, and all individuals remain in good health to date. altogether, as a fully human mab, m . shows promise as a potential prophylactic or therapeutic agent against henipavirus infection, and appears to be suitable for controlled safety trials in humans. to fully characterize the binding epitope [ ] , as well as the binding and recognition mechanism, we determined and here present the crystal structure of the complex between the globular head domain of hev-g and the fab domain of m . , a close derivative of m . , featuring an identical heavy chain and a similar light chain. the structure reveals the molecular mechanism of neutralization and cross-reactivity and provides a basis for further improvement of m . , including efficacy enhancement and escape mutant prevention. additionally, the presented structural information may aid the development of a henipavirus vaccine or other specific entry inhibitors. the head domain of hev-g (residues - ) was produced using the baculovirus expression system, and the fab domain of m . was expressed in hb cells. the protein complex was generated by mixing hevsg with hmab in a : . molar ratio followed by a hour incubation and purification by size-exclusion chromatography (sec). the peak fractions containing the complex were collected and used for crystallization. we obtained two crystal forms of the m . /hev-g complex and used molecular replacement to determine the structures at . Å resolution (in space group p ) and at . Å resolution (in space group i ) (table s ). there is one : complex per asymmetric unit in both crystal forms. the overall structures in the two crystal forms are very similar ( figure s ) and the region containing the hev-g molecules and the complementarity determining region- of the heavy chain (cdr-h ) of the fabs can be superimposed with an r.m.s.d of . Å for ca atoms, while the two fab structures, excluding just cdr-h , can be superimposed with an r.m.s.d of . Å for ca atoms. the difference in the two structures is in the angle between cdr-h and the rest of the fab, which consequentially causes a slight difference in the buried m . /hev-g interface area: Å in crystal form i , and Å in crystal form p . however, most interface residues in the core region are the same in both crystal forms. for the remainder of this report and figures, we use the p structure. the fab binds a similar area on hev-g ( figure a ) as ephrin-b , which is composed of a hydrophobic central cavity and a hydrophilic rim. interestingly, cdr-h of the fab approaches the hev-g central cavity in a similar angle and from the same direction as the g-h loop of ephrin-b ( figure b) . the interface involves fab residues mostly located on cdr-h , as well as three cdr-h residues (l , g and i ), one cdr-h residue (n ) and one cdr-l residue (r ) (figure ). the long protruding cdr-h ( residues) adopts a b-hairpin conformation, including a stalk and a tip (q -y ). the central hydrophobic hev-g/fab contacts since their initial emergence, henipaviruses have continued to cause spillover events in both human and livestock populations, posing significant biothreats. currently there are no licensed or approved therapies for treatment of henipavirus infection and the human case mortality rates average . %. we used x-ray crystallography to determine the high-resolution structures of the hendra virus g glycoprotein in complex with a cross-reactive neutralizing human monoclonal antibody. the structures provide detailed insight into the mechanism of hev and niv neutralization by this potent and clinically-relevant human monoclonal antibody that is currently in development for use in humans. this monoclonal antibody was recently shown to be an effective post-exposure therapy in nonhuman models of lethal hendra virus infection. indeed, it has already been used in four people on a compassionate use request, three in australia and one in the united states, as a therapeutic agent. furthermore, we identified and characterized two escape mutants generated in vitro and evaluated their mechanism of escape. our results serve as a blueprint for further optimization of this antibody and for the development of novel entry inhibitors and vaccines. this report also supports the additional pre-clinical data required for eventual licensure by detailing the antibody's mechanism of henipavirus neutralization. are formed by embedding the tip of cdr-h into the hydrophobic hev-g receptor-binding cavity. a simulated annealing omit electron density map of this region is illustrated in figure s . among the eight residues on the tip of cdr-h , l is surrounded by q , a , n , y , i , i and e of hev-g; p is surrounded by t , a , p , t , q of hev-g; p is surrounded by e , w , y of hev-g; s is surrounded by l and w of hev-g; and y is surrounded by t , c , t , s and e of hev-g ( figure a and c ). in addition, the side chain of q , h and p of the fab are stacked together, with q , which forms the top layer of the stack and hydrogen bonds to q of hev-g. the surrounding hydrophilic hev-g/fab contacts involve residues on cdr-h , cdr-l and cdr-h . notably, r on cdr-l forms salt-bridges with e and d of hev-g; hydrogen bonds are also formed between n on cdr-h and e of hev-g, as well as between r , e , y , y and y on cdr-h and y , s , t , q and t of hev-g, respectively ( figure a) . all hev-g residues engaged by m . are listed in figure s . interestingly, fab binding does not induce any significant conformational changes in hev-g and the r.m.s.d in ca positions between unbound and fab-bound hev-g is . Å . some previously identified hev-g mutations that were reported to affect the m or m . binding [ , ] are not part of the m . epitope ( figure s ) suggesting that those mutations might affect the overall hev-g structure. the hev-g head domain and fab are both strictly monomeric, but, interestingly, when these two proteins are mixed together, they first form a hetero-dimeric complex that oligomerizes further in solution. indeed the sec assays indicate that hours after mixing more than % of the complex migrates at a position corresponding to twice its original size in the gel-filtration column. an explanation of this phenomenon is provided by the crystal packing of the complex, where two copies of fab and hev-g assemble into a heterotetramer ( figure s ). it should be noted that the same : heterotetrameric fab/hev-g complex assembly is observed in both crystal forms. as illustrated in figure s , the heterotetrameric m . /hev-g assembly is generated by a two-fold crystallographic symmetry axis in which the two fab molecules contact each other burying approximately Å surface area on each side, while the two hev-g molecules remain separate. importantly, there is an additional contact area between the fab light chains elbow region and the hev-g molecule of the interacting complex, in which a further , Å are buried in each binding partner ( figure a , blue region). thus, a total Å surface area is occluded on each side of the interface between the two : complexes. within the : heterotetrameric complex, m . and hev-g form a more extensive interacting interface, rendering a more stable assembly. as shown in figs. a and b, the total hev-g surface region involved in the hev-g/m . interaction is almost the same as the one in the hev-g/ephrin-b , b interaction. however, the physiological relevance of this tetrameric assembly, and resulting cross-linking of mab/g complexes on the viral membrane, needs to be studied further. ephrin-b and m . both interact with the receptor-binding surface of hev-g, which includes a central hydrophobic cavity and surrounding hydrophilic rim. cdr-h of m . resembles the g-h loop of ephrin-b in both its shape and the insertion angle into the hev-g cavity ( figure ) . most of the g-h loopcontacting residues of hev-g also participate in the cdr-h binding (figure c, d). unlike ephrin, the binding of m . does not cause any significant conformational changes in hev-g. interestingly, although the tips of the g-h loop and cdr-h both target the pockets in the hev-g central cavity, each of them uses slightly different residues and anchoring strategies. as shown in figure d , from left to right, f , p , l and w on the ephrin g-h loop insert into four hydrophobic hev-g pockets, occupying half of the hev-g cavity; while l , p and p on the m . cdr-h insert into the first three of these same pockets ( figure c ). among these, ephrin p and p of cdr-h are strikingly similar. additionally, the insertion of cdr-h further embeds s and y into the other half of the hev-g cavity. the side chain of h of cdr-h extends in the same direction as w of the g-h loop but does not reach the fourth pocket. instead, it is embedded in a groove defined by q , w , and e of hev-g. a hydrogen bond between q of cdr-h and q of hev-g further locks the h in this position, preventing the withdrawal of cdr-h from the binding cavity. since the s contacting resides on hev-g are l and w , mutation of s to a or v could presumably enhance the interaction between m . and hev-g. in summary, cdr-h binds to hev-g utilizing a very high affinity lock-and-key mode without inducing conformational changes in hev-g. structure of hendra g with a neutralizing antibody plos pathogens | www.plospathogens.org comparison of m . binding to niv-g and hev-g although m mab was originally isolated against hev-g, it demonstrated a more potent neutralization capacity against niv than hev. indeed, in vitro binding measurements using biolayer infetrometry (table s ) document that both m . and m . display higher binding affinities for niv-g than for hev-g. due to the high similarity between niv-g and hev-g, presumably m . binds to niv-g and hev-g in a very similar manner, but an examination of the structure highlights some small structural differences that may explain the increased affinity of m for niv-g. upon superimposition of niv-g to the m . -bound hev-g, we found that only three residues at the mab contacting regions are different. among them, t and y in hev-g, which are part of the binding pockets for p and p of cdr-h , are replaced by two hydrophobic residues, valine and phenylalanine, in niv-g ( figure ). increasing hydrophobicity in this area very likely strengthens the predominantly hydrophobic interaction between the g protein and m . , enhancing its neutralizing activity. the panel of m derivatives was generated by light chain shuffling and heavy chain random mutagenesis. among them, m . was reported to have an equal or slightly higher affinity to henipavirus g glycoproteins in comparison to the others [ ] . biolayer interferometry (table s ) , on the other hand indicates that m . actually has slightly higher binding affinities to both niv-g (k d : . nm) and hev-g (k d : . nm) than m . (k d : . nm and nm, respectively). the primary sequences of m . and m . are overall highly similar, featuring an identical heavy chain (which provides all but one of the binding residues) and different light chain amino acid that are not part of the m . /hev-g interface ( figure s ) (but indirectly account for the small differences in binding affinities). as all hev-g contacting residues in m . are conserved in m . , the structural information obtained from the m . /hev-g complex could also be applied to explain the mechanism of m . neutralization. in the neutralization assay we performed, the efficiency of the m . mab to wild type niv and hev is three folds higher than that of the m . fab and m . fab, highlighting the importance of dimerization conferred by the fc region. notably, the efficiency of the m . fab is within the same range or slightly lower than that of the m . fab (figure ) , suggesting that the neutralization efficiency may also be affected by the other factors, such as protein stability or flexibility. to further detail and characterize the binding of the hmab to the virus, infectious niv and hev were used to generate antibody neutralization escape mutants by incubating and culturing high titers of virus in the presence of hmabs m . (fab fragment) or m . (fab fragment and mab). after passages, the resulting virus stocks were plaque purified and tested for neutralization efficacy. the g and f glycoprotein genes from a minimum of five plaques of each escape variant were sequenced in order to identify mutations associated with the antibody escape phenotype. for the niv escape mutant, all ten plaques of both the m . and m . escape mutants contained a single amino acid change at location v i. hev mutants that escaped m . neutralization all contained a single amino acid mutation at location d n ( figure s ). the m . and m . cloned virus stocks of these escape mutants of niv and hev, in contrast to wild-type niv and hev, were no longer neutralized by m . at antibody concentrations exceeding mg/ml (figure ). in addition, the cloned virus stocks of the escape mutants were then analyzed in single round growth assays in comparison to wild-type hev and niv on vero e , hela-usu-ephrin-b and hela-usu-ephrin-b cells ( figure ). both neutralization escape mutants grew as efficiently and to equal titers as the wild-type virus in vero e cells. noticeably however, during passaging, the m . and m . neutralization resistant viruses were relatively slow in developing cytopathic effects (cpe). whole genome sequence was performed to identify any additional mutations in these two escapes, as compared to their parent strains (sequencing report is attached in supporting materials as report s ). the hev escape variant contains another silent mutation in the p gene, in addition to the d n mutation in the g gene; while the niv escape variant contains mutations in the n gene ( end), m gene and l gene, in addition to the v i mutation in the g gene. thus, the escape is certainly due to the mutation in the g gene in case of the d n hev escape mutant. whole genome analysis of the parent virus stocks and the escape mutants and identification of all snps, indicates that the likely reason for the slow appearance of cpe in the presence of m . was the need for resistant virus amplification to levels sufficient for cell-cell fusion. combined with binding affinity measurements (table s ) , the hev-g/m . structure provides clues to the escape mechanism of the escape mutants ( figure c and d) . interestingly, the affinity of the g proteins to both antibodies and ephrin-b was increased by the v i mutation in niv-g, and decreased by the d n mutation in hev-g. residue v i is located at the bottom of the niv-g cavity, interacting with p of ephrin-b , b and p of m . . the additional methyl group of i would likely result in a more intimate interaction with both the cdr-h and g-h loops resulting in a lower k d value due to a decreased dissociation (k off ) rate. furthermore, ephrinb binding benefits slightly more than both antibodies from the v i substitution. intriguingly, d is located on the b s -s loop of the g protein, which is outside of the receptor/mab binding region, suggesting the d n mutation affects the fab/g-protein interaction through an indirect pathway ( figure s ). d forms salt-bridges with two residues on b s , r and k . the d n mutation would likely cause a conformational change in b s , causing re-arrangement of several m . -contacting residues including i , y and i , thus hindering the insertion with both the cdr-h and g-h loops. indeed, the observed association rates (k on , table s ) of this mutant to both antibodies and ephrinb decreased similarly. however, such a conformational change would affect the overall ephrin binding less, and it seems a similar rearrangement takes place even in the wild-type g protein ( figure s ) [ , ] . indeed, i , y and i are the pocket-forming residues for f of ephrinb ( figure d ). the elimination of the two salt-bridges resulting from the d n substitution might even have a slight stabilizing effect on the g-h loop insertion. accordingly, the observed dissociation rate (k off ) for binding of the hev-g mutant (d n) to ephrin-b was slightly decreased, while the dissociation rates for binding the antibodies were increased. in summary, both mutations in the g protein favor ephrin-b binding as compared to mab binding, consistent with their neutralization-escape phenotypes. targeting the viral surface spike proteins has been a powerful strategy in the development of neutralizing mabs. similar to m targeting the henipavirus g proteins, a number of potent antibodies have been developed against the spike (s) glycoprotein of the sars-associated coronavirus, the hemagglutinin glycoprotein of influenza virus and the envelope glycoprotein of hiv. the epitopes recognized by these antibodies are often functionally associated with the viral entry mechanism (e.g. attachment and membrane fusion) to reduce the occurrence of escape mutants. for instance in hiv, the epitopes targeted by neutralizing antibodies are located in four regions: receptor binding site (rbs), fusion associated membrane-proximal external region (mper) region, conserved glycan structures and glycan associated loop regions, while in influenza, the epitopes are located in the fusion associated stem region and sialic acid binding pocket region [ ] [ ] [ ] . in henipaviruses, as in all members of the paramyxovirus family, the attachment and fusion functions are exerted by two different proteins, which renders as possible epitope locations the rbs, the fusion-related regions, and sites associated with transducing the fusion-triggering signal from the attachment to the fusion proteins. in the past years, crystal structures of complexes between viral rbs and neutralizing antibodies have been determined, including m and r targeting the sars s glycoprotein rbs [ , ] , ch and c targeting the influenza virus sialic acid binding pocket [ , ] , and b , hj , vrc , nih - , a , bnc , vrc-pg and vrc-ch targeting the hiv- cd -binding site (reviewed in [ ] ). interestingly, in many of the examples above, the antibody cdr region mimics the conformation of the binding region of the cellular receptor (either protein or carbohydrate). amongst them, most similar to our m . /hev-g structure are the structures of ch and c targeting the influenza virus sialic acid binding site, which is a conserved shallow groove. all three mabs use only cdrh to bind their target groove, but compared to ch , c contacts a larger conserved region in the rbs, without interacting with the surrounding variable regions, which accounts for its greater neutralization breadth. the same strategy could be applied to further improve m . / . the m . / antibodies feature a long cdr-h ( residues in kabat numbering), adapting a b-hairpin, providing an interesting example of how antibodies circumvent obstacles in reaching the targeted epitope. one of the challenges in viral epitope targeting is that the epitopes are sometimes hidden, either behind heavy glycosylation or deep in a cavity. another example of a long cdr-h forming a b-hairpin is the antibody against hiv ( residues) [ ] . the extreme cases in this category are the hiv neutralizing antibodies pg and pg , which contain a -residue axe-shaped cdr-h [ , ] . of the many tested therapeutic strategies to prevent and/or treat infection and disease caused by the henipaviruses in a variety of well-characterized animal models, few have been effective [ , ] . recently, the only post-exposure therapeutic option that is highly effective in animal models with clear potential for future approved human use applications has been the hmab m . [ , ] . the reported success of m . in a nonhuman primate model of hev infection has been particularly encouraging, and the m . exhibited an excellent distribution half-time (, day) and elimination half-time (, days) in the agm. no evidence of hev-specific pathology was observed in any of the m . -treated animals and no infectious hev could be recovered. this study revealed that hmab m . prevented wide-spread hev dissemination in virus challenged subjects, and was the first successful post-exposure in vivo therapy against hev and the first in a nonhuman primate [ ] . during the hev spillover occurrence in queensland, australia, there were two individuals that were considered to be at high risk of hev infection [ ] . the m . hmab was requested by australian health authorities and administered to the two individuals as a compassionate use therapeutic option even though no human safety testing has been carried out and it was not recommended for use in humans. in this instance, m . was administered to the individuals prior to any hev diagnosis or onset of clinical disease [ ] with doses (, mg/kg) sufficient to achieve a high serum concentration, and to date both individuals remain healthy and no evidence of hev infection has been reported. the antibody appeared well tolerated when administered which was not unexpected since m . is a human mab. the m . hmab is now in further pre-clinical development stages in both the united states and australia. as part of our continued characterization of m . we sought to provide the molecular details of its ephrin receptor blocking activity by determining the crystal structure of a (nearly identical) m . derivative, m . , which possesses the same crossreactive neutralizing and henipavirus g binding activity with an identical heavy chain sequence and g glycoprotein binding loop in the cdr domain. the structure reported here of the hev-g/ m . complex reveals the molecular mechanism underling the exceptional cross-reactivity and neutralizing potency of these antibodies. the binding of the hmab to the g glycoprotein involves a single loop of its heavy chain with hydrophobic amino acid residues occupying the same pockets in g that the ephrin receptors engage during receptor binding. it is clear now that the central cavity on the henipavirus g glycoprotein receptor-binding face is vital for viral attachment and infection. from the crystal structure of the m . /g protein complex, we know that blocking access to this cavity is a feasible and efficient way of inhibiting henipavirus attachment and infection. specific peptide or small-molecule inhibitors for the viral attachment glycoprotein can be designed based on the structural data. for example, the existing pockets in the g glycoprotein cavity can be used as targets in structure-based computational screens. another approach would be to screen compound libraries using a protein interaction primary assay, and then optimize the initial hits to better fit the binding cavity. the data presented here are consistent with the initial steps of the henipavirus entry models proposed earlier based on the analysis of the g glycoprotein and the ephrin receptor/g glycoprotein complex structures [ , [ ] [ ] [ ] . of further importance, the new hmab . /g complex structure provides important information and leads for potential antibody improvement in two regards: increasing the antibody's affinity to the g glycoprotein in order to obtain even higher efficiency, and manipulating the interacting interface in order to reduce the potential of occurrence of escape mutants. the difficulty of the second aspect lies in the observation that the affinity of the attachment proteins to their receptors is not strictly correlated with the infection efficiency of henipaviruses. thus, mutations that affect the henipavirus g glycoprotein binding affinities to ephrin receptors and mabs to similar degrees could still allow potential escape. we indeed observed that even though there was a remarkable overlap between the m . epitope and the receptor binding region of henipavirus g, two escape mutant variants of hev and niv, containing g glycoprotein mutations d n and v i respectively, were identified. in vitro manipulation, such as repeated passaging of virus and allowing replication in the presence of a neutralizing antibody is routinely used as an approach to generate escape variants that can then be examined as a means to map epitopes and detail mab neutralization mechanisms. however, it should be emphasized that the appearance of m . escape variants has not been observed in any of the in vivo efficacy testing against hev or niv to date, and this is likely explained by the fact that very high doses of mab are utilized, similar to mab dosing used in people in the prophylactic treatment of rsv infection with f (synagis/palivizumab) [ ] . in addition, the effectiveness of m . appears to be by virtue of its ability to slow the progression and dissemination of virus within the challenged host, allowing the host an effective window in which to mount its own innate and adaptive immune response that eventually prevents lethal disease outcome. taken together, the success of hmab m . in vivo as an effective post-exposure treatment against henipavirus disease in two different well-characterized animal models (the ferret and nonhuman primate), along with the new detailed structural findings on its viral g glycoprotein binding features that help explain its superior cross-reactive neutralizing activity, will facilitate efforts aimed at obtaining approved human use application to treat accidental exposure to hev or niv infection. soluble head domain (amino acid residues - ) of hev-g was cloned into a pgp vector and expressed in the baculovirus expression system (bd biosciences). the plasmid was transfected into sf cells using cellfectin (invitrogen) and baculo-gold linearized baculovirus dna (bd biosciences). the virus was then amplified in sf cells for three rounds to reach the proper titer before applying to hi cells for final expression (in : volume infection ratio). the infected hi cells were harvested hours after infection. the cell media containing the hev-g protein was purified using ion-exchange and size-exclusion chromatography (ge biosciences). soluble fab was expressed and purified as described [ , ] . the hev-g/m . complex was obtained by mixing the two proteins in a : . molar ratio and was passed through a superdex column (ge biosciences). the fractions containing both proteins were collected and concentrated to mg/ml in hbs buffer ( mm hepes ph . , mm kcl). the initial crystallization condition was obtained with wizard iii (emerald biosystems) and pro-complex (qiagen) screens using robot screening (ttp labtech's mosquito). after several rounds of optimization using hanging drop vapor diffusion at room temperature, two crystals forms were obtained in conditions: % peg , . m (nh ) citrate, and % peg , . m hepes ph . , . m mgcl . crystals were frozen in liquid nitrogen with % glycerol as cryo-protectant. diffraction data were collected at beamline ne-cat id- of the advanced photon source at argonne national laboratory. data images were processed using program hkl . the structures were determined by molecular replacement with pdb id d (niv-g) and rz (hmab against gp of hiv virus). phaser [ ] , coot [ ] and phenix refine in the program suite phenix [ ] were used for structure determination, model building and refinement. the details of the crystallographic analysis are presented in table s . neutralization resistant niv and hev mutants were generated by incubating tcid of each virus with mg or mg of mabs m . (niv) and m . (niv or hev) respectively, in ml media for h at uc and then inoculated onto vero e cells in the presence of mabs at the same concentration. the development of cytopathic effect (cpe) was monitored over h and progeny viruses harvested. mab treatment was repeated two additional times with cpe developing slowly with each passage. passage viruses were plaque purified in the presence of mabs and neutralization resistant viruses were isolated. experiments were performed in duplicate and the glycoprotein and fusion protein genes of individual plaques from each experiment were sequenced. the neutralization titers between wild type and the neutralization resistant virus were determined by micro neutralization assay. briefly mab m . and fabs m . and m . were serially diluted two-fold, and incubated with tcid of the wild type (wt) and neutralization resistant niv or hev for h at uc. virus and antibodies were then added to a -well plate with vero e /well in wells per antibody dilution. wells were checked for cpe days post infection and the % neutralization titer was determined as the mab concentration at which at least % of wells showed no cpe. growth curves were performed by inoculating cell cultures with niv, hev and their escape mutants at a multiplicity of infection (moi) of for h, after which the cells were washed times with pbs and overlaid with medium. virus samples were obtained at various time points after infection and stored at uc until viral titers were determined by tcid . the binding kinetics of the wild type or mutant g proteins to both antibodies (m . and m . ) or to ephrin-b were measured by bio-layer interferometry on a blitz instrument (fortebio). ni-nta biosensors were used to immobilize the hexa-histidine fused antibodies and ephrin-b proteins. kinetic parameters (k on and k off ) and affinities (k d ) were calculated from a non-linear fit of the blitz instrument data using the blitz software. whole genome sequencing and analysis ml of each virus was mixed with ml trizol ls and rna was extracted following the manufacturers guidelines. illumina truseq cdna libraries were prepared from total rna, omitting the polya selection step. each library was subjected to half a miseq run using a cycle kit, paired end sequencing. a quality control tool for high throughput sequence, fastqc, a java stand-alone program was downloaded from babraham bioinformatics institute: http://www.bioinformatics. babraham.ac.uk/projects/fastqc/ and each fastq file was checked for quality. resulting wt hev and wt niv reads were mapped to their respective reference genomes, nc_ and nc_ , using clc genomics workbench v . . , using default parameters. consensus sequence was extracted for each and used as the reference genome to which the reads resulting from sequencing the mutant samples were mapped. consensus sequence for each mutant was extracted and aligned to the wt using clc genomics workbench v . . , and default parameters. henipavirus g attachment glycoprotein sequences were aligned in clc main workbench v . . using default parameters (gap open cost = ; gap extension cost = ). all molecular representations were produced with pymol (delano scientific llc). figures were prepared using adobe illustrator, adobe photoshop. figure s comparison of the fab/hev-g structures in the two crystal forms. a: the m . /hev-g complex structures in the two crystal forms were superimposed using the fab as a reference. b: the complex structures were superimposed using hev-g as a reference. fab and hev-g are colored in blue and grey in the p hendra and nipah viruses: different and dangerous fields virology the natural history of hendra and nipah viruses infection of humans and horses by a newly described morbillivirus human hendra virus encephalitis associated with equine outbreak human hendra virus infection causes acute and relapsing encephalitis hendra and nipah: lethal zoonotic paramyxoviruses henipavirus vaccine development new insights into the hendra virus attachment and entry process from structures of the virus g glycoprotein and its complex with ephrin-b nipah virus outbreak with person-to-person transmission in a district of bangladesh date palm sap linked to nipah virus outbreak in bangladesh person-to-person transmission of nipah virus in a bangladeshi community recurrent zoonotic transmission of nipah virus into humans nipah encephalitis, human -bangladesh: (jipurhat). international society for infectious diseases modes of paramyxovirus fusion: a henipavirus perspective entry and fusion of emerging paramyxoviruses henipavirus mediated membrane fusion, virus entry and targeted therapeutics potent neutralization of hendra and nipah viruses by human monoclonal antibodies exceptionally potent cross-reactive neutralization of nipah and hendra viruses by a human monoclonal antibody a neutralizing human monoclonal antibody protects against lethal disease in a new ferret model of acute nipah virus infection a neutralizing human monoclonal antibody protects african green monkeys from hendra virus challenge a novel model of lethal hendra virus infection in african green monkeys and the effectiveness of ribavirin treatment host cell recognition by the henipaviruses: crystal structures of the nipah g attachment glycoprotein and its complex with ephrin-b crossneutralization of influenza a viruses mediated by a single antibody loop structural insights into key sites of vulnerability on hiv- env and influenza ha human antibodies that neutralize hiv- : identification, structures, and b cell ontogenies structural basis of neutralization by a human anti-severe acute respiratory syndrome spike protein antibody, r structure of severe acute respiratory syndrome coronavirus receptor-binding domain complexed with neutralizing antibody broadly neutralizing human antibody that recognizes the receptor-binding pocket of influenza virus hemagglutinin crystal structure of human antibody reveals conserved features of quaternary structure-specific antibodies that potently neutralize hiv- crystal structure of pg and chimeric dissection with somatically related pg : structure-function analysis of two quaternary-specific antibodies that effectively neutralize hiv- structure and function of broadly reactive antibody pg reveal an h subdomain that mediates potent neutralization of hiv- therapeutics and vaccines against hendra and nipah viruses henipavirus outbreaks to antivirals: the current status of potential therapeutics hendra virus, equine -australia ( ): (queensland) human exposure structural basis of nipah and hendra virus attachment to their cell-surface receptor ephrin-b crystal structure and carbohydrate analysis of nipah virus attachment glycoprotein: a template for antiviral and vaccine design dimeric architecture of the hendra virus attachment glycoprotein: evidence for a conserved mode of assembly a systematic review of compliance with palivizumab administration for rsv immunoprophylaxis phaser crystallographic software coot: model-building tools for molecular graphics phenix: a comprehensive python-based system for macromolecular structure solution the authors wish to thank nick fera, uniformed services university, for preparation of the m . fab protein; and the staff of the ne-cat beamline id- at the advanced photon source of argonne national laboratory, for crystal data collection. key: cord- -sz xj o authors: menzel, nicolas; fischl, wolfgang; hueging, kathrin; bankwitz, dorothea; frentzen, anne; haid, sibylle; gentzsch, juliane; kaderali, lars; bartenschlager, ralf; pietschmann, thomas title: map-kinase regulated cytosolic phospholipase a activity is essential for production of infectious hepatitis c virus particles date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: sz xj o hepatitis c virus (hcv) has infected around million individuals. current therapies have limited efficacy and are fraught with side effects. to identify cellular hcv dependency factors, possible therapeutic targets, we manipulated signaling cascades with pathway-specific inhibitors. using this approach we identified the mapk/erk regulated, cytosolic, calcium-dependent, group iva phospholipase a (pla g a) as a novel hcv dependency factor. inhibition of pla g a activity reduced core protein abundance at lipid droplets, core envelopment and secretion of particles. moreover, released particles displayed aberrant protein composition and were -fold less infectious. exogenous addition of arachidonic acid, the cleavage product of pla g a-catalyzed lipolysis, but not other related poly-unsaturated fatty acids restored infectivity. strikingly, production of infectious dengue virus, a relative of hcv, was also dependent on pla g a. these results highlight previously unrecognized parallels in the assembly pathways of these human pathogens, and define pla g a-dependent lipolysis as crucial prerequisite for production of highly infectious viral progeny. approximately million people are chronically infected with hepatitis c virus (hcv) [ ] . without treatment, at least % of patients will develop liver cirrhosis and of these, approximately % will progress to liver cancer within ten years [ ] . hcv is the sole member of the genus hepacivirus within the family of flaviviridae. its plus strand rna genome encodes a polyprotein that is flanked by non-translated regions. proteolytic processing releases ten viral proteins which coordinate viral rna replication and particle assembly. the non-structural proteins ns , ns a, ns b, ns a and ns b in conjunction with cellular co-factors are both necessary and sufficient to catalyze genome replication [ ] . core protein, envelope protein and (e , e ) reside in the very n-terminal portion of the viral polyprotein and compose the virus particle encasing the rna genome. these proteins are essential for virus assembly. interestingly, the ion channel protein p , and the ns protease also contribute functions to the production of infectious viral progeny [ , ] . lipid droplets have been recognized as an essential cellular organelle for production of infectious hcv progeny [ ] . during virus production core protein resident on lipid droplets recruits viral proteins and rna, which is an essential prerequisite for virus production [ ] . in turn, core protein is loaded onto these cellular organelles through an interaction with diacylglycerol acyltransferase- (dgat- ) [ ] , an enzyme which catalyzes the final step in the biosynthesis of triglycerides that is essential for lipid droplet biogenesis [ ] . in addition, host factors involved in the biosynthesis and secretion of human lipoproteins have emerged as essential cofactors for virus production. specifically, apolipoprotein b (apob), apolipoprotein e (apoe) and microsomal triglyceride transfer protein (mttp) were shown to contribute to virus production [ , , ] . likely as a consequence, infectious hcv is a ''lipo-viro particle'' rich in cholesteryl esters and comprising viral proteins, apob and apoe [ , ] . cells constantly respond to environmental changes by sensing these alterations through dedicated receptors and associated signaling cascades that reprogram cellular processes. such signaling-dependent modifications may also influence important cellular hcv dependency factors regulated by these pathways thus providing a lead for identification of novel and possibly druggable host factors crucial for hcv. using this approach we show that mitogen-activated protein kinase (map kinase) regulated enzymatic activity of pla g a is crucial for production of infectious hcv progeny highlighting the intricate involvement of host cell lipids and lipid-modifying enzymes in the replication of this virus. the mapk/erk signaling pathway is involved in hcv assembly/release cultured cells respond to multiple stimuli by growth factors and hormones present in serum-containing culture media. therefore, to reduce the complexity of our experimental system we developed a transient virus replication assay and cultured cells in serum-free conditions. when transfecting our infectious firefly luciferase reporter virus genome luc-jc [ ] into highly permissive huh- . human hepatoma cells [ ] cultured in the presence or absence of serum, we measured comparable levels of luciferase activity to h after transfection ( figure s a ). likewise, transduction of luciferase activity upon inoculation of naïve cells with culture fluid from the transfected cells was similar ( figure s b ). therefore, in this transient time course viral rna replication and production of infectious progeny particles was comparable in both serum-free and serum-containing conditions. to identify new host factors involved in hcv replication and/ or virus production we used pathway-specific inhibitors of central signal transduction cascades including akt/pkb, mtor, wnt, jnk and mapk/erk (figure and data not shown). to reveal rapid, signal-mediated changes of rna replication and virus production we added the inhibitors h post transfection during the logarithmic growth phase of cells. one hour later culture medium was replaced with fresh medium with or without inhibitors and virus production as well as rna replication was assessed h later. this procedure which is summarized in figure a ensures that specifically infectivity of particles produced and released during inhibitor treatment -and thus blockade of the respective signaling cascade -is evaluated. among the inhibitors tested, u a selective inhibitor of the mapk/erk pathway, substantially decreased the production of infectious virus particles as is evident from the dose-dependent reduction of luciferase activity in the inoculated cells ( figure b) . interestingly, u did not measurably impede rna replication and only affected hcv particle production when added in serum-free medium. this latter finding may be related to a much more efficient blockade of the mapk/erk pathway in serum-free conditions compared to serum-containing medium that is evident from a lower level of phosphorylated erk and erk in the presence of the drug when serum was absent ( figure c ). collectively, these results suggested that the applied doses of u efficiently prevented phosphorylation of erk under serum-free conditions thus impeding production of infectious hcv progeny. interestingly, pd and sorafenib which inhibit the mapk/erk pathway upstream of u [ ] [ ] also reduced production of infectious hcv particles ( figure s ) further confirming the role of mapk/ erk signaling in hcv morphogenesis. notably, at least under serum free conditions, these inhibitors also reduced rna replication. this may either be linked to a more potent suppression of mapk/erk signaling or due to inhibition of additional signaling events. to confirm these findings, we analyzed the impact of u on the production of infectious wildtype jc particles [ ] in transfected cells ( figure d and e). congruent to our findings with reporter viruses, treatment of cells with u reduced both extracellular and intracellular levels of infectious hcv particles ( figure d ). although intracellular levels of core and ns a were moderately reduced in the absence of serum ( figure e ), addition of u did not further reduce abundance of viral proteins suggesting that the inhibitor did not prevent rna translation or rna replication. we also tested if presence of u interferes with hcv cell entry by adding the drug to infectious reporter virus particles that had been produced in the absence of the inhibitor ( figure s ). since hcv infection was not decreased, we concluded that addition of u does not prevent hcv cell entry but interferes with production of infectious progeny particles under serum-free conditions. the mapk/erk regulated pla g a is involved in production of infectious hcv besides activating transcription factors in the nucleus, erk / also directly regulate the activity of cytoplasmic enzymes. therefore, we searched for cellular factors that are regulated by the mapk/erk pathway and operate at the er, the presumed site of hcv particle production. based on these criteria we focused on pla g a, which is activated by mapk/erkdependent phosphorylation [ ] and recruited to the er by ca + ions [ , ] , as a possible candidate host factor that may be responsible for the observed u -dependent blockade of hcv particle production. in line with our finding that u only inhibited hcv in the absence of serum, phosphorylation of pla g a was selectively inhibited under these conditions and not affected when serum was present (figure a ). to investigate if pla g a activity is relevant for the production of infectious hcv particles we treated luc-jc transfected cells with pyrrolidine- (py- ), a specific inhibitor of this type of phospholipase [ , ] . irrespective of culturing these cells in the presence or absence of serum, we observed a strong and dose-dependent inhibition of production of infectious particles resulting in a more than -fold reduction of luciferase transduction at and mm of py- , respectively ( figure b ). similar to u , rna replication and cell entry were not affected by py- ( figure b and data not shown). moreover, accumulation of hcv proteins in cells transfected with authentic hcv was not changed by addition of py- ( figure c ). however, the human genome encodes more than phospholipase a s. these enzymes cleave fatty acids at the c atom of phosphoglycerides and thus modulate membrane properties. among all pla s only pla g a, which is recruited to perinuclear membranes by ca + and activated by extracellular stimuli via the mitogen activated protein kinase pathway, specifically cleaves lipids with arachidonic acid. metabolism of arachidonic acid yields prostaglandins and leukotriens, important lipid mediators of inflammation. we show that inhibition of pla g a produces aberrant hcv particles and that infectivity is rescued by addition of arachidonic acid. our results suggest that a specific lipid (arachidonic acid) is essential for production of highly infectious hcv progeny, likely by creating a membrane environment conducive for efficient incorporation of crucial host and viral factors into the lipid envelope of nascent particles. strikingly, pla g a is also essential for production of highly infectious dengue virus (denv) particles but not for vesicular stomatitis virus (vsv). these observations argue that hcv and denv which unlike vsv produce particles at intracellular membranes usurp a common host factor (pla g a) for assembly of highly infectious progeny. these findings open new perspectives for antiviral intervention and highlight thus far unrecognized parallels in the assembly pathway of hcv and denv. [ ] and seeded into replicate tissue culture plates. at h post transfection (hpt), medium was changed to culture conditions with or without fcs. inhibitors (e.g. u ) were added into the medium at hpt. one hour later, medium was replaced by medium containing given doses of the inhibitor. finally, at hpt cells and culture fluids were analyzed. (b) hcv rna replication in cells prepared as in (a) was measured by using a luciferase reporter assays (top panel). the release of infectious particles was titers of both extracellular as well as intracellular infectivity were strongly impaired by -and -fold, respectively ( figure d ). interestingly, py- also inhibited production of infectious genotype a, a and a hcvcc particles, indicating that pla g a activity is important for hcv virus production across different hcv genotypes ( figure s ). intracellular phospholipase a enzymes comprise cytosolic, ca + -dependent enzymes (cpla ) as well as the structurally similar ca + -independent lipases (ipla ) [ ] . to investigate if ipla activity contributes to hcv particle production we treated luc-jc transfected cells with bromenol lactone (bel) a specific inhibitor of ipla [ , ] . however, bel neither affected hcv rna replication nor production of infectious particles ( figure e ), supporting the notion that specifically the pla g a is involved in the hcv life cycle. to determine whether utilization of pla g a activity is unique to hcv or common to other enveloped viruses, we analyzed production of infectious vsv and denv in the presence of py- . in case of vsv, a member of the family rhabdoviridae which assembles infectious virus particles at the plasma membrane [ ], we used a replication competent reporter virus (designated vsv*m q ) that expresses a gfp transgene from an additional transcriptional unit placed between the g and l genes [ ] . interestingly, py- treatment of vsv*m q infected huh- . cells did neither affect intracellular level of gfp ( figure a ) nor production of infectious vsv progeny particles ( figure b ) indicating that unlike for hcv, production of infectious vsv particles did not rely on pla g a activity. in contrast, production of infectious denv, a relative of hcv from the genus flavivirus that is thought to assemble at intracellular membranes [ ] , was heavily impaired by py- treatment ( figure ). strikingly, like for hcv, rna replication was not affected ( figure c ) and release of particles was only moderately reduced as is evident from ca. -fold lower copy numbers of viral rna in the culture fluid of py- treated cells compared to mock treated denv infected cells ( figure d ). importantly, when we quantified the infectivity of released particles using a limiting dilution assay we noted an approximately , -folder lower infectivity titer for particles produced in the presence of py- as compared to particles produced in the absence of the compound ( figure e ). since py- did not grossly inhibit cell entry ( figure f ) we concluded that inhibition of pla g a activity via py- primarily impairs infectivity of released particles ( figure e ). in summary these results indicate that pla g a is a key host enzyme required for efficient release and high infectivity of hcv and denv, but not vsv particles. to corroborate our finding that pla g a is involved in production of infectious hcv particles we knocked down expression of the enzyme in hcv transfected cells using rna interference ( figure ) . surprisingly, despite decreased abundance of pla g a in sirna-transfected cells ( figure a ), we observed at best a moderate reduction of the total cellular pla g a activity as determined by a commercial enzymatic test ( figure b ). in line with the result of the enzymatic test, knock down of pla g a did not impede production of infectious hcv particles ( figure c ). in contrast, reduction of virus titer correlated again with pla g a inhibition upon treatment with py- ( figures b and c) . to confirm that indeed pla g a was directly contributing to hcv particle production rather than alternative enzymes which may share a similar enzymatic activity, we combined sirna treatment with application of py- . under these conditions the reduction of pla g a within cells should increase the susceptibility of hcv to treatment with py- because due to lower abundance of the host factor a lower level of the drug should be sufficient to prevent hcv particle production. as expected, sirna and py- treatment did not decrease hcv rna replication ( figure d and e). in fact rna-replication moderately increased in cells that were treated this way compared to cells receiving a scrambled sirna and no py- ( figure e ). despite of this we found significantly lower levels of infectious virus particles secreted from cells receiving the pla g a-specific sirna and or mm py- as compared to cells treated with the scrambled sirna and these drug doses ( figure e ). collectively, these data indicate that sirna treatment does not sufficiently suppress pla g a enzyme activity to limit hcv production in huh- . cells. however, when adding the pla g a-specific inhibitor to these cells, knock down of pla g a increased the susceptibility to the drug, arguing that the abundance of enzymatically active pla g a is important for production of infectious particles. arachidonic acid (aa) restores efficient production of infectious hcv in the absence of pla g a activity mammals encode genes for more than phospholipase a s (pla -s) and related enzymes which are further divided into several classes [ ] . these enzymes share the property of hydrolyzing the sn- position of membrane glycerophospholipids to release free fatty acids and lysophospholipids. among pla -s only the pla g a displays selectivity for cleaving phospholipids carrying aa at the sn- position [ , ] . while local release of aa modifies membrane properties including curvature and fluidity [ , , ] , this lipid is also a precursor for production of bioactive prostaglandins (pgs) and leukotriens (lts) which play essential roles in inflammatory reactions. in fact, production of pgs and lts is reduced by ca. % in pla g a deficient mice highlighting the dominant role of this phospholipase for generation of these lipid mediators [ , ] . given these circumstances we wanted to distinguish if properties of the pla g a-derived cleavage products (aa and/or lysophosphatidic acid) directly contribute to hcv particle production, or if these molecules may indirectly promote virus production through activating inflammatory reactions. since aa is further metabolized by lipoxygenases and cyclooxygenases to yield prostaglandins and leukotrienes we determined by inoculation of naïve cells with culture fluids collected at hpt and determination of luciferase activity in cells h after inoculation (bottom panel). data are shown as means +/ sd of three independent experiments (the dotted line represents background luciferase activity in mock infected cells). (c) analysis of erk / expression and phosphorylation in luc-jc transfected and u -treated cells. cell lysates were collected hpt. erk proteins were detected using erk-and phospho-erk-specific antibodies (bottom and top panel, respectively). (d) cells were transfected with jc rna and subjected to the assay described in (a). culture fluids and cells were harvested hpt and extracellular and intracellular infectivity was determined using a limiting dilution assay. intracellular infectious particles were collected by three repetitive cycles of freeze and thaw. (e) aliquots of cell lysates were analyzed for expression of ns a, core and actin using mono-specific antibodies. doi: . /journal.ppat. .g assessed whether inhibition of aa metabolism by application of (s)-flurbiprofen or nordihydroguaiaretic acid (ndga), inhibitors of cyclooxgygenases and lipoxygenases, respectively, prevents efficient hcv particle production. however, neither drug modulated hcv rna replication or decreased production of hcv particles ( figure s ). in fact high doses of (s)-flurbiprofen even slightly increased levels of infectious hcv ( figure s a ). these results argue against the notion that aa metabolites and their biological activities are crucial for production of infectious hcv. next, we analyzed if application of aa or related fatty acids restores production of hcv particles in the presence of the pla g a inhibitor py- . remarkably, we observed a pronounced and dose-dependent restoration of infectious particle production in the presence of aa ( figure a ). importantly, , , , -eicosatetraynoic acid (etya) a derivative of aa with four triple bonds at positions , , , and of the acyl backbone did not restore virus production ( figure b ). likewise further natural fatty acids with , , or double bonds at various positions did not relieve the blockade of virus production caused by addition of the pla g a inhibitor ( figure s ). among all tested lipids only aa itself and to a moderate level , -dehydro aa ( , -dha) restored virus production ( figure a and c). notably, aa did not increase hcv cell entry since the infectivity of particles produced in the absence of the drug was not stimulated by addition of aa during cell entry ( figure s ). next, we investigated if repression of denv infectious particle production is also relieved by addition of aa. as is depicted in figure s , we noted a trend that high doses of aa partially restore production of infectious denv progeny in the presence of py- . however, the rescue of infectious virus production was moderate and not statistically significant. to investigate if in the context of hcv, addition of aa truly rescues the blockade of pla g a enzymatic activity and not simply over-stimulates production of hcv particles, we applied aa to hcv transfected cells in the absence of py- . interestingly, under these conditions we observed a moderate increase of hcv infectivity ( figure d ). this finding mirrors the moderate gain of infectivity when cells were treated with flurbiprofen that prevents aa metabolism. since both treatments are expected to increase availability of aa, these data suggesting that availability of aa may be sub-saturating in non-treated hcv transfected cells. collectively, these results indicate that specific properties of aa, which is created by cleavage of glycerophospholipids carrying this lipid at the sn- position by pla g a, are important for production of highly infectious hcv progeny particles. pla g a contributes to association of core with lipid droplets and to core protein envelopment pyrrolidine has been described as precursor for compounds against the ns -protease and rna-polymerase of hcv [ ] . as the pla g a-specific inhibitor py- shares a heterocyclic ring with pyrrolidine we wanted to exclude that py- may prevent virus production indirectly by inhibiting the viral protease or polymerase. both enzymes contribute to active rna replication complexes and may be required to feed in newly synthesized viral rna into assembling virus particles. to address this, we treated jc transfected cells with a polymerase inhibitor ( -c-methyladenosine; cma), a protease inhibitor (boceprevir), with py- , or with quinidine, the latter being a class i antiarrhythmic drug recently found to inhibit production of infectious hcv [ ] . as expected, only the rna-replication inhibitors ( cma and boceprevir) reduced abundance of hcv rna in transfected cells ( figure s a ) dose dependently. while at the used doses (well beyond the ic for all compounds) all drugs moderately impaired release of hcv core protein ( - -fold), only py- and quinidine strongly reduced infectivity of particles ( figure s c ) to levels more than -fold lower compared to the dmso control. thus, it is unlikely that indirect effects of py- on rna-replication are responsible for the reduced amount of secreted particle and their impaired infectivity. rather these findings argue that py- directly interferes with hcv assembly and the infectivity of released particles. to investigate how py- impairs hcv assembly, we investigated the subcellular localization of core, and pla g a in the presence or absence of py- . adipose differentiation-related protein (adrp), a host protein interacting with the surface of lds was stained in parallel. since we were unable to detect endogenous pla g a with commercial antibodies, we created a stable huh- . cell line ectopically expressing a gfp-tagged pla g a protein. as is shown in figure s we did not see gross changes of the distribution of these proteins during the short term py- treatment. moreover, localization of gfp-pla g a did not differ between cells expressing hcv proteins and those cells that were not positive for hcv. these findings provide preliminary evidence that localization of epitope tagged pla g a is not influenced by hcv. more work, ideally with untagged pla g a will be needed to fully resolve the localization and trafficking of this protein in the presence or absence of hcv and py- . next we assessed the amount of intracellular core protein that is resistant to proteolysis by proteinase k. reasoning that core protein that is surrounded by a membrane should be protected from digestion by the protease, this assay estimates the number of core protein that has acquired a lipid envelope. since among members of the family flaviviridae virus particle envelopment depends on expression of functional glycoproteins and as for hcv deletion of e -e abrogates production of infectious progeny [ ] , we used a jc mutant carrying a deletion of e -e genes as a control and reference. as expected, when the protease was added to cell lysates prepared by repetitive cycles of freeze together with detergent (triton x- ), the viral protein was completely degraded ( figure a ). however, when cell lysates were incubated with the protease in the absence of detergent, a substantial amount of core protein resisted digestion indicating protection by a membrane envelope. notably, the amount of protected core protein was approximately -fold lower in py- treated compared to dmso treated jc transfected cells, resembling the phenotype figure . inhibition of pla g a by py- impairs infectivity of hcv. (a) u prevents phosphorylation of pla g a in the absence of serum. cells were cultured in presence or absence of serum and the u -assay was carried out as described in figure a . cell lysates were analyzed using antibodies specific for pla g a or the s -phosphorylated enzyme. (b) luc-jc -transfected cells were treated with indicated doses of py- as outlined in figure a . the influence on rna replication (left panel) and production of infectious particles (right panel) was determined as described in the legend to figure . data are shown as means +/ sd of three independent experiments (the dotted line represents background luciferase activity in mock infected cells). (c) analysis of ns a and core protein levels in jc -transfected cells in the presence or absence of py- . (d) secreted and intracellular infectious hcv was quantified using a limiting dilution assay. (e) an ipla -specific inhibitor does not impede hcv rna replication or virus production. huh- . cells were transfected with luc-jc and treated with given doses of bel, an ipla -specific inhibitor, using the procedure outlined in figure a . luciferase activity was measured in transfected (left panel) and in the inoculated cells (right panel). data are shown as means +/ sd of three independent experiments (the dotted line represents background luciferase activity in mock infected cells). doi: . /journal.ppat. .g of cells transfected with jc de e ( figure a and b), arguing that py- had decreased the amount of enveloped core protein structures. since trafficking of core protein to lipid droplets (lds) [ ] is crucial for assembly of infectious progeny particles we analyzed the influence of pla g a on the accumulation of core protein on lds. to this end, we prepared lds from jc -transfected and py- treated cells and analyzed the abundance of core protein on the surface of this cellular organelles. the quality of our ld preparation was monitored by detection of actin (cytosol), adrp, calreticulin (er) and golgi matrix protein (golgi) in total lysates and the ld fraction ( figure c ). importantly, calreticulin and golgi matrix protein were below the detection limit of our assay in the ld fraction whereas adrp was readily detected thus confirming that our procedure successfully enriched cellular lds. notably, py- moderately increased the total cellular level of core protein but at the same time reduced the abundance of core in the ld fraction of the cell lysate. this difference evident by western blot was further confirmed using a core-specific elisa demonstrating a ca. -fold higher core protein amount in the lysate of py- -treated cells and about -fold lower levels in the ld fraction. collectively, these data indicate that py- -dependent inhibition of pla g a impedes association of core with lds which in turn may limit core protein envelopment and particle release. in principle inhibition of pla g a by py- may reduce infectivity by preventing assembly/release of particles and/or by altering particle properties including the association with lipoproteins. therefore, we used ultracentrifugation of hcv particles through density gradients to analyze the amount and density of virus particles released from cells treated in the presence or absence of py- ( figure ). using this approach we noted that the distribution of hcv core protein-containing structures throughout the density gradient was essentially unchanged with peak core protein levels in fractions with a density of . - . g/ml irrespective of py- treatment ( figure a) . notably, the overall amount of released core protein was moderately reduced (ca. - fold; figure a and c) when particles were produced in the presence of the pla g a inhibitor. most strikingly, inhibitor treatment heavily decreased the infectivity of released hcv particles by -to -fold ( figure a ). to elucidate, why particles produced in the presence of py- are less infectious, we analyzed their protein composition. to this end we transfected jc carrying a flag-epitope tag at the n-terminus of e [ ] into huh- . cells where endogenous apoe was silenced by a specific shrna and replaced by ectopic expression of an ha-tagged, shrna resistant, human apoe gene ( figure s ). this approach enabled us to monitor co-precipitation of viral and host factors with an apoe-specific or a virus envelope-specific antibody. notably, treatment of transfected cells with py- reduced the amount of secreted apoe and core protein by ca. % and %, respectively ( figure b and c, respectively) . therefore, we normalized the culture fluids to equal quantities of apoe or core protein before the apoe-specific or flag-specific co-precipitation. remarkably, py- treatment reduced the level of core protein co-precipitating with apoe by -fold ( figure b) . likewise, inhibition of pla g lowered the association of apoe and core with the flag-tagged e by ca. -fold ( figure c ). in summary, inhibition of mapk-dependent pla g a activity by py- moderately decreased the titer of released hcv particles but heavily impaired infectivity of both intracellular and extracellular particles likely through gross changes of their protein composition. in this study we manipulated key cellular signaling cascades to identify host cellular hcv dependency factors. we report that blockade of the mapk/erk cascade by a well-established specific inhibitor (u ) potently repressed production of infectious hcv progeny ( figure ). making reasonable assumptions (activation by erk, function at the er membrane), we focused on pla g a, an erk-regulated host enzyme that is recruited to the er-membrane by ca + , as possible new hcv dependency factor for hcv assembly. our further experiments provide three lines of evidence supporting our conclusion that the erk-dependent activation of pla g a is crucial for production of infectious progeny: first, we show that py- impedes production of infectious hcv in a dose-dependent fashion ( figure ) . notably, phospholipase a enzymes are subdivided into several classes including secreted pla s (spla s), ca + -dependent cytosolic (cpla s), ca + -independent cytosolic (ipla s), platelet-activating factor acetylhydrolases (paf-ahs), lysosomal pla s and the most recently identified adipose-specific pla [ ] . importantly, py- potently inhibits pla g a (the a isoform of the ca + -dependent cytosolic pla s, also named cpla a) in various in vitro assays [ ] . in contrast it interferes with pla g b (cpla b) and pla g c (cpla c) only at very high doses, probably by a non-specific mechanism, and it does not inhibit the secreted spla s [ ] . congruently, bel, a ''suicide substrate'' and specific inhibitor of ipla s with a . , fold selectivity for ipla s over cpla s [ ] , did not impede hcv particle production ( figure ) . second, using rna interference we show that reduction of the abundance of pla g a enzyme increased susceptibility of hcv to inhibition by py- ( figure ) . surprisingly, we did not observe an influence of the knock down of pla g a on hcv particle production ( figure ) . however, we note that in spite of clearly reduced abundance of the enzyme in sirna-treated cells we measured only a small decline of pla g a enzyme activity. it is possible that the active, phosphorylated pla g a enzyme has a relatively long half-life which may preclude reduction of the active enzyme to a level that limits hcv infectious particle production under these experimental conditions. third, we observed an almost complete restoration of hcv infectivity upon supplementing py- treated cells with aa ( figure ) . importantly, among all pla enzymes, only the pla g a displays a preference for cleaving glycerophospholipids carrying the polyunsaturated aa at the sn -position [ ] . moreover, related fatty acids including etya which differs from aa only by triple-bonded c-atoms in place of the double-bonded c-atoms in aa, were unable to restore virus production ( figure and s ). even -dha (with a single triple-bonded c atom) only partially compensated production of infectious hcv in the presence of py- indicating that highly specific properties of aa, the cleavage product of pla g a, are crucial for production of infectious hcv progeny. aa is the precursor for biologically active lipid mediators including prostaglandins, thromboxane and leukotrienes collectively termed eicosanoids. these molecules are synthesized from aa through the cyclooxygenase and lipoxygenase pathways and play important roles during inflammation. however, since inhibitors of both pathways of aa metabolism did not impede production of infectious hcv particles ( figure s ), we believe that the properties of aa itself rather than indirect effects caused by aa-metabolites are important for hcv. our data support the conclusion that pla g a activity is relevant for hcv assembly in two principal ways. first, inhibition of pla g a decreased the amount of core protein associated with lipid droplets and reduced the level of core protein that is protected from proteolytic digestion ( figure ). the latter finding may indicate a lower level of intracellular core protein that is encased in membranes -possibly within virus particles -and therefore protected from proteolysis. moreover, we observed reduced levels of extracellular hcv particles ( - -fold; figure ). it is currently unclear why inhibition of pla g a reduces the level of core protein at lds. in principal several mechanisms account for this including an increase of core assembly and subsequent unloading from lds or a reduced trafficking of core to lds possibly due to aberrant processing of the protein by signal peptide peptidase cleavage. however, since we observed lower levels of secreted virus particles we consider it unlikely that increased assembly and protein unloading from lds is responsible. moreover we did not detect an overt processing defect of core in the presence of py- (figure and ) . notably, gubern et al. have shown that mapk-dependent phosphorylation of pla g a at ser is necessary for biogenesis of lipid droplets [ , ] . therefore, it is tempting to speculate that blockade of pla g a by py- reduces lipid droplet biogenesis, thus limiting abundance of core protein at these organelles which are essential for hcv particle production [ ] . assuming that core protein has to be unloaded from lds to drive budding and virus production which is consistent with the recent findings of counihan et al. [ ] , it is reasonable to suggest that reduced core protein levels at lds may decrease membrane envelopment and particle release. while aa (the product of the pla g a-catalzed triglyceride cleavage) is apparently not required for lipid droplet biogenesis [ ] , it nevertheless seems to be essential for the second prominent influence of pla g a on hcv, i.e. the production of highly infectious hcv progeny. remarkably, both hcv and denv produced and released in the presence of py- were approximately -fold less infectious as compared to viruses assembled in the absence of the drug (figures and ). since addition of py- to particles generated in the absence of the drug, did not impede cell entry (data not shown and figure ), we exclude that py- interferes with cell entry of hcv or denv. rather, our results indicate that particle properties are altered when py- is present. while the density spectrum of released hcv particles was unchanged, coprecipitation with apoe-or envelope-specific antibodies provide firm evidence that blockade of pla g a disturbs the composition/ structure of released hcv particles. specifically, we noted -fold reduced levels of core co-precipitating with anti-apoe and likewise -fold lower amounts of apoe and core co-precipitating with the envelope protein-specific pull down. these results argue that either less particles are directly associated with apoe or that particles contain lower levels of apoe. since apoe is important for infectivity of hcv particles [ , ] , a defect in the loading of this protein onto hcv particles may explain their reduced infectivity. in the envelope-specific pull down mediated by the flag-tagged viral e protein, we observed both -fold lower apoe and also core protein. on one hand this may indicate lower abundance of both proteins in secreted enveloped hcv particles. on the other hand, this may reflect incorporation of lower numbers of glycoprotein complexes into the viral envelope and in turn reduced precipitation efficiency. unfortunately, due to insufficient sensitivity of currently available envelope protein detection systems, we are currently unable to distinguish between these two possibilities. nevertheless, these results argue that blockade of pla g a by py- prevents normal loading of viral (core and envelope proteins e e ) and host proteins (apoe) onto nascent hcv particles. as a consequence, virus attachment or the interaction with entry factors or membrane fusion might be inefficient. notably, recent evidence suggests that aa and other poly-unsaturated fatty acids increase membrane fluidity [ , , ] . thus, pla g a activity in the vicinity of particle production may modify membrane characteristics including curvature and fluidity. these altered properties may disturb virus budding and/or the trafficking of viral and host-derived components to the site of virus assembly and thus result in the production of particles with disturbed stoichiometry, aberrant envelope composition, and poor infectivity. a more detailed proteomic and lipidomic comparison between particles produced in the presence or absence of py- should help to clarify this in the future. our finding that hcv and denv particle assembly depend on pla g a activity while vsv apparently does not rely on this host factor indicates fundamental differences between the assembly of enveloped vsv particles compared to hcv and denv. it remains to be shown how exactly pla g a contributes to production and release of infectious denv particles. while it has been reported that denv may also usurp lds for its assembly [ ] , welsch and colleagues showed that denv assembly sites are physically linked to rna replication sites on modified er figure a . (d) knock down of pla g a was monitored by western blotting. (e) hcv rna replication was measured in cell lysates (top panel) and release of infectious particles was determined by inoculation of naïve huh- . cells (bottom panel) and luciferase assays. the box plots in panels (b) and (e) as well as in the following figures visualize the full distribution of the data; the central horizontal line in each box indicates the value of the median, whereas the box represents the range between the lower and upper quartile of the data, i.e. the area in which the central % of the measurements lie. the whiskers extend from the quartiles to the minimum and maximum measurements, respectively. statistical significance of difference between means is indicated using asterisks (*): n.s -not significant, * marginally significant (p# . ), ** significant (p# . ), *** highly significant (p# . ). doi: . /journal.ppat. .g structures [ ] . unlike for hcv, aa did not consistently restore virus production of denv supporting the notion that pla g a may participate in hcv and denv morphogenesis through different mechanisms. more work will be needed to fully understand the role of this host factor for these two viruses. along these lines it will be interesting to analyze if other enveloped viruses (e.g. coronaviruses) that like hcv and denv assemble progeny particles at intracellular membranes depend on pla g a as well. such studies could in the future reveal common replication mechanisms between hcv and denv (and figure . arachidonic acid restores production of infectious hcv particles in py- -treated huh- . cells. at hpt given doses of (a) arachidonic acid (aa), (b) , , , -eicosatetraynoic acid (etya) or (c) , -dehydro arachidonic acid ( , -dha) were added to the medium of luc-jc transfected cells. rna replication and virus production in the presence or absence of py- was determined as described in figure a . data are shown as means +/ sd of three independent experiments (the dotted line represents background luciferase activity in mock infected cells). (d) rna replication and virus production in cells treated as above except that medium was only supplemented with aa (and not with py- ). doi: . /journal.ppat. .g figure . pla g a activity contributes to the association of core with lipid droplets and the membrane envelopment of core. cells were treated with py- as outlined in figure a . (a) freeze and thaw lysates of these cells were prepared as described in materials and methods and were left untreated or were incubated with proteinase k in the presence or absence of triton x- . the abundance of core protein was determined by western blot. cells transfected with a jc mutant lacking the coding region of e and e served as control. (b) the % of core protein protected from proteolytic digestion was determined by chemiluminescence imaging and evaluated by using the imagej software. mean values of two independent experiments are shown. (c) total cell lysates were subjected to western blots for detection of core, adrp, calreticulin and golgi matrix protein (left panel). in parallel equal amounts of total cell lysates were used for preparation of lipid droplets by ultracentrifugation. lipid droplet associated proteins were analyzed by western blotting (right panel). (d) the amount of core protein in the total lysates and the ld fractions was determined by using a core-specific elisa. statistical significance of differences of means: n.s -not significant, * marginally significant (p# . ), ** significant (p# . ), *** highly significant (p# . ). doi: . /journal.ppat. .g possibly other viruses) that may provide valuable insights into conserved assembly pathways of enveloped viruses. finally, inhibitors of the pla g a which have been pursued and brought into clinical development for treating inflammatory diseases may prove useful as antiviral therapeutics for the treatment of chronic hcv infection and possibly other viral diseases. antibodies were obtained from the following companies: apolipoprotein b (millipore); pla g a (abcam); p(s )-pla g a, erk / , p-erk / (cell signaling); actin (sigma- . hcv particles produced in the presence of py- display aberrant protein composition. (a) virus particles produced in the presence or absence of py- were separated by using ultracentrifugation and an iodixanol step gradient. ten fractions were collected from the bottom, and core protein abundance (left panel) and infectivity titers (right panel) were determined. one example of two independent experiments is given. (b) flag-jc rna [ ] was transfected into huh- . -ha-apoe cells ( figure s ) and cells were treated with py- as outlined in figure a . the total amount of apoe secreted from transfected cells was determined using an elisa (top panel). virus containing culture fluid of py- and untreated cells were normalized for equal quantities of apoe and precipitated with apoe-specific antibodies. the amount of co-precipitated core protein was determined by elisa and is expressed relative to the untreated control. data are shown as means +/ sd of three independent experiments (c) flag-jc transfected huh- . -ha-apoe cells were treated as in (b). the amount of secreted hcv core protein was determined by elisa (left panel) and normalized to equal amounts of core prior to precipitation with flag-specific antibodies. the amount of co-precipitated apoe and core was determined by western blot and elisa, respectively and is expressed relative to the untreated control. data are shown as means +/ sd of three independent experiments. doi: . /journal.ppat. .g pd (cell signaling); sorafenib (bay - , alexa biochemicals); proteinase k (roche); cpla a-sirna (thermo); etya; , dehydro arachidonic acid; (s)-bromoenol lactone; ndga (cayman); quinidine; arachidonic acid (sigma); flag-agarose (sigma). huh- . cells were grown in dulbecco's modified minimal essential medium (dmem; life technologies) supplemented with mm l-glutamine, nonessential amino acids, u/ml of penicillin, mg/ml of streptomycin, and % fetal calf serum (dmem w/fcs). the plasmids pfk-luc-jc and pfk-jc , encoding the genotype a/ a chimera jc with or without the firefly luciferase reporter gene have been described [ , ] . chimeric hcv constructs pfk-h /c encoding the genotype a/ a chimera [ ] , ps /jfh (a c) encoding the genotype a/ a chimera [ ] , and psa /jfh (c g-a g) encoding the genotype a/ a chimera have been described [ ] . for the shrna-mediated knockdown of apoe expression, the vector plenti- -u -ec-ep [ ] which contains a blasticidine resistance gene was modified to harbor an shrna specific to the untranslated region of human apoe ( -gccgaagcctg-cagccatgcg- ). as control, a construct containing a nontargeting shrna was used. the lentiviral plasmid pwpi hapoe-linker-ha-gun encodes the human apoe variant with an ha tag added to the -end via a glycine-glycine-serine-glycine linker in the self-inactivating pwpi vector [ ] which comprises a gfp-ubiquitin-neomycinphosphotransferase fusion protein as selectable marker. the gene encoding pla g a (thermo scientific, cdna clone mhs ) was n-terminally fused to a gfp-tag and subcloned into the pwpi vector. the creation of a huh- . -cell line expressing gfp-pla g a fusion protein is described below. finally, pfk-jc -de -e was created by a pcr-based strategy. in this construct the entire e and e coding region is deleted and the core coding region is directly fused in frame to the coding region of p . sequence information is available upon request. hcvcc particles and firefly luciferase hcv reporter virus were generated as reported previously [ ] . luciferase reporter virus infection assays were carried out as described [ ] . for inhibitor assays, cells were pre-treated with cellular or viral inhibitors to completely abolish enzyme activity h after transfection of hcv-rna. one hour later, supernatants were removed and fresh medium with inhibitors was added to harvest newly synthesized virus in a period of six hours. at hpt, supernatants and cell lysates were used for the infection of naïve huh- . cells or subjected to assays as described below. lipid droplets were isolated according to a published protocol [ ] with minor modifications. briefly, jc -transfected cells of -mm plates were scraped into ml pbs and pelleted by centrifugation at g. cells were resuspended in ice-cold hlm buffer ( mm tris-hcl (ph . ); mm edta) with protease inhibitors (complete mini; roche) and incubated for min on ice. cells were homogenized by eight gentle strokes with a potter-elvehjem tissue homogenizer and nuclei were removed from lysates by low-speed centrifugation ( g). density of the post-nuclear supernatant was adjusted with sucrose ( % final) and samples were loaded below a discontinuous sucrose gradient ( , , %). flotation of lipid droplets through the gradient was achieved by centrifugation at , g for min, uc. the white band containing lipid droplets at the top of gradient was collected and proteins were characterized by immunoblotting or core elisa. confluent jc -transfected cells were suspended in ml pk buffer ( mm tris-hcl (ph . ); mm cacl ; mm dtt) and homogenized by five freeze-and-thaw cycles. the lysate was divided into three ml fractions and treated with or without mg/ml proteinase k for one hour on ice. as control, the third sample additionally containted % (v/v) triton x- during protease digestion. the reactions were stopped by mm pmsf for min on ice and addition of laemmli buffer. samples were analysed by immunoblotting. huh- . cells were transfected with sirnas specific to pla g a (d- - , - ; thermo) or scramble sirna ( ; ambion) in a forward transfection procedure according to the manufacture's protocol (rnaimax; life technologies). to achieve an efficient knock-down, cells were transfected twice within h and re-seeded in -wells at a density of . cells per well. cells were infected with a -fold concentrated stock of luc-jc virus and h later the inhibitor assay was performed as described above. the efficiency of pla g a knock-down was verified by immunoblotting. viral rna was prepared from infected cells using a nucleo spin rnaii kit (macherey-nagel) according to the manual's instructions. ml of the rna sample was used for hcv-specific quantitative reverse transcription-pcr (qrt-pcr) analysis using a lightcycler device (roche). hcv-specific qrt-pcrs were conducted in duplicate measurements as published [ ] to normalize for equal quantities of total rna in the samples, the gapdh-specific mrna was detected in parallel employing gapdh-specific oligonucleotides (s-gapdh, -gaaggt-gaaggtcggagtc- ; a-gapdh, -gaagatggtgat-ggg atttc- ) and a gapdh-specific probe (tib molbiol), -gapdh-bbq probe ( -lc -caagcttcccgttct-cagcct-bbq- ). reactions were performed in three stages by using the following conditions: stage (rt), min at uc; stage (initial denaturation), s at uc; stage (amplification), cycles of s at uc and s at uc. the amount of hcv rna was calculated by comparison to serially diluted in vitro transcripts and normalized to the amount of gapdh, which served as a housekeeping gene. hcv core protein within cell lysates and culture fluids was quantified with a commercially available diagnostic kit (architect anti-hcv; abbott). one mg of total rna or / of rna extracted from ml cell culture supernatant was reverse transcribed into cdna using the high capacity cdna reverse transcription kit (applied biosys-tems) following the manufacturer's protocol. quantitative rt-pcr was performed on an abi prism sequence detection system (applied biosystems). the reaction was carried out in a final volume of ml, including . ml green dye master mix (p.j.k., kleinblittersdorf), ml cdna template, . ml primer mix ( mm each), ml rnase-free sterile water. reactions were carried out using the following settings: uc: minr [ uc: secr uc: secr uc: sec]. the amounts of denv rna were calculated from a standard curve derived from serially diluted in vitro transcripts of known concentration. primers used for the amplification were: sdv - , -gcccttctgttcacaccatt- and asdv - , -ccacatttgggcgtaagact- . to quantify core protein, cell culture supernatants or immunoprecipitated flag-jc particles were diluted in pbs/ % triton in a ratio of : . the core elisa was performed with a commercially available diagnostic kit (architect anti-hcv, abbott). apoe was quantified according to the manufacturer's instructions (mabtech). density gradient centrifugation was performed as described recently [ ] . briefly, viruses were separated by overnight centrifugation through a % to % iodixanol step gradient at , g. ten fractions of ml were collected from the bottom and analyzed for virus infectivity, core protein levels, and viral rna copies. following py- treatment, aliquots of cell culture supernatants were subjected to core or apoe elisa in order to equilibrate the volumes for immunoprecipitations. to capture flag-jc particles or ha-apoe, equilibrated supernatants were mixed with either ml flag-agarose or mg anti-ha antibody and ml g-protein agarose (roche) overnight at uc in gentle rotation. immunoprecipitated proteins were washed three times in pbs, eluted with ml pbs/ % triton for min at uc and analyzed by core elisa or immunoblotting. phospholipase a activity was measured according to the manufacture's protocol (enzchek phospholipase a kit; life technologies). in brief, huh- . cells were harvested from -mm wells, resuspended in ml enzchek pla reaction buffer and disrupted by sonication. to avoid any measurement of ipla activity, bromenol lactone was added to all samples at a concentration of mm. liposomes were prepared with the enzchek phospholipase a substrate and mixed with lysates at a ratio of : to give a total volume of ml. samples were transferred in -wells and pla g a activity was monitored by the intensity increase of a single wavelength at nm in a fluorescence microplate reader (flx ; biotek). for the generation of stable huh- . -ha-apoe cells, lentiviral gene transfer was used as described before [ ] . endogenous apoe expression in huh- . cells was silenced using plenti- -u -ec-ep [ ] which contains a blasticidine resistance gene and an shrna specific to the -untranslated region of human apoe ( -gccgaagcctgcagccatgcg- ). subsequently, apoe expression was restored by transduction with pwpi hapoe-linker-ha-gun described above. lentiviral particles were generated by transfection of pcmv dr. , pcz vsv-g and a derivative of either plenti- -u -ec-ep or pwpi at a ratio of : : into t cells. lentiviral particles were collected h post transfection and used to transduce target cells. selection was carried out in the presence of mg/ml blasticidine or . mg/ml g . for generation of huh- . -gfp-pla ga cells, huh- . cells were transduced with lentiviruses carrying the pwpi-gfp_pla g a vector. transduced cells were selected in the presence of mg/ml blasticidine. the protocol for immunostaining was carried out as described previously (frenzen, hueging, steinmann, plos pathogens april volume issue e ). immunostainings of core and adrp proteins were performed at dilutions of : respectively : . texas red and cy- secondary antibodies were used at dilutions of : . statistical data analysis was performed using the free statistical environment r. data were initially visualized using histograms, boxplots and qq-plots, and normality of the distributions was assessed. statistical significance of differences was then calculated using welch's t-test if data were sufficiently well approximated by a normal distribution, or using the wilcoxon rank sum test as a non-parametric alternative for non-normal data. p-values were calculated and statistical significance reported as highly significant (***) if p# . , significant (**) if p# . , and marginally significant (*) if p# . . differences were considered not significant (n.s.) for p. . . figure a . hcv rna replication in cells was measured by using a luciferase reporter assays (top panels). the release of infectious particles was determined by inoculation of naïve cells with culture fluids collected at hpt and determination of luciferase activity in cells h after inoculation (middle panels). data are shown as means +/ sd of three independent experiments (the dotted line represents background luciferase activity in mock infected cells). the bottom panels display erk / expression and phosphorylation in luc-jc transfected and inhibitor treated cells. erk proteins were detected as described in figure . (tif) figure s influence of mapk/erk inhibitor u on hcv cell entry. luc-jc particles prepared in the presence or absence of fcs were supplemented with the given dose of u or left untreated. virus suspensions were incubated with huh- . cells for h at uc. subsequently, unbound particles as well as the inhibitors were removed and cells were cultured in fcs-containing culture fluid until the analysis of hcv infection h later. data are shown as means +/ sd of three independent experiments. (tif) figure s py- impedes production of infectious hcv across different hcv genotypes. cells were transfected with indicated chimeric hcv genomes encoding structural proteins of genotype a, a or a, and subsequently treated with py- as described in figure a . production of infectious progeny was quantified using a limiting dilution assay. two independent experiments are shown in the two panels. mean values of six replicates +/ sd of the replicates are given. (tif) figure s blockade of arachidonic acid metabolism by inhibition of cyclooxygenases and lipoxygenases does not impede production of infectious hcv. luc-jc transfected huh- . cells were treated with given doses of (s)-flurbiprofen (a) or ndga (b) as outlined in figure a . rna replication in transfected cells and release of infectious particles was determined by luciferase asssays. data are shown as means +/ sd of three independent experiments (the dotted line represents background luciferase activity in mock infected cells). (tif) figure s fatty acids with varying degree of unsaturation are unable to restore virus production in py- treated huh- . cells. luc-jc -transfected cells were loaded with given lipids hpt and subsequently subjected to the py- inhibition assay outlined in figure a . hcv rna replication and virus production was determined by luciferase assays in cells treated with different fatty acids data are shown as means +/ sd of three independent experiments (the dotted line represents background luciferase activity in mock infected cells). (tif) figure s arachidonic acid does not increase hcv cell entry. luc-jc particles were supplemented with aa or left untreated. virus suspensions were incubated with huh- . cells for h at uc. subsequently, unbound particles as well as the inhibitors were removed and cells were cultured in fcscontaining culture fluid until the analysis of hcv infection h later. data are shown as means +/ sd of three independent experiments. (tif) figure s influence of aa production of infectious denv particles in the presence or absence of py- . cells were transfected with a denv rna and treated as described in figure a . infectivity of released particles was determined by inoculation of naïve huh- . cells. statistical significance of differences of means: n.s -not significant, * marginally significant (p# . ), ** significant (p# . ), *** highly significant (p# . ). (tif) figure s hcv protease or polymerase inhibitors do not impede production of infectious particles in the transient assay. given drugs were applied to jc -transfected huh- . cells as outlined in figure a [ ] . rna replication (left) and production of infectious particles (right) was monitored using luciferase assays (means +/ sem are shown). (tif) evolving epidemiology of hepatitis c virus aging of hepatitis c virus (hcv)-infected persons in the united states: a multiple cohort model of hcv prevalence and disease progression replication of subgenomic hepatitis c virus rnas in a hepatoma cell line hepatitis c virus p protein is crucial for assembly and release of infectious virions hepatitis c virus p and ns proteins are essential for production of infectious virus the lipid droplet is an important organelle for hepatitis c virus production efficient hepatitis c virus particle formation requires diacylglycerol acyltransferase- thematic review series: glycerolipids. dgat enzymes and triacylglycerol biosynthesis hepatitis c virus production by human hepatocytes dependent on assembly and secretion of very low-density lipoproteins cellular determinants of hepatitis c virus assembly, maturation, degradation, and secretion human apolipoprotein e is required for infectivity and production of hepatitis c virus in cell culture characterization of low-and very-low-density hepatitis c virus rna-containing particles biochemical and morphological properties of hepatitis c virus particles and determination of their lipidome characterization of the early steps of hepatitis c virus infection by using luciferase reporter viruses highly permissive cell lines for subgenomic and genomic hepatitis c virus rna replication targeting the raf-mek-erk mitogen-activated protein kinase cascade for the treatment of cancer sorafenib blocks the raf/mek/erk pathway, inhibits tumor angiogenesis, and induces tumor cell apoptosis in hepatocellular carcinoma model plc/prf/ construction and characterization of infectious intragenotypic and intergenotypic hepatitis c virus chimeras ) cpla is phosphorylated and activated by map kinase a novel arachidonic acid-selective cytosolic pla contains a ca( +)-dependent translocation domain with homology to pkc and gap a calcium-dependent mechanism for associating a soluble arachidonoyl-hydrolyzing phospholipase a with membrane in the macrophage cell line raw . a pyrrolidine-based specific inhibitor of cytosolic phospholipase a( )alpha blocks arachidonic acid release in a variety of mammalian cells pyrrolidine inhibitors of human cytosolic phospholipase a( ) recent progress in phospholipase a research: from cells to animals to humans regulation and inhibition of phospholipase a suicide inhibition of canine myocardial cytosolic calcium-independent phospholipase a . mechanism-based discrimination between calcium-dependent andindependent phospholipases a fusion-active glycoprotein g mediates the cytotoxicity of vesicular stomatitis virus m mutants lacking host shut-off activity composition and three-dimensional architecture of the dengue virus replication and assembly sites processive interfacial catalysis by mammalian -kilodalton phospholipase a enzymes on product-containing vesicles: application to the determination of substrate preferences cytosolic phospholipase a effects of fatty acid unsaturation numbers on membrane fluidity and alpha-secretase-dependent amyloid precursor protein processing the modification of mammalian membrane polyunsaturated fatty acid composition in relation to membrane fluidity and function eicosatetraynoic and arachidonic acid-induced changes in cell membrane fluidity consonant with differences in computer-aided design-structures role of cytosolic phospholipase a in allergic response and parturition reduced fertility and postischaemic brain injury in mice deficient in cytosolic phospholipase a optimization of novel acyl pyrrolidine inhibitors of hepatitis c virus rnadependent rna polymerase leading to a development candidate a cell protection screen reveals potent inhibitors of multiple stages of the hepatitis c virus life cycle production of infectious hepatitis c virus in tissue culture from a cloned viral genome group iva phospholipase a is necessary for the biogenesis of lipid droplets ccaat/enhancer binding proteins are not required for hiv- entry but regulate proviral transcription by recruiting coactivators to the long-terminal repeat in monocytic cells trafficking of hepatitis c virus core protein during virus particle assembly apolipoprotein e on hepatitis c virion facilitates infection through interaction with low-density lipoprotein receptor infectivity of hepatitis c virus is influenced by association with apolipoprotein e isoforms dengue virus capsid protein usurps lipid droplets for viral particle formation robust hepatitis c genotype a cell culture releasing adapted intergenotypic a/ a (s /jfh ) viruses highly efficient jfh -based cell-culture system for hepatitis c virus genotype a: failure of homologous neutralizing-antibody treatment to control infection the zinc finger antiviral protein acts synergistically with an interferon-induced factor for maximal activity against alphaviruses lentiviral vectors interfering with virus-induced cd down-modulation potently block human immunodeficiency virus type replication in primary lymphocytes hepatitis c virus hypervariable region modulates receptor interactions, conceals the cd binding site, and protects conserved neutralizing epitopes isolation of lipid droplets from cells by density gradient centrifugation a novel diagnostic target in the hepatitis c virus genome low ph-dependent hepatitis c virus membrane fusion depends on e integrity, target lipid composition and density of virus particles a thirdgeneration lentivirus vector with a conditional packaging system we are grateful to takaji wakita for jfh and to jens bukh for the j cf strain, to marc p. windisch for boceprevir, to timothey tellinghuisen for cma, to charles rice for the ns a-specific antibody e and huh- . cells, to darius moradpour for the core-specific antibody c - , to john maclauchlan for the adrp-specific antibody and to gert zimmer for vsv*m q . we would also like to thank all members of the department of experimental virology for helpful suggestions. key: cord- -o m kvw authors: sedeyn, koen; schepens, bert; saelens, xavier title: respiratory syncytial virus nonstructural proteins and : exceptional disrupters of innate immune responses date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: o m kvw human respiratory syncytial virus (rsv) is the most important cause of acute lower respiratory tract disease in infants worldwide. as a first line of defense against respiratory infections, innate immune responses, including the production of type i and iii interferons (ifns), play an important role. upon infection with rsv, multiple pattern recognition receptors (prrs) can recognize rsv-derived pathogen-associated molecular patterns (pamps) and mount innate immune responses. retinoic-acid-inducible gene-i (rig-i) and nucleotide-binding oligomerization domain-containing protein (nod ) have been identified as important innate receptors to mount type i ifns during rsv infection. however, type i ifn levels remain surprisingly low during rsv infection despite strong viral replication. the poor induction of type i ifns can be attributed to the cooperative activity of unique, nonstructural (ns) proteins of rsv, i.e., ns and ns . these viral proteins have been shown to suppress both the production and signaling of type i and iii ifns by counteracting a plethora of key host innate signaling proteins. moreover, increasing numbers of ifn-stimulated genes (isgs) are being identified as targets of the ns proteins in recent years, highlighting an underexplored protein family in the identification of ns target proteins. to understand the diverse effector functions of ns and ns , goswami and colleagues proposed the hypothesis of the ns degradasome (nsd) complex, a multiprotein complex made up of, at least, ns and ns . furthermore, the crystal structure of ns was resolved recently and, remarkably, identified ns as a structural paralogue of the rsv matrix protein. unfortunately, no structural data on ns have been published so far. in this review, we briefly describe the prrs that mount innate immune responses upon rsv infection and provide an overview of the various effector functions of ns and ns . furthermore, we discuss the ubiquitination effector functions of ns and ns , which are in line with the hypothesis that the nsd shares features with the canonical s proteasome. introduction human respiratory syncytial virus (rsv) is a negative strand rna virus belonging to the family pneumoviridae. rsv is a major cause of acute lower respiratory tract infections in the pediatric population and is increasingly recognized as an important cause of severe respiratory disease in the elderly [ ] [ ] [ ] . despite the major clinical impact of rsv on human health worldwide, no approved vaccine or effective antiviral therapy is available. although rsv infections do evoke humoral and cellular immune responses required to clear infection, these responses do not provide strong protection against a subsequent infection with rsv. early during infection, viral replication is sensed by the host's innate immune system, which leads to the production of type i and iii interferons (ifns), which is followed by the induction of an array of genes that code for antiviral proteins. in addition, ifn will recruit and activate innate leukocytes, including antiviral monocytes and natural killer (nk) cells, and stimulate the adaptive immune response [ ] . rsv has evolved a marvelous set of mechanisms to subvert this canonical antiviral response of the mammalian host, most notably by virtue of its nonstructural (ns) and ns proteins. the innate immune system is an important early line of defense against pathogens. this system comprises pattern recognition receptors (prrs) that can recognize pathogen-associated molecular patterns (pamps). activated prrs can induce the expression of cytokines, e.g., type i and iii ifns, which mount an antiviral state in an autocrine and paracrine fashion. upon rsv infection, airway epithelial cells, macrophages, and dendritic cells (dcs) are the main inducers of innate immune responses. several toll-like receptors (tlrs) have been identified that can act as prrs for rsv-derived pamps (fig ) . tlr , - , and - , for example, have been implicated in the induction of cytokines and chemokines upon rsv infection [ , ] . a role for tlr , which is well known to respond to lipopolysaccharide, as a prr for rsv is currently debated. although some groups reported an impaired innate immune response in tlr -deficient mice [ - ], ehl and colleagues could not confirm a role for a tlr -mediated immune response upon rsv infection [ ] . in addition, tlr might even play a role in tempering immune responses upon rsv infection [ ] . moreover, type i ifn production by macrophages and dcs from wild-type and tlr , - , - , - , and - knockout mice is very similar following rsv infection [ , ] . finally, alveolar macrophages are the primary source of type i ifn in rsv-infected mice, and tlrs do not play a crucial role in this response [ , ] . in contrast, retinoic-acid-inducible gene-i (rig-i)-like receptors (rlrs) are important for the induction of type i and possibly type iii ifns upon recognition of rsv (fig ) . type i ifn expression is strongly hampered in the absence of mitochondrial antiviral-signaling protein (mavs), the adaptor protein for rig-i and melanoma differentiation-associated protein (mda ) [ , ] . early on, gene expression ablation studies revealed that rig-i is the most important rlr to detect rsv, which is supported by the observation that rsv mrna could be coimmunoprecipitated with rig-i but not with mda [ - ]. later, however, both rig-i and mda were found to colocalize with rsv genomic rna and the n protein [ ] . interestingly, whereas rig-i partially localizes to rsv-induced inclusion bodies, mda and mavs were found nearly exclusively in these inclusion bodies, thereby dramatically blunting ifn-β induction. a third class of prrs, the nucleotide-binding oligomerization domain-like receptors (nlrs), is also important for the recognition of rsv. nucleotide-binding oligomerization domain-containing protein (nod ) can be activated by intact genomic single-stranded rna (ssrna) and is involved in the induction of ifn-β in mice in a mavs-dependent way (fig ) [ ]. cytoplasmic dna sensors (cdss), such as z-dna binding protein (zbp ) and cyclic gmp-amp synthase (cgas), are well known to induce innate immune responses upon recognition of pathogen-derived double-stranded dna (dsdna) or even rna [ ] [ ] [ ] . a contribution of cdss in innate sensing of rsv replication has not yet been reported. in contrast to other respiratory viruses, i.e., influenza virus and human respirovirus (formerly named human parainfluenza virus ), nasal washes from rsv-infected infants hardly firstly, tlr- , - , - , - , and - (marked in green) are involved in the production of cytokines and chemokines upon rsv infection. secondly, rig-i and possibly mda (rlrs, marked in red), are important in the induction of type i ifns. thirdly, nod (marked in dark blue) also induces type i ifns upon rsv infection. currently, there is no evidence for a role of the cdss (marked in purple), which signal through the er-associated sting protein, as prrs during rsv infection. activation of the rlrs or nod induces their association with the mitochondrial-associated mavs, which recruits the adaptor proteins traf or traf . via the traf adaptor, the kinases ikkε and tbk are subsequently activated, which phosphorylate and activate the transcription factors irf and irf . via the traf adaptor, kinases are activated, i.e., the ikk kinase complex, jnk, and p mapk, which phosphorylate and activate multiple transcription factors such as nf-κb, c-jun, and atf , respectively. activation of tlrs leads to the recruitment of adaptor proteins, e.g., myd , ticam , tirap, and tram. these adaptors can signal via traf or traf . the transcription factors activated by prr signaling ultimately induce expression of cytokines, chemokines, and ifns. above each prr, the confirmed or likely rsv-derived pamp is depicted. atf , activating transcription factor ; cds, cytoplasmic dna sensor; er, endoplasmic reticulum; ifn, interferon; ikk, inhibitor of nuclear factor kappa-b kinase; ikkε, inhibitor of nuclear factor kappa-b kinase subunit epsilon; irf , interferon regulatory factor ; irf , interferon regulatory factor ; jnk, c-jun n-terminal kinase; mapk, mitogen-activated protein kinase; mavs, mitochondrial antiviral-signaling protein; mda , melanoma differentiation-associated protein ; myd , myeloid differentiation primary response protein myd ; nf-kb, nuclear factor-kappa b; nod , nucleotide-binding oligomerization domain-containing protein ; pamp, pathogenassociated molecular pattern; prr, pattern recognition receptor; rig, retinoic-acid-inducible gene-i; rlr, rig-i-like receptor; rsv, respiratory syncytial virus; sting, stimulator of interferon protein; tbk , tank binding kinase ; ticam , toll/interleukin- receptor domain-containing adapter molecule ; tirap, toll/ interleukin- receptor domain-containing adapter protein; tlr, toll-like receptor; traf , tumor necrosis factor receptor-associated factor ; traf , tumor necrosis factor receptor-associated factor ; tram, toll-like receptor adaptor molecule. https://doi.org/ . /journal.ppat. .g contain ifn-α and -β [ - ]. apparently, this virus has evolved ways to outsmart the canonical mammalian antiviral response. two unique viral proteins, ns and ns , are responsible for the suppression of ifn induction and signaling. human and bovine rsv strains that lack ns and/or ns have been explored as live-attenuated vaccine candidates. such viruses are strongly attenuated in in vivo rsv infection models (cotton rats, calves, and chimpanzees) as well as in human but, at least in calves and chimpanzees, retain their ability to induce antibody responses [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . positioned proximal on the negativestranded rna genome, ns and ns are the most abundantly transcribed viral genes. recently, the crystal structure of ns was determined, revealing that this protein is composed of a β-sandwich flanked by α-helices [ ] . interestingly, the d structure of ns is very similar to the n-terminal domain of rsv m despite the complete absence of any primary sequence homology. both ns and ns strongly reduce the induction of type i and iii ifns upon rsv infection [ , [ ] [ ] [ ] [ ] [ ] . infection with recombinant viruses lacking ns or ns , separately or combined, suggests that these proteins function individually and cooperatively to suppress ifn induction. they do so by targeting multiple proteins of the signaling cascade that starts with the recognition of pamps by prrs and ends with the induction of ifn gene expression by several transcription factors. a widespread strategy of viruses to dampen innate immune responses is to counteract one of the early signaling steps in rlr-mediated ifn induction, i.e., the interaction of rig-i or mda with mavs [ ] [ ] [ ] [ ] . likewise, rsv prevents the interaction of rig-i with its adaptor mavs. in rsv-infected hep- cells and a cells overexpressing ns , ns was shown to interact with mavs (fig ) [ ] . by binding to mavs, ns could dose-dependently prevent the interaction between rig-i and mavs in a cells. furthermore, ban and colleagues demonstrated that ectopically expressed ns in hek t cells interacts with the pry-spry domain of e ubiquitin/ifn-stimulated gene (isg ) ligase tripartite motif-containing protein (trim ). this domain is responsible for the interaction of trim with rig-i [ ] . ns binding to the pry-spry domain suppresses k -linked polyubiquitination of rig-i by trim , which is essential for its downstream interaction with mavs (fig ) . proteins of other respiratory viruses also target the pry-spry domain of trim , which highlights the evolutionary importance of trim in mammalian antiviral defense. by binding the pry-spry domain of trim , the ns protein of influenza a virus and the nucleocapsid protein of severe acute respiratory syndrome virus also counteract trim -mediated rig-i ubiquitination [ , ] . ns can interact with the n-terminal domain of rig-i, both upon overexpression in hek t cells and during rsv infection in a cells (fig ) [ ] . as such, overexpressed ns disrupts the binding of rig-i with mavs; however, this has not yet been confirmed for endogenous ns expressed during an rsv infection. in addition to counteracting the interaction between rig-i and mavs, ns may also influence rig-i expression, although the reported findings seem conflicting. rig-i expression in a cells is strongly reduced in the presence of rsv ns , either expressed separately or in the context of an rsv infection [ ] . boyapalle and colleagues, however, reported that rig-i expression in a cells is reduced following infection with an ns -deficient rsv [ ] . this is surprising, because rig-i is itself an isg. possibly, this discrepancy is caused by the different multiplicity of infection (moi) used by these groups (moi and , respectively). in contrast to rig-i, mavs appears to be resistant to ns -and/or ns -mediated down-regulation [ ] . all together, these results highlight that the interaction of rig-i with mavs is suppressed by both ns and ns and that rig-i expression itself might be reduced by ns . currently, it is not clear whether ns and/or ns can disturb the interaction of mda or nod with mavs and whether they impact mda or nod expression levels. type i and iii ifn responses are inhibited by ns and ns at multiple levels, both during the induction of type i and iii ifns (left panel) and during ifn-induced signaling (right panel). ns and ns can form a so-called "ns degradasome" complex that is stabilized by mitochondria via mavs. the nsd complex is thought to contain hps, including the proteasome α subunit and other as yet unidentified proteins. ns and ns prevent the interaction of rig-i with mavs in different ways. ns binds to the pry-spry domain of trim , which is responsible for the interaction of trim with rig-i. as such, ns prevents the trim -mediated k -linked polyubiquitination of rig-i, which is necessary for the subsequent interaction of rig-i with mavs ( ). moreover, ns directly interacts with rig-i ( ) and ns interacts with mavs ( ) to suppress binding of rig-i to mavs. whether the interaction of ns with mavs also prevents the interaction of nod with mavs is currently unclear. ns reduces protein expression of traf and ikkε ( ), whereas ns modestly reduces traf and induces ikkε and tbk ( ). ns subsequently inhibits irf and irf by different proposed mechanisms ( ). ns reduces irf and irf protein expression and prevents the interaction between irf and cbp, thereby lowering the binding of the irf -cbp complex to the ifn-β promoter. ns , and to a lesser extent ns , enhances the activation and nuclear translocation of nf-κb ( ). these ns /ns effector functions ( - ) synergistically reduce the production of type i and iii ifns. furthermore, ns and ns also suppress type i and iii ifn receptor-mediated signal transduction. ns induces mir- a expression, which targets the mrna coding for ifnar , one of the subunits of the type i ifn receptor ( ). ns and ns may induce expression of socs proteins (socs and ), which negatively regulate the tyrosine kinases jak and tyk , which are important to transmit signaling from the type i and iii ifn receptors ( ). ns inhibits jak / tyk -mediated activation of stat / by reducing stat protein levels ( ) and by reducing stat phosphorylation ( ). some groups, however, reported that stat expression can also be reduced by ns (see text) ( ). ns and ns counteract the anti-inflammatory activity of the gr, although the exact mechanism is debated. in one model, ns interacts with the nuclear translocator ipo , which competes with gr for its nuclear translocation ( ). recent evidence suggests that the ns proteins may also counteract antiviral effector functions of isgs, e.g., ns degrades the oasl, ifit , and ifitm proteins, whereas ns degrades mapk ( ). full and dashed lines indicate robust and moderate inhibitory or stimulatory effector functions, respectively. cbp, creb binding protein; gr, glucocorticoid receptor; hp, host protein; ifit, interferon-induced protein with tetratricopeptide repeats; ifitm, interferon-induced transmembrane protein; ifn, interferon; ifnar, interferon alpha/beta receptor; ikkε, inhibitor of nuclear factor-kappa b kinase subunit epsilon; ipo , importin- ; irf , interferon regulatory factor ; isg, ifn-stimulated gene; jak, janus kinase; mapk , mitogen-activated protein kinase ; mavs, mitochondrial antiviral-signaling protein; ns, nonstructural; oasl, - -oligoadenylate synthase-like protein; rig-i, retinoic-acid-inducible gene-i; rlr, rig-i-like receptor; rsv, respiratory syncytial virus; socs, suppressor of cytokine signaling; stat, signal transducer and activator of transcription; tbk , tank binding kinase ; trim , tripartite motif-containing protein; tyk, tyrosine kinase . https://doi.org/ . /journal.ppat. .g association of rlrs with mavs leads to the recruitment of the adaptors tumor necrosis factor receptor-associated factor (traf ) and - (traf ). whereas traf activates the downstream serine/threonine kinases inhibitor of nuclear factor-kappa b kinase subunit epsilon (ikkε) and tank binding kinase (tbk ), traf activates the downstream kinases ikk, c-jun n-terminal kinase (jnk), and p mitogen-activated protein kinase (p mapk) (fig ) . the effect of ns and ns on traf , ikkε, and tbk is currently inconclusive. some groups reported that ns , either overexpressed or expressed during an rsv infection in a cells, reduces the expression levels of both traf and ikkε (fig ) [ , ] . ren and colleagues, however, observed no difference in endogenous and recombinant traf and ikkε expression in a cells in the presence or absence of ns [ ] . possibly, the different strains (rsv long versus a ) or experimental timing used explain these opposing observations. overexpression of ns in a cells modestly reduces and slightly enhances traf and ikkε expression levels, respectively (fig ) [ , ] . these effects of ns were, however, not observed with an rsv strain that lacks ns . ling and colleagues also observed that overexpressed ns enhances overexpression of ikkε and tbk in hek t cells (fig ) [ ] . to our knowledge, no data have been published on the possible effect of ns on the expression of tbk . coexpression of ns and ns in a cells reduces recombinant ikkε expression, suggesting that the inhibitory effect of ns is dominant over the enhancing effect of ns [ ] . these results suggest that ns suppresses traf and ikkε expression, although more evidence is needed to confirm this hypothesis. ectopic expression of ns slightly suppresses traf expression and enhances ikkε and tbk expression, although these effects need to be confirmed during an rsv infection. whether ns and/or ns affect the traf adaptor has not been investigated yet. some evidence indicates that rsv may indeed affect traf expression. in different models, traf expression has been shown to be down-regulated by the microrna (mirna) mir- a [ , ] . interestingly, eilam-frenkel and colleagues found that mir- a expression was up-regulated in rsv-infected hep- cells [ ] . future research may elucidate whether rsv can downregulate traf expression via up-regulating mir- a and whether this is mediated by ns and/or ns . activated ikkε and tbk phosphorylate the transcription factors interferon regulatory factor (irf)- and irf , which induces conformational changes that allow the formation of homo-and heterodimers of irf and - that translocate to the nucleus. phosphorylation of inhibitor of kappa b (iκb), e.g., by the canonical ikkα/β/γ complex, initiates its degradation with subsequent release and nuclear translocation of nuclear factor-kappa b (nf-κb). the irf and nf-κb transcription factors are essential for the induction of type i and iii ifns (fig ) . several observations highlight that ns and ns can impair the activation and effector functions of irf transcription factors. initial work with recombinant rsv strains lacking one or both ns proteins in a cells highlighted that ns and ns prevent nuclear translocation of irf , especially late (> hours post infection) in the rsv replication cycle [ , , ] . this might, in part, be explained by the ability of ns to directly reduce the recombinant expression of irf and irf (fig ) [ ] . in addition, by preventing the formation of the irf /creb binding protein (cbp) complex in the nucleus, ns may also inhibit irf -dependent gene expression downstream of the nuclear translocation of irf (fig ) [ , ] . it is important to note that this ns effector function has only been demonstrated upon overexpression in hek t cells, so additional confirmation in an rsv infection is necessary. in a and vero cells, nf-κb activation and nuclear translocation occur early after rsv infection and are clearly enhanced by ns and, to some extent, by ns (fig ) [ , ] . in addition to the canonical iκb phosphorylation and subsequent degradation, rsv-induced nf-κb activation involves p ser phosphorylation via the rig-i/mavs/traf /ikkβ signaling pathway [ ] . although nf-κb contributes to the induction of type i and iii ifns, nf-κb also strongly induces the expression of anti-apoptotic genes. in the context of an rsv infection, the induction of an anti-apoptotic cellular environment by nf-κb likely dominates the contribution of nf-κb over the induction of ifn, which is primarily controlled by irf / activation. taken together, ns and ns suppress the induction of type i and iii ifns by targeting multiple proteins of the signaling cascade that leads to type i or iii gene activation, i.e., rig-i, mavs, traf , ikkε, tbk , irf , and irf . although some of these effector functions have been confirmed in rsv-infected cells, others have so far only been identified with overexpression of ns or ns alone. so, future research is needed to elucidate the biological relevance of these recombinant responses, particularly because ns can relocate ns to the mitochondria [ ] . binding of type i and iii ifns to their heterodimeric receptor complex induces a janus kinase (jak)-signal transducer and activator of transcription (stat) signaling cascade that induces an antiviral state by expression of isgs. compared with wild-type rsv, rsv strains that lack ns and/or ns are more sensitive to type i ifn treatment [ , ] . thus, rsv ns proteins also have effector functions downstream of the induction of ifn synthesis to suppress ifninduced antiviral responses. in rsv-infected a cells, ns induces the expression of the mirna mir- a, which targets the mrna coding for interferon alpha/beta receptor (ifnar ) by an unknown mechanism (fig ) [ ] . as a result, the ifnar protein levels are reduced, which decreases responsiveness to type i ifns of rsv-infected cells and thus favors rsv replication. the induction of an antiviral state by type i and type iii ifns requires the phosphorylation of stat and stat proteins in the proximity of the type i and iii receptors. two tyrosine kinases, jak and tyrosine kinase (tyk ), are responsible for the phosphorylation and activation of stat proteins after activation of the type i and iii ifn receptor ( fig ) . for now, there is no evidence that ns or ns may alter the levels and activation status of these kinases. several groups reported that the phosphorylation and total protein levels of tyk are not altered by the expression of ns and/or ns [ ] [ ] [ ] . whether this also accounts for jak is currently not clear. infection of airway epithelial cell lines with human metapneumovirus, a respiratory virus closely related to rsv, does reduce both jak and tyk protein levels [ ] . additionally, jak kinase activity is regulated by a negative feedback loop that consists of the suppressor of cytokine signaling (socs) family, with socs and being the strongest inhibitors of jak kinases. it has been reported that ns and/or ns may regulate the expression of socs and/or socs , thereby enhancing rsv replication [ ] [ ] [ ] [ ] . heterodimers of phosphorylated stat and transcription factors are important for the induction of isgs. consequently, numerous viral proteins counteract stat and proteins by using different strategies, e.g., by preventing stat / heterodimer formation (nipah and hendra virus nucleoprotein), by suppressing stat / nuclear translocation (ebolavirus vp ), or by degrading stat and/or (paramyxovirus v protein) [ ] [ ] [ ] . both stat and are targets for rsv. in human tracheobronchial epithelial cells and a cells, infection with rsv slightly increases total stat protein levels, likely because stat proteins are themselves isg products [ - , , ] . stat phosphorylation induced by exogenous type i ifn, however, is clearly reduced by ns but not by ns (fig ) [ [ ] [ ] [ ] ] . whether ns also reduces stat phosphorylation during an rsv infection has not been reported yet. in contrast to stat , both phosphorylated and nonphosphorylated stat protein levels are clearly reduced by rsv infection [ , , ] . whether ns , ns , or both reduce stat levels is currently debated. based on overexpression experiments and infections with rsv strains lacking ns and/or ns , several groups reported that ns , but not ns , reduces stat levels in vitro (fig ) [ , , , ] . others, however, also observed reduced stat levels after overexpression of ns (fig ) [ , ] . lo and colleagues reported that recombinant coexpression of ns and ns reduces stat levels stronger than ns alone, although expression of ns alone did not affect stat levels [ ] . in addition to reducing total stat levels, rsv also seems to suppress nuclear translocation of the residual stat proteins, an effect that depends on ns and/or ns [ ] . so far, the possible impact of rsv ns proteins on stat and expression levels in vivo has not yet been demonstrated. because mouse stat appears resistant to ns , primary human airway epithelial cell cultures or experiments in nonhuman primates may be required to confirm that ns -mediated stat reduction also occurs in vivo [ ] . evidence is rising that the ns proteins also suppress the antiviral activity of at least some isg products. cells that express myxovirus resistance protein a (mxa) or cells pretreated with type i ifn only modestly limit rsv replication [ ] . moreover, ectopic expression of ns and ns in hek -derived cells has been shown to degrade certain isg products, i.e., - oligoadenylate synthetase-like protein (oasl), interferon-induced protein with tetratricopeptide repeats (ifit ), interferon-induced transmembrane protein (ifitm ), and mapk , in a selective manner (fig ) [ , ] . an important remark, however, is that these effector functions have not yet been validated during an rsv infection. as stated by ribaudo and barik, a comprehensive screen of all known isgs will likely unravel additional substrates of ns and/or ns [ ] . ns and ns affect the induction of apoptosis and cell shedding. ns and ns individually and cooperatively delay apoptosis in rsv-infected cells, with ns being stronger than ns in doing so [ ] . the early expression of ns and ns after infection activates the antiapoptotic -phophoinositide-dependent protein kinase (pdk)-rac serine/threonine-protein kinase (akt)-glycogen synthase kinase (gsk) pathway [ , , ] . later in infection (> hours), activation of this pathway drops, and the incidence of apoptosis increases [ ] . by delaying apoptosis, ns and ns may facilitate prolonged rsv replication with increased viral yields. a typical hallmark of severe disease following rsv infection is the obstruction of the smaller airways by plugs consisting of infiltrating immune cells, mucus, and shed infected epithelial cells. in primary human airway epithelial cells and in an in vivo hamster model, it has been shown by experiments with a set of elegant recombinant virus constructs that rsv ns is necessary and sufficient to induce shedding of infected epithelial cells [ ] . interestingly, cell death, associated with nuclear changes that are indicative of apoptosis, of infected epithelial cells only occurred after these cells were detached from the epithelial layer. moreover, shedding of infected epithelial cells coincided with reducing viral titers. all together, these results suggest that expression of ns early after infection delays apoptosis and induces changes in cell morphology that ultimately result in shedding of infected cells in the airway lumen. these shed and detached infected epithelial cells die. as shedding (and ultimately clearance by mucociliary transport) of infected epithelial cells reduces rsv viral titers, it seems that the bulk of viral spread precedes cell shedding. possibly, (early) changes in cell morphology that ultimately lead to shedding of infected epithelial cells may facilitate rsv production and spreading. as such, ns plays a pivotal role in the production and spreading of rsv virions. ns and ns counteract the anti-inflammatory activity of the glucocorticoid receptor. although rsv-induced bronchiolitis is characterized by a (severe) inflammatory response, the use of anti-inflammatory glucocorticoids has shown no clinical benefits against (severe) disease [ ] [ ] [ ] . based on experiments with different transformed and primary cell cultures as well as mice, several groups concluded that rsv can inhibit the anti-inflammatory activity of glucocorticoids via the glucocorticoid receptor (gr) [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the exact mechanism that accounts for this inhibition, however, is debated. one group demonstrated that rsv blocks glucocorticoid-mediated gr activation in a , beas- b, and primary human small airway epithelial cells but not in the monocytic thp- cell line, suggesting that the effect is restricted to rsv-infected epithelial cells [ , ] . further mechanistic analysis in a cells suggested that ns and ns reduce the binding of the gr to gr-responsive promoters, without affecting gr total protein levels and nuclear translocation [ , , ] . interestingly, knockdown of mavs in a cells lowered dexamethasone-induced transforming growth factor β (tgf-β)-stimulated clone (tsc ) domain family member (also known as glucocorticoid-induced leucine zipper protein [gilz]) mrna expression to a level similar to that of an rsv infection [ ] . these results suggest that mavs plays a role in glucocorticoidinduced gr activation in a cells and that ns and ns may indirectly suppress gr activation through inhibition of mavs (see "ns and ns interfere with rig-i"). xia and colleagues used beas- b and primary differentiated human bronchial epithelial cells grown at an air-liquid interface to demonstrate that rsv suppresses glucocorticoidinduced gr activation by a mechanism that involves up-regulation of tgf-β expression [ ] . a model was proposed in which viral infection is sensed by tlr , which induces tgf-β that subsequently activates the type i tgf-β receptor activin receptor-like kinase (alk ). alk activation subsequently counteracts glucocorticoid activity through an unknown mechanism. in accordance with the aforementioned study, no difference in total grα protein levels or nuclear translocation were observed upon rsv infection. interestingly, blocking alk with the selective inhibitor sb and reducing tgf-β activity by tranilast (a compound used to manage a wide variety of diseases, including inflammatory diseases) could subvert the rsvmediated suppression of glucocorticoid activity. although xia and colleagues did not investigate the role of ns or ns , ns may contribute to rsv-induced tgf-β expression by up-regulating the transcription factor kruppel-like factor (discussed in the next paragraph) [ ] . another study concluded that rsv ns prevents gr nuclear translocation [ ] . this finding was based on experiments with a cells, mouse lung tissue, but also analysis of nasopharyngeal aspirates from rsv-infected infants. moreover, expression of grα and importin- , which is important for gr nuclear entry, was reduced at the mrna and protein level in mouse lungs, whereas grβ and ipo expression at the mrna level were reduced in the nasopharyngeal aspirates upon rsv infection. mechanistically, ns was found to directly interact with ipo and can thus compete with gr for ipo binding. the capacity of rsv to suppress the anti-inflammatory effect of glucocorticoids through ns and ns are in line with the ineffectiveness of glucocorticoid treatments off severely ill rsv patients. in addition to mir- a, ns proteins also regulate the expression of the mirnas mir- , let- i, and mir- b [ , , ] . in rsv-infected a cells, ns suppresses mir- expression by up-regulating the transcription factor kruppel-like factor , which drives the expression of tgf-β [ ] . remarkably, inhibition of mir- was shown to actually repress rsv replication in a cells [ ] . this apparent discrepancy could be explained by the difference in time points after infection that were analyzed ( day versus days post infection). possibly, ns suppresses the rsv-induced up-regulation of mir- within hours post infection, whereas later on, mir- expression may be up-regulated to enhance viral replication. investigating mir- expression levels beyond hours post infection and the impact of ns on these levels may result in a better understanding of the role of mir- in rsv replication. in normal human bronchial epithelial cells, let- i and mir- b expression is up-regulated during an rsv infection by a type i ifn and nf-κbdependent mechanism, respectively, and further increased in the absence of ns or ns [ ] . this can be expected for let- i, as the ns proteins suppress the type i ifn response. moreover, the mir- b promoter can be activated by the nf-κb family member p , which is activated during an rsv infection through a rig-i/mavs/traf /ikkβ signaling pathway [ , ] . by counteracting the interaction between rig-i and mavs (see "ns and ns interfere with rig-i"), the ns proteins may dampen this pathway, leading to reduced activation of p and subsequent expression of mir- b. to our knowledge, the effect of ns and/or ns on the rsv-induced activation of p has not been investigated yet. this could readily be tested by comparing total levels of activated p between cells infected with wild-type rsv and ns deletion strains. whether the ns protein-mediated suppression of let i and mir- b favors or counteracts rsv replication is currently unclear. ns and ns interfere with the adaptive immune response. the ns proteins also play a role in the development of adaptive immune responses during rsv infection, partly as a consequence of their capacity to suppress type i and iii ifn levels. in addition to airway epithelial cells, rsv can infect dcs and activate prrs by pamps [ ] . munir and colleagues investigated the activation status of isolated human monocyte-derived myeloid dcs upon rsv infection by quantifying several maturation markers, including cluster of differentiation (cd) , , , , and [ ] . infection with wild-type and ns-deficient rsv strains highlighted that ns , and to a lesser extent ns , suppress the maturation of human dcs. by using an ifnar -blocking antibody, this suppression was shown to partially depend on the capacity of the ns proteins to counteract the production of type i ifns. moreover, in vivo pulmonary conventional dc activation, as measured by the up-regulation of cd and cd , was strongly hampered in rsv-infected mice that are deficient for mavs, an essential signaling protein for rsv-induced type i ifn production [ ] . taken together, these results highlight that type i ifns play a role in rsv-induced dc maturation. as such, the pleiotropic effector functions of ns and ns to counteract the production of type i ifns likely account for the reduced dc maturation by ns and ns . in a follow-up report, munir and colleagues investigated the consequence of reduced dc maturation by ns and ns on subsequent activation of t-cell responses by co-cultivation of rsv-infected human monocyte-derived dcs and autologous t cells [ ] . deletion of ns was found to promote the proliferation and activation of cd + cd + t cells and t-helper cells, cell populations that can counteract rsv, and to suppress the activation of il- -producing cd + t cells. remarkably, none of these effects on t cells appeared to depend on type i ifn. possibly, il- β and il- α play a role as these cytokines were suppressed in dcs by ns in a type i ifn-independent manner. in contrast, a study by kotelkin and colleagues in mice identified that ns , and not ns , suppresses cd + t-cell responses in a type i ifndependent manner [ ] . remarkably, although rsv-induced activation of dcs in mavs -/-and mavs -/myeloid differentiation primary response protein myd (myd ) -/mice was strongly impaired, these mice could still mount comparable cd + t-cell responses as wildtype mice [ ] . this may in part be explained by the approximately -fold higher viral loads in mavs -/mice compared with wild-type mice. in addition, a small residual population of cd -positive pulmonary dcs was still present in these mice that could migrate to the draining mediastinal lymph node to activate naive cd + t cells. as mavs -/-myd -/mice are still functional for toll/interleukin- receptor domain-containing adapter molecule (ticam ), the adaptor of tlr , these dcs may have matured after the activation of tlr by rsvderived double-stranded rna (dsrna) [ ] . live-attenuated rsv vaccines with a targeted deletion in ns have been tested for use in infants [ ] . whereas a strain with only ns deleted was insufficiently attenuated, other ns deletion strains with additional mutations were found to be over-attenuated, highlighting the importance of a right balance between attenuation and immunogenicity. currently, phase i clinical trials with other ns deletion strains (clinicaltrials.gov identifiers: nct and nct ) and one with an ns deletion (clinicaltrials.gov identifier: nct ) are ongoing. the primary endpoints of these ongoing phase i trials with the live-attenuated rsv strains that lack either ns or ns are measures for safety, vaccine virus infectivity, and the induction of rsv-neutralizing titers. to our knowledge, cellular immune responses after immunization with these live-attenuated rsv vaccines have not been investigated. taking into account the small size of ns and ns , it is remarkable how many host functions are affected by these rsv proteins. goswami and colleagues hypothesized that ns and ns form a large ( to kda) degradative complex, which they called the ns degradasome (nsd) [ ] . in a cells, this complex could selectively degrade particular innate immune signaling proteins of which rig-i was also confirmed as a substrate of nsd complexes isolated from rsv-infected cells. in this respect, it would be interesting to assess whether other reported ns target proteins are also substrates of the proposed nsd complexes in rsv-infected cells. the nsd complex appears to be metastable, and its activity is enhanced by mitochondria via mavs, possibly through stabilizing the nsd complex [ ] . interestingly, in mavs-deficient cells stimulated with ifn-α, wild-type rsv is almost equally attenuated as rsv lacking ns and ns , confirming the importance of mavs for ns protein-mediated suppression of type i ifn responses. in line with these results, ns mainly localizes to mitochondria and seems to recruit ns towards the mitochondria [ ] . furthermore, ns target proteins, but not other innate proteins, relocate from the cytoplasm to the mitochondria upon expression of ns and ns [ ] . all together, these results support the hypothesis of the formation of a mitochondrialassociated nsd complex to selectively degrade innate immune proteins in rsv-infected cells. although the exact composition of the nsd complex is still unresolved, the presence of the α subunit from the s core proteasome sparked the idea that the nsd complex might act in a similar way as the host s proteasome [ ] . in line with this hypothesis, ns protein-induced stat and oasl degradation is (partially) blocked by the proteasome inhibitors mg and lactacystin [ , , , , ] . in contrast, ns protein-induced traf and ikkε degradation appears insensitive to mg [ ] . proteasome-like activity of the nsd complex is further supported by the identification of ns and ns as potential inducers of host protein ubiquitination [ , ] . whereas ns is a putative elongin b/c-cullin / -socs box-type e ubiquitin ligase, ns does not contain an elongin c binding consensus sequence [ , , ] . interestingly, an rsv strain with mutated ns residues that appear essential for ubiquitination activity and ns -mediated stat degradation is nearly equally attenuated as an ns deletion strain [ ] . these results suggest that ns and ns may induce ubiquitination of host proteins, which are then degraded by the nsd complex. it is currently not known, however, if ns and/or ns selectively mark innate immune proteins for degradation, which would explain the selective degradative activity of the nsd complex. recently, the crystal structure of ns was determined and revealed a remarkable structural homology with the n-terminal domain of the matrix protein of rsv [ ]. one clear difference between ns and m is the presence of an α-helix (called α ) at the c-terminus of ns . a truncated ns lacking this helix or mutation of residues that are important for the interaction of helix α with the rest of ns partially abrogates ns -mediated suppression of ifn-β induction and attenuates recombinant rsv strains. these results confirm that helix α is important for at least some ns -mediated effector functions. unravelling the crystal structure of ns and the composition of the nsd complex would greatly enhance our understanding of the remarkable diverse effector functions of ns and ns . moreover, ns and/or ns may be attractive targets for antiviral therapy. intranasal administration of nanoparticles carrying a plasmid encoding an ns -targeting small interfering rna (sirna) in mice can reduce lung viral titers, airway hyperresponsiveness, and pulmonary inflammation, both in a prophylactic as well as therapeutic setting [ ] . a recent small compound high-throughput screen revealed candidate inhibitors of ns , highlighting that ns could be targeted by a small compound drug [ ] . in humans, rsv infections induce remarkably low levels of ifn-α and -β compared with other respiratory viruses. this is largely the consequence of unique viral proteins, ns and ns , that strongly suppress the induction and signaling of ifn. in recent years, evidence is rising that, in addition, antiviral activities of isgs may be hampered by the ns proteins. ns and ns exert additional functions, such as delaying cell apoptosis, and loss of ns is associated with a stronger adaptive immune response and reduced in vivo viral replication. ns and ns are thought to form a so-called "ns degradasome" complex, which may function as a proteasome-like complex that selectively degrades a plethora of innate immune proteins. further insight in the composition of the nsd and the resolution of the structure of ns will likely help to explain the remarkable diverse effector functions of ns and ns . some effector functions, however, have so far only been identified in artificial cell systems and should be interpreted carefully. large-scale protein-protein interaction screens, preferentially performed using multiple complementary protein-protein interaction techniques in the context of an rsv infection, will generate a more comprehensive list of host proteins that interact with ns and/or ns . such new knowledge, combined with co-crystal structure analysis of rsv ns and ns in complex with host protein factors, will be instrumental to design antiviral drugs that impact on the rsv-host interface and thereby complement directly acting antivirals. global burden of acute lower respiratory infections due to respiratory syncytial virus in young children: a systematic review and meta-analysis global, regional, and national disease burden estimates of acute lower respiratory infections due to respiratory syncytial virus in young children in : a systematic review and modelling study estimates of the global, regional, and national morbidity, mortality, and aetiologies of lower respiratory infections in countries nonstructural proteins ns and ns of bovine respiratory syncytial virus block activation of interferon regulatory factor a novel mechanism for the inhibition of interferon regulatory factor- -dependent gene expression by human respiratory syncytial virus ns protein replacement of the respiratory syncytial virus nonstructural proteins ns and ns by the v protein of parainfluenza virus nonstructural proteins and of respiratory syncytial virus suppress maturation of human dendritic cells hepatitis c virus protease ns / a cleaves mitochondrial antiviral signaling protein off the mitochondria to evade innate immunity disruption of innate immunity due to mitochondrial targeting of a picornaviral protease precursor mutual antagonism between the ebola virus vp protein and the rig-i activator pact determines infection outcome influenza a virus ns targets the ubiquitin ligase trim to evade recognition by the host viral rna sensor rig-i respiratory syncytial virus ns protein colocalizes with mitochondrial antiviral signaling protein mavs following infection human respiratory syncytial virus ns targets trim to suppress rig-i ubiquitination and subsequent rig-i-mediated antiviral signaling. viruses the severe acute respiratory syndrome coronavirus nucleocapsid inhibits type i interferon production by interfering with trim -mediated rig-i ubiquitination molecular mechanism of influenza a ns -mediated trim recognition and inhibition human respiratory syncytial virus nonstructural protein ns antagonizes the activation of beta interferon transcription by interacting with rig-i viral degradasome hijacks mitochondria to suppress innate immunity respiratory syncytial virus nonstructural proteins decrease levels of multiple members of the cellular interferon pathways microrna- a induction during influenza h n virus infection targets and regulates traf levels in human nasal epithelial cells (hnecs) extracellular vesicles containing mir- a attenuate experimental colitis by targeting traf and irak microrna - p, mir-let- c- p, mir- and mir- - p are differentially expressed in respiratory syncytial virus (rsv) persistently infected hep- cells effects of nonstructural proteins ns and ns of human respiratory syncytial virus on interferon regulatory factor , nf-kappab, and proinflammatory cytokines nonstructural proteins of respiratory syncytial virus suppress premature apoptosis by an nf-kappab-dependent, interferon-independent mechanism and facilitate virus growth multiple functional domains and complexes of the two nonstructural proteins of human respiratory syncytial virus contribute to interferon suppression and cellular location respiratory syncytial virus non-structural protein facilitates virus replication through mir- a-mediated inhibition of interferon-alpha receptor specific inhibition of type i interferon signal transduction by respiratory syncytial virus respiratory syncytial virus nonstructural protein specifically inhibits type i interferon signal transduction respiratory syncytial virus nonstructural proteins ns and ns mediate inhibition of stat expression and alpha/beta interferon responsiveness human metapneumovirus inhibits ifn-beta signaling by downregulating jak and tyk cellular levels respiratory syncytial virus (rsv) attachment and nonstructural proteins modify the type i interferon response associated with suppressor of cytokine signaling (socs) proteins and ifn-stimulated gene- (isg ) rsv replication is attenuated by counteracting expression of the suppressor of cytokine signaling (socs) molecules respiratory syncytial virus ns protein degrades stat by inducing socs expression respiratory syncytial virus nonstructural proteins upregulate socs and socs in the different manner from endogenous ifn signaling identification of paramyxovirus v protein residues essential for stat protein degradation and promotion of virus replication ebola virus vp targets a unique nls binding site on karyopherin alpha to selectively compete with nuclear import of phosphorylated stat nipah and hendra virus nucleoproteins inhibit nuclear accumulation of signal transducer and activator of transcription (stat ) and stat by interfering with their complex formation ifnbeta-dependent increases in stat , stat , and irf mediate resistance to viruses and dna damage interferome v . : an updated database of annotated interferon-regulated genes respiratory syncytial virus ns protein degrades stat by using the elongin-cullin e ligase respiratory syncytial virus strain a is resistant to the antiviral effects of type i interferons and human mxa '- '-oligoadenylate synthetase-like protein inhibits respiratory syncytial virus replication and is targeted by the viral nonstructural protein the nonstructural proteins of pneumoviruses are remarkably distinct in substrate diversity and specificity role of p /nf-kappab functional balance in respiratory syncytial virus-induced inflammation response rsv-encoded ns promotes epithelial cell shedding and distal airway obstruction a randomized, doubleblind, placebo-controlled trial of dexamethasone in severe respiratory syncytial virus (rsv) infection: effects on rsv quantity and clinical outcome the effect of high dose inhaled corticosteroids on wheeze in infants after respiratory syncytial virus infection: randomised double blind placebo controlled trial effect of dexamethasone on respiratory syncytial virus-induced lung inflammation in children: results of a randomized, placebo controlled clinical trial epithelial cells infected with respiratory syncytial virus are resistant to the anti-inflammatory effects of hydrocortisone respiratory syncytial virus represses glucocorticoid receptor-mediated gene activation respiratory syncytial virus (rsv) suppression of glucocorticoid receptor phosphorylation does not account for repression of transactivation poly i:c and respiratory syncytial virus (rsv) inhibit glucocorticoid receptor (gr)-mediated transactivation in lung epithelial, but not monocytic, cell lines the respiratory syncytial virus (rsv) nonstructural proteins mediate rsv suppression of glucocorticoid receptor transactivation glucocorticoid insensitivity in virally infected airway epithelial cells is dependent on transforming growth factor-beta activity respiratory syncytial virus nonstructural protein blocks glucocorticoid receptor nuclear translocation by targeting ipo and may account for glucocorticoid insensitivity human respiratory syncytial virus non-structural protein ns modifies mir- expression via transforming growth factor-beta respiratory syncytial virus regulates human micrornas by using mechanisms involving beta interferon and nf-kappab respiratory syncytial virus modifies micrornas regulating host genes that affect virus replication nf-kappab p -dependent transactivation of mirna genes following cryptosporidium parvum infection stimulates epithelial cell immune responses primary human mdc , mdc , and pdc dendritic cells are differentially infected and activated by respiratory syncytial virus respiratory syncytial virus interferon antagonist ns protein suppresses and skews the human t lymphocyte response the ns protein of human respiratory syncytial virus suppresses the cytotoxic t-cell response as a consequence of suppressing the type i interferon response respiratory macrophages and dendritic cells mediate respiratory syncytial virus-induced il- production in tlr -or tlr -dependent manner. int immunopharmacol identification of respiratory syncytial virus nonstructural protein residues essential for exploitation of the host ubiquitin system and inhibition of innate immune responses mutation of the elongin c binding domain of human respiratory syncytial virus non-structural protein (ns ) results in degradation of ns and attenuation of the virus inhibition of respiratory syncytial virus infection with intranasal sirna nanoparticles targeting the viral ns gene modular cell-based platform for high throughput identification of compounds that inhibit a viral interferon antagonist of choice key: cord- -vblgotjn authors: sawicki, stanley g; sawicki, dorothea l; younker, diane; meyer, yvonne; thiel, volker; stokes, helen; siddell, stuart g title: functional and genetic analysis of coronavirus replicase-transcriptase proteins date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: vblgotjn the coronavirus replicase-transcriptase complex is an assembly of viral and cellular proteins that mediate the synthesis of genome and subgenome-sized mrnas in the virus-infected cell. here, we report a genetic and functional analysis of temperature-sensitive (ts) mutants of murine hepatitis virus mhv-a that are unable to synthesize viral rna when the infection is initiated and maintained at the non-permissive temperature. both classical and biochemical complementation analysis leads us to predict that the majority of mhv-a orf a replicase gene products (non-structural proteins nsp –nsp ) form a single complementation group (cistron ) while the replicase gene products encoded in orf b (non-structural proteins nsp –nsp ) are able to function in trans and comprise at least three, and possibly five, further complementation groups (cistrons ii–vi). also, we have identified mutations in the non-structural proteins nsp , nsp , nsp , nsp , nsp , and nsp that are responsible for the ts phenotype of eight mhv-a mutants, which allows us to conclude that these proteins are essential for the assembly of a functional replicase-transcriptase complex. finally, our analysis of viral rna synthesis in ts mutant virus-infected cells allows us to discriminate three phenotypes with regard to the inability of specific mutants to synthesize viral rna at the non-permissive temperature. mutant la ts appeared to be defective in continuing negative-strand synthesis, mutant alb ts appeared to form negative strands but these were not utilized for positive-strand rna synthesis, and mutant alb ts was defective in the elongation of both positive- and negative-strand rna. on the basis of these results, we propose a model that describes a pathway for viral rna synthesis in mhv-a -infected cells. further biochemical analysis of these mutants should allow us to identify intermediates in this pathway and elucidate the precise function(s) of the viral replicase proteins involved. coronaviruses are positive-strand, enveloped rna viruses that infect vertebrates and are associated mainly with respiratory and enteric disease. they have long been recognized as important pathogens of livestock and companion animals, and they are a common cause of respiratory tract infections in humans [ ] [ ] [ ] . more recently, a coronavirus has been identified as the causative agent of sars, a form of atypical pneumonia in humans with a case fatality ratio of approximately % [ ] . clearly, there is an urgent need to develop new strategies to prevent or control coronavirus infections, and understanding the biology, replication, and pathogenesis of these viruses is an essential part of this process. murine hepatitis virus, strain a (mhv-a ), is a group ii coronavirus with a genome of approximately , nucleotides. the genomic rna encodes the structural proteins of the virus, non-structural proteins involved in viral rna synthesis (the nsp or replicase proteins), and proteins that are non-essential for replication in cell culture but appear to confer a selective advantage in vivo (accessory proteins) [ ] . in the mhv-a -infected cell, the expression of the replicase protein genes is mediated by translation of the genomic rna, and the expression of the structural protein genes is mediated by the translation of a set of -coterminal subgenomic mrnas. the subgenomic mrnas are produced by a unique mechanism that involves discontinuous transcription during negative-strand rna synthesis [ ] [ ] [ ] . the organization and expression of the mhv-a genome are illustrated in figure . the proximal open reading frames (orf) of mhv-a genomic rna (orf a and orf b) are translated to produce two large polyproteins, pp a and pp ab, with calculated molecular masses of . and . kilodaltons, respectively. translation of the larger pp ab involves programmed (À ) ribosomal frameshifting [ ] . during or after synthesis, these polypeptides are extensively processed by three virusencoded proteinases to produce a membrane-bound replicase-transcriptase complex [ ] . cleavage of the replicase polyproteins is predicted to result in end-products; nsp -nsp encoded in orf a and nsp - encoded in orf b [ ] . these proteins have been shown, or are predicted to have multiple enzymatic functions, including papain-like proteases (nsp ), adenosine diphosphate-ribose -phosphatase (nsp ), c-like cysteine proteinase (nsp ), rna-dependent rna polymerase (nsp ), superfamily helicase (nsp ), exonuclease (nsp ), endoribonuclease (nsp ), and s-adenosylmethionine-dependent -o-methyl transferase (nsp ) [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the crystallographic structures of sars coronavirus nsp and nsp have been determined and are likely to be similar for mhv-a [ ] [ ] [ ] . in the course of an infectious cycle, the mhv-a replicase-transcriptase complex amplifies the genomic rna and synthesizes subgenomic mrnas. amplification of the genomic rna involves full-length negative-strand templates, and the synthesis of subgenomic mrna involves subgenomelength negative-strand templates [ , ] . the structures engaged in the replication and transcription of positivestrand mhv-a rna have been characterized [ ] . approximately % of the replicating and transcribing structures that accumulate in infected cells are multi-stranded intermediates (replicative and transcriptive intermediate rna, ri/ti rna) and % are found in structures with only one or very few nascent strands (native replicative and transcriptive forms, rf/tf rna). although the structures engaged in negative-strand rna synthesis have not yet been characterized, it is known that mhv negative-strand templates are unstable and turn over during viral replication [ ] . the cis-acting rna elements involved in the different phases of mhv rna synthesis have been studied quite extensively. it has been shown that -and -utr, as well as [ ] . the hatched box represents the common leader sequence and the hatched circle represents the programmed (À ) frameshifting element. the translation products of the genome and sub-genomic mrnas are depicted and the autoproteolytic processing of the orf a and orf a/orf b polyproteins into non-structural proteins nsp to nsp is shown. a number of confirmed and putative functional domains in the non-structural proteins are also indicated: cl, c-like cysteine proteinase; exon, exonuclease; hel, superfamily helicase; mt, s-adenosylmethioninedependent -o-methyl transferase; neu, endoribonuclease; pl , papain-like protease ; pl , papain-like protease ; pol, rna-dependent rna polymerase; x, adenosine diphosphate-ribose - coronaviruses infect both humans and animals and are associated mainly with respiratory and enteric diseases. the recent outbreak of sars emphasizes the need to develop new strategies to control these infections. this paper focuses on the proteins involved in the replication of the coronavirus genome and the production of viral mrnas in the host cell. these so-called replicase-transcriptase proteins are likely to make good targets for the development of anti-coronaviral drugs. the approach used here is to analyze conditional, temperature-sensitive mutants of murine hepatitis virus that are normal at c (the permissive temperature) but are unable to replicate and transcribe viral rnas at . c (the restrictive temperature). by identifying the genetic changes responsible for these temperature-sensitive mutations and by analyzing the precise nature of the defect in rna synthesis at the restrictive temperature, the authors are able to propose a model that describes a pathway for viral rna synthesis in the infected cell. further analysis of these mutants should allow the elucidation of the precise function(s) of the viral proteins involved. -utr-adjacent regions of the genome are required for mhv replication and transcription [ , ] . also, studies on mhv, and other nidoviruses, have shown the critical role of the so-called transcription-regulating sequence (trs) element in the discontinuous phase of the transcription process [ , [ ] [ ] [ ] [ ] . these data show that the stability of the leader-trs/body-trs duplex, which forms during the discontinuous extension phase of negative-strand template synthesis, is an important determinant of subgenomic mrna abundance. however, it is also evident from these studies that the regions flanking the trs elements have a profound affect on the amounts of subgenomic mrnas that are produced. in the context of the discontinuous-extension model [ ] , this is explained as different degrees of ''attenuation'' at each of the trs elements during negative-strand synthesis. in contrast, there is still very little known about the structure, functions, and interactions of viral and cellular proteins in the replicase-transcriptase complex as it is engaged in different modes of rna synthesis. as mentioned above, bioinformatic and biochemical studies have identified a number of (putative) enzymatic activities associated with individual coronavirus replicase proteins, and a number of cellular proteins have also been implicated as components of the mhv replicase-transcriptase complex [ ] [ ] [ ] . however, the essential nature of some of these cellular proteins has been questioned [ ] , and further work is needed to determine the exact protein composition of the coronavirus replicase-transcriptase complex and how the composition is altered, or how the proteins are modified to regulate the different activities of the complex. in order to address these sorts of questions, we have embarked upon a detailed analysis of temperature-sensitive (ts) mutants of mhv-a that are unable to synthesize viral rna when the infection is initiated and maintained at the non-permissive temperature. the essential feature of these mutants is that they are likely to be defective in different aspects of viral rna synthesis and a detailed characterization of their genotype and phenotype should provide insights into the mechanisms of rna synthesis, the functions of individual viral replicase proteins, and the protein-rna and protein-protein interactions that regulate the activity of the replicase-transcriptase complex. these conditional-lethal mutants may also be used in a cis-trans test to define the number of complementation groups, or cistrons, that contribute to a specific phenotype. this sort of analysis can also provide valuable insight into the possible pathways that polyproteins must travel to assume functional configurations and has been used with success for other rna viruses [ ] . the mhv-a mutants that we study have been produced in a number of laboratories over a period of years [ ] [ ] [ ] . they have been selected to have a low efficiency of plaque formation at the non-permissive temperature compared with the permissive temperature and hence a reversion frequency indicative of single point mutations. in this study, we describe a complementation analysis, and by sequence analysis of both ts virus and revertants, we identify the causal mutation for eight of these mutants. we also describe a more detailed phenotype for selected mutants and suggest a model that describes the different modes of rna synthesis during coronavirus replication and transcription. table s lists the ts mutants of mhv-a used in our collection. all the ts mutants failed to form plaques or synthesize viral rna when infection was initiated and maintained at the non-permissive temperature. while many mutants failed to form plaques at c, other mutants formed plaques at c and were considered leaky. this included alb ts that produced pin-prick-sized plaques after d at c (compared with the wild-type [wt] a virus, which produced uniform plaques of - mm in diameter) and wü ts , wü ts , and wü ts , which produced smaller than wt plaques at c. however, even for these mutants, the ts defects responsible for their rna-negative phenotype appeared to be caused by a single point mutation because each ts mutant possessed a characteristic low reversion frequency between À and À per average base [ ] . the virus produced at c by alb ts , wü ts , wü ts , and wü ts was also ts, i.e., the efficiency of plating (eop) was less than À . for most mutants, the revertant virus obtained from plaques formed at the non-permissive temperature had properties identical to wt mhv-a . one exception was alb ts , which produced equal numbers of revertant viruses causing a -sized plaques and revertant viruses with noticeably smaller plaques ( figure s ). we isolated revertant viruses from a large (a -sized) plaque (alb r l ) and a small plaque (alb r s ) for sequence analysis (see below). some of the ts mutants did not produce revertant viruses (e.g., la ts , alb ts ) or produced revertant viruses that were markedly different from the parental mhv-a virus. we began our complementation analyses using alb ts , la ts , and alb ts because they each had a distinct ts viral rna synthesis phenotype (see below). cells were singly infected or doubly infected with two ts mutants and the cells and medium were harvested after the completion of a single round of replication, i.e., h post-infection (hpi) at c. we also confirmed that if infection with a ts virus alone was allowed to proceed for up to h at c, and then the culture shifted to c and the virus harvested at hpi, the titer we obtained was low (; plaque-forming units [pfu]/ml). thus, this protocol prevented the production of revertant virus by a second round of replication. complementation was measured by determining the complementation index (ci) as described in materials and methods. by definition, if the mutations are in the same cistron, the viruses will not complement each other. on the other hand, if the mutations are in different cistrons, the mutants will complement each other and progeny ts virus will be recovered. the results of six individual crosses between alb ts and la ts are shown in table . all of these crosses failed to show complementation. the average ci value was . ( . . sd), which is the theoretical value for two mutants with mutations in the same cistron [ ] . this ci value was obtained using only the titers determined at c and was not corrected for the presence of revertants (or recombinants) as was done by others [ , ] . we found it unnecessary to make this correction because it did not significantly change the ci value (at most a decrease of one tenth) and whether or not the mutants scored as able to complement one another. from these results, we concluded that alb ts and la ts had a mutation in the same cistron and were, therefore, in the same complementation group. we next determined if alb ts would complement alb ts or la ts . as shown in table , in three separate experiments alb ts clearly complemented both alb ts and la ts . therefore, the mutation in alb ts was in a different cistron than the mutations in alb ts and la ts , thus identifying a second complementation group. in a series of further experiments, we extended our complementation analysis to include alb ts , wü ts , wü ts , and wü ts . using the same assay, we found that alb ts complemented alb ts but failed to complement alb ts or la ts . thus, we conclude that alb ts was in the same complementation group as alb ts and la ts . finally, we found that wü ts , wü ts , and wü ts were in a different complementation group(s) from that of either alb ts or alb ts , and thus, these mutants defined at least a third complementation group. in our analysis of the ts mutants of mhv-a described above, values for the ci were always less than two or more than five and thus readily interpreted as positive or negative without correction for the presence of revertants or recombinants. however, from the results we obtained, it was clear that recombination did occur when there was complementation. the eop of the virus harvested from cells co-infected with two complementing viruses was usually ; À , and not the eop of the individual ts mutants, which was À - À . this result is in contrast to similar experiments using sindbis virus in complementation assays, where we obtained similar eops to the input viruses when assaying the progeny from complementing ts mutants (unpublished data). we took these results to indicate that complementation allowed recombination in mhv. this finding provided the means to develop a more convenient and more rapid method of determining complementation for mhv-a ts mutants. we reasoned that because recombination appeared to be driven by complementation, biochemical complementation (i.e., viral rna synthesis) might be detected in cells co-infected with complementing ts mutants, but not in cells infected with ts mutants in the same complementation group. we devised such an assay. cells were infected at the permissive temperature and were then re-fed with medium prewarmed to the non-permissive temperature and containing dactinomycin to inhibit dna-dependent rna synthesis and h-uridine to label viral rna. the infected cells were incubated until - hpi at c to c or - hpi at c, and rna synthesis was measured by the incorporation of h-uridine into acidprecipitable material. figure a shows the results of single and double infection with the alb ts , alb ts , alb ts , and la ts mutants. the data show that at c, the mutants alb ts , alb ts , and la ts were not able to rescue the rnanegative phenotype of each other and thus, the three mutants were in the same complementation group. in contrast, alb ts mutants cells were mock-infected or infected with mhv-a , one of the ts mutants, or with a mixture of two ts mutants. the cells were incubated at c in medium containing dactinomycin and h-uridine and, at hpi, h-uridine incorporation into trichloroacetic acid-precipitated rna was determined. cells were infected with: m, mock-infected; a , mhv-a ; a , alb ts ; a , alb ts ; a , alb ts ; a , alb ts ; l , la ts ; w , wü ts ; w , wü ts ; w , wü ts ; a xa , alb ts and alb ts ; a xl , alb ts and la ts ; a xa , alb ts and alb ts ; a xl , alb ts and la ts ; a xa , alb ts and alb ts ; l xa , la ts and alb ts ; a x a , alb ts and alb ts ; a xl , alb ts and la ts ; a xa or a xa , alb ts and alb ts ; a xw , alb ts and wü ts ; a xw , alb ts and wü ts ; a xw , alb ts and wü ts ; a xw , alb ts and wü ts ; a xw , alb ts and wü ts ; a xw , alb ts and wü ts ; w xw , wü ts and wü ts ; w xw , wü ts and wü ts ; w xw , wü ts and wü ts . doi: . /journal.ppat. .g ts was able to rescue the rna-negative phenotype of alb ts , alb ts , and la ts and thus, was the sole member of a separate complementation group. this result is identical to that obtained using classical complementation assays and served to validate the new method. the assay was as specific as classic genetic complementation, which measures progeny virus production, but is less time-consuming. using this assay, we were able not only to confirm the prediction of at least three complementation groups that were obtained using classical complementation procedures but also to identify a fourth complementation group. the results are presented in figure b and c and show that mutants alb ts , wu ts , wu ts , and wu ts define not one but two additional complementation groups. we found alb ts and wü ts belong to the same complementation group based on their failure to complement each other's defects. however, both of these mutants complemented wü ts and wü ts , which did not complement each other. finally, we extended this assay to include the full collection of mutants that we have available and table summarizes the complementation patterns of the rna-negative ts mutants of mhv-a assayed to date. the numbers shown in table represent the percentage of viral rna synthesis found for the mixed mutant-infected cells compared to a virus-infected cells at c. a value less than zero means the h-uridine incorporation was less than that obtained from mockinfected cells. with this type of assay, we took less than % of the mhv-a incorporation as indicating failure to complement and greater than % as evidence of positive complementation. based on these results, it was possible to assign a further ten mutants (alb ts , ts , and ts , and ts ; ut ts and ts ; la ts and ts ; and nc ts and ts ) to the same complementation group as alb ts , alb ts , and la ts and, it was possible to assign mutant ut ts to the same complementation group as wü ts and wü ts . thus, it was possible to assign the entire collection of rnanegative ts mutants of mhv to one of four complementation groups, which we have tentatively named cistrons i, ii, iv, and vi based on the locations identified for their causal point mutations (see below). this numbering scheme leaves open the possibility of finding two additional complementation groups (cistrons iii and v) in the future that would represent gene products of orf b (see below). the entire coding region of the replicase genes (orf a and orf b) was sequenced for each of eight ts mutant/revertant pairs. in each case, a single nucleotide change was identified as the mutation responsible for the ts mutant phenotype. using the numbering that we have assigned to the infectious cdna clone of the mhv-a genome [ ] (genbank accession number ay ), the nucleotide changes compared to wt mhv-a were identified as: alb ts , figure a) . we also identified a number of nucleotide differences between mutants isolated in different laboratories, but in no case did they correlate with the ts phenotype. with the exception of the alb r s revertant, all of the revertants we isolated were true, i.e., they were genetically and phenotypically identical to the wt mhv-a . the alb r s revertant was a pseudorevertant in that the nucleotide at position had reverted from a! c, which resulted in a substitution of tyr with arg. this radical substitution was reflected in a small plaque phenotype ( figure s ). all of the nucleotide changes responsible for the ts mutant phenotype were non-synonymous mutations. the amino acid substitutions are shown in figure b . conservative substitutions were identified in nsp and nsp of the alb ts and la ts mutants, respectively. moderately conservative substitutions were identified in nsp and nsp of the alb ts and alb ts mutants, respectively. and radical substitutions were identified in nsp of the alb ts and wü ts mutant, as well as nsp of the wü ts and wü ts mutants [ ] . a comparison of the predicted replicase protein sequences from different coronaviruses showed that there was, by and large, conservation of the amino acids that were substituted in the proteins with a ts phenotype. for example, the gln residue of nsp , the his residue of nsp , and the cys residue of nsp appear to be well conserved in group i, ii (including sarscov), and iii coronaviruses. in contrast, the asn residue of nsp is only found in mhv strains, although in the majority of other coronaviruses, it is substituted by an aspartic acid. finally, it is possible, with different degrees of confidence, to predict the structural environment in which the residues in question are found. on the one hand, it is highly likely that the phe residue of nsp is located in an extended area that connects the a-helices b and c in the carboxyl-terminal domain iii of nsp , the c-like proteinase. this conclusion is based upon the similarity in the sequences of coronavirus nsp proteins and the crystallographic structures that have been solved for the transmissible gastroenteritis virus (tgev), sarscov, and hcov- e nsp proteins [ , , ] . on the other hand, programs that predict protein secondary structure [ ] indicate that the gln residue of nsp , the his residue of nsp , and the cys residue of nsp are located in disordered loop structures, while the asn residue of nsp and the leu residue of nsp are involved in a-helices. obviously, more definitive structural data will be needed to confirm these predictions. we focused our phenotypic analysis on the eight mhv-a ts mutants that had been genotyped and began by measuring ''total'' viral rna synthesis in infected cells prior to and following shift from the permissive to the non-permissive temperature. this analysis was done after h of incubation at c, a time at which the replicase-transciptase complex produces mainly (. %) positive-strand rna, and ; % of the maximum rate of rna synthesis has been reached. mutant virus-infected cells were shifted to c at hpi and a duplicate set was left at c. both sets of cultures were labeled for h with h-uridine in the presence of lg per ml of cycloheximide (ch) to monitor the replicase-transcriptase activity at the time of shift. the results are shown in figure . in mhv-a infected cells, the amount of huridine incorporation doubled, as expected, when the temperature was increased by c. the group i mutants had about the same level of viral rna synthesis at both temperatures, while in the group ii, iv, and vi mutantinfected cells, viral rna synthesis diminished by % or more in the hour following temperature shift. we interpret this to mean that mutations in replicase proteins encoded in orf a appeared to confer temperature-sensitivity to the viral replicase-transcriptase complex, but once it had formed at c, its positive-strand synthetic activity was relatively resistant to higher temperature. in contrast, mutations in orf b-encoded proteins, namely nsp , nsp , and nsp appeared to affect the positive-strand synthetic activity of already-formed replicase-transcriptase complexes. we then went on to analyze the phenotypes of three ts mutants in more detail. alb ts . the phenotype described above for group ii, iv, and vi mutants would be consistent with a defect in any stage of positive-strand rna synthesis. in the case of mutant alb ts , however, we have shown that the ts lesion is located in nsp , the viral rna-dependent rna polymerase subunit. this suggested to us that the alb ts might be defective in the elongation phase of rna synthesis. to analyze the phenotype of alb ts in more detail, rna synthesis in alb ts -infected cells was determined using h pulse labels with h-uridine in the presence of dactinomycin, given between - hpi at c or between - hpi at c ( figure a ). at c, alb ts -infected cells incorporated only mock levels of h-uridine, as expected for an rna-negative ts mutant. in contrast, cells infected with wt mhv-a or with alb r (a revertant of alb ts ) made rna at high rates and at identical times. at c, alb ts was defective in viral rna synthesis and never reached the levels of viral rna synthesis shown by wt mhv-a or alb r. these results are consistent with our finding that, at c, the plaques formed by alb ts were smaller that those formed by wt mhv-a . analysis by gel electrophoresis of the species of positive-strand rna made in alb ts -infected cells at c showed the typical pattern of seven rnas (genome and six subgenomic mrnas), although the six subgenomic mrnas were reduced equally in amount relative to the genome rna when compared to alb r infected cells (unpublished data). we conclude that alb ts not only produced less overall rna compared to wt mhv-a and alb r, even at the permissive temperature, but also under-produced all of the subgenomic mrna species relative to the genome rna. we also examined the ability of alb ts -infected cells to continue viral rna synthesis after shift from c to c at hpi ( figure b ). this allowed us to follow the activity at c of the viral rna-dependent rna polymerase that was made and assembled at c. at this time, alb ts rna synthesis was at its maximum rate and rna synthesis by wt mhv-a and alb r was declining. the results show that a shift to c led to the rapid loss of rna synthesis by alb ts but not by wt mhv-a or alb r. this result is consistent with a failure of the viral rna-dependent rna polymerase to continue transcription at the non-permissive temperature. we concluded alb ts had a ts defect in elongation, although we do not know if elongation is directly affected or if the amino acid change in nsp affects its interaction with an as yet unknown, but essential protein. we have also shown that, as expected, alb ts is unable to synthesize negative-strand rna at the non-permissive temperature (unpublished data). alb ts and la ts . although both alb ts and la ts are unable to synthesize viral rna when the infection is initiated and maintained at the non-permissive temperature, the data shown in figure suggests that they are not significantly impaired in their ability to synthesize positive-strand rna at this temperature. this conclusion is strengthened by the results shown in figure a , which demonstrate the kinetics of overall viral rna synthesis in alb ts and la ts virusinfected cells after shifting the incubation temperature from c to c at hpi. with wt mhv-a , viral rna synthesis increased rapidly within the first min after temperature shift, consistent with the synthesis of both additional negative-strand templates and their nascent positive-strand product. the addition of ch at the time of shift resulted in a constant rate of viral rna synthesis for at least h. as we know that negative-strand synthesis in mhv-a -infected cells is short-lived and stops within min of the inhibition of protein synthesis [ ] , we deduce that the addition of ch prevented the synthesis of new viral proteins, which in turn prevented the formation of additional replicase-transcriptase activity and negative-strand templates. in cells infected with complementation group i ts mutants alb ts and la ts , viral rna synthesis continued at c at the level ongoing at the time of temperature shift ( figure a ). this meant that the replicase-transcriptase complexes assembled at c continued to function at c in the synthesis of positive-strand rna. however, unlike a infected cells, the group i mutants did not increase their rates of rna synthesis after shift to non-permissive temperature, indicating that no new active complexes were formed. this phenotype resembled that seen with mhv-a -infected cells treated with ch, and we conclude that the complementation group i mutants are defective in their ability to form active replicase-transcriptase complexes at c but retain the positive-strand synthesis activity of the complexes formed at the permissive temperature. at least two possibilities could account for a failure of group i ts mutants to form fully competent replicasetranscriptase complexes at the non-permissive temperature. either no new negative-strand templates were made, i.e., a defect in negative-strand synthesis, or, if they were made, they could not be used as templates for positive-strand synthesis. the latter phenotype has been observed for certain alphavirus mutants [ ] , which were called conversion-defective mutants. to distinguish between these two possibilities, it is necessary to shift the ts mutant-infected cells to the nonpermissive temperature and determine their ability to continue negative-strand rna synthesis. mutants that fail to continue negative-strand rna synthesis would be defective in this step, while mutants that continued to make negative strands would be designated as conversion-defective mutants. cells infected with wt mhv-a , alb ts , and la ts were shifted from c to c at h (alb ts ) or h (la ts ) post-infection and were pulse-labeled with h uridine at c. then, viral negative-strand templates in replicating and transcribing structures were purified free of single-stranded rna, and the incorporation of radioactivity into negativestranded rna was measured by nuclease protection assays. in this assay, the results are expressed as the percentage of the h-uridine incorporated into the negative-stranded component of the purified, nuclease-resistant rna cores of the replicative-transcriptive structures. as these structures represent double-stranded rna, if %- % of the total incorporation in the core rna is found in negative strands, it means that %- % of the negative strands that were active as templates during the pulse period had been made during this same period. this occurred when negative-strand synthesis was measured early in the infection cycle, when viral rna synthesis was ; % of the maximum [ ] . figure b shows that in wt mhv-a -infected cells, negative-strand synthesis continues following a shift from c to c at a time when the amount of viral rna synthesis is ; % of maximum. this is seen by the similar high values of %- % of the labeled rf rna being in negative strands for successive min pulse-periods in the absence of ch. also, as shown previously, continued negativestrand synthesis in mhv-a -infected cells is dependent on continued translation and abruptly declines in the presence of ch. in the case of la ts , the percentage of h-uridine incorporated in negative strands declined abruptly after shifting to c and this decline was the same in the absence or the presence of ch. with alb ts , negative-strand synthesis continued during the - min and the - min pulse-periods in the absence of ch but was inhibited in the presence of ch. for this mutant, to find that negativestrand synthesis continued at c without an increase in the rate of positive-strand synthesis, as seen for mhv-a , was consistent with alb ts having a ts defect affecting the ability of the negative-strand templates to be efficiently used at c. thus, we conclude that la ts was defective in continuing negative-strand synthesis after shift to c and alb ts displayed what appears to be a conversion phenotype. taken together with the complementation analysis, the identification of the mutations responsible for the ts phenotypes of alb ts , alb ts , alb ts , alb ts , la ts , wü ts , wü ts , and wü ts leads to a number of important conclusions. first, our data strongly suggest that most of the replicase gene products of orf a are cis-active and form a single complementation group (cistron i) encompassing, at least, the nsp to nsp coding region. if correct, our conclusion must mean that a large proportion of nsp -nsp proteins function as a polyprotein, if only initially or transiently, or they associate as a cis-acting complex before they are proteolytically processed. we favor a model in which a pp a-related polyprotein represents a large modular scaffolding protein that displays binding sites for orf bencoded pp ab processing products. while the large number of mutants that fall into cistron i clearly suggest that it is extensive and polygenic, it is not yet clear if all of the orf aencoded proteins function in cis. we are aware that the arterivirus equine arteritis virus orf a-encoded protein nsp can function in trans [ ] and it has recently been shown that the mhv-a orf a-encoded protein nsp is nonessential for virus replication [ ] . the genetic analysis of further mhv-a ts mutants will be needed to define the precise boundaries of mhv-a cistron i. second, our results suggest that the replicase gene products encoded in orf b (i.e., nsp -nsp ) are diffusible and thus assemble and function in viral rna synthesis after cleavage from pp ab. this also leads us to the prediction that there will be five cistrons in orf b, each corresponding to one of the proteolytic cleavage products, and we have designated them tentatively as cistrons ii-vi in a to direction (nsp , cistron ii; nsp , cistron iii; nsp , cistron iv; nsp , cistron v; and nsp , cistron vi). the idea that the mhv-a orf b-encoded replicase proteins function in trans is consistent with the results of brockway et al., who have shown that a green fluorescent protein-tagged mhv-a nsp is able to diffuse into the replicase-transcriptase complex if expressed individually in virus-infected cells [ ] . however, we would also like to note that our data does not exclude the possibility that some of the orf b-encoded proteins may function as intermediates, rather than the end products of proteolytic cleavage. for example, functional proteins comprising nsp /nsp , nsp /nsp , nsp /nsp , nsp /nsp as well as nsp /nsp /nsp could all be accommodated as single cistrons based upon our complementation data. this would lead to the prediction of either three or four cistrons encoded in orf b. the idea that a number of the enzymes involved in coronavirus rna synthesis may be linked not only functionally, i.e., sequentially in a concerted reaction pathway, but also structurally (i.e., as multifunctional proteins) is also suggested by other studies. for example, ziebuhr and colleagues [ ] have shown that -o-ribose-methylated rna substrates are resistant to cleavage by the sars-coronavirus endoribonuclease (nsp ), indicating a functional link with the s-adenosylmethionine-dependent -o-methyl transferase (nsp ). we are currently searching for further ts mutants that might help resolve this issue and we are attempting to trans-complement ts mutants with cell lines that constitutively express orf b-encoded replicase proteins. despite these reservations, the genetic data do allow us to conclude that not only nsp , the c-like cysteine proteinase, and nsp , the putative rna-dependent polymerase (as might have been predicted), but also nsp , the putative mhv exonuclease, nsp , the putative mhv -o-methyltransferase, nsp , and nsp are essential for the assembly of a functional replicase-transcriptase complex. in contrast to most other positive-stranded rna virus, the viral replicase-transcriptase complex of coronaviruses (and most other nidoviruses) functions to amplify the genome via a full-length negative-strand intermediate and to produce, via a discontinuous process, subgenome-length negative-strand templates that are then copied directly into subgenomic mrna. how the replicase-transcriptase complex accomplishes these various activities is not understood in any detail. for example, it is not known whether the same replicase-transcriptase complex functions to produce fulllength and subgenome-length rna or how the conversion from negative-to positive-strand rna synthesis is regulated. does the analysis of mhv-a ts mutants help us to understand these complex processes? we have shown previously that negative-and positivestrand rna synthesis occurs simultaneously throughout mhv-a infection but that negative-strand synthesis is short-lived, i.e., its synthesis halts within several minutes after protein synthesis is inhibited [ ] . this contrasts with positive-strand synthesis, which continues unabated for h and then gradually declines and disappears about h after the inhibition of translation. these observations suggest that unprocessed forms of the replicase polyprotein(s) might function in negative-strand synthesis and that cleavage of the nascent polyprotein inactivates the negative-strand activity of the replicase, as it does for alphaviruses [ , ] . the replicasetranscriptase activity for positive-strand synthesis can be restarted after the block of translation is reversed [ ] but, for this to happen, new negative-strand templates need to be synthesized. in other words, it appears that the coronavirus replicase-transcriptase complex ages, losing both its negativestrand templates and its activity. this interpretation fits well with our genetic analysis of the mutants la ts , alb ts , and alb ts , which shows that they all fall into a single complementation group. it is also consistent with our proposal that the replicase proteins encoded in orf a are expressed and function as a polyprotein, or that they assemble as a cis-acting complex before they are proteolytically processed. it is also worth noting that in vivo protein labeling experiments indicate that proteolytic processing of both mhv-a orf a and mhv-a orf b-encoded replicase proteins is measured in hours rather than minutes [ ] [ ] [ ] and that the fully processed c-like cysteine proteinase is first detected several hours post-infection [ ] , a time at which the rate of viral rna synthesis is already increasing rapidly [ ] . the idea that the mhv replicase-transcriptase complex is active in negative-strand rna synthesis before pp a is extensively processed also fits well with our detailed phenotypic analysis of cistron i mutants. in the case of la ts , negative-strand synthesis was inhibited after shift to the non-permissive temperature and, in time, this leads to a decline in positive-strand rna synthesis (unpublished data). thus, at the non-permissive temperature, la ts could not sustain positive-strand synthesis, nor replace or replenish aging replicase-transcriptase complexes. the causal mutation in la ts would substitute a glu for the gln residue of wt nsp . as noted above the gln residue is conserved in group i, ii (including sarscov), and iii coronaviruses and its substitution with glu might prevent the proper folding of pp a into a conformation that would allow it to participate in the formation of a replicase-transcriptase complex with negative-strand activity. it would be interesting to determine if, at the non-permissive temperature, nsp of la ts could associate with nsp , nsp , nsp , nsp , or nsp . also, it was curious that la ts had a very low reversion frequency of ; À . why certain bases fail to revert at the typical frequency of À to À is unknown but may be indicative of a region of the genome that is transcribed with higher fidelity than other regions. alternatively, this low reversion frequency may be an inherent property of the la ts replicase-transcriptase complex. in contrast to la ts , alb ts appeared to be able to continue to form negative strands after shift to the nonpermissive temperature, but these negative strands were not converted into templates for positive-strand synthesis. we speculate that alb ts has a ts defect in the conversion of the replicase-transcriptase complex from one able to synthesize negative strands to one able to synthesize positive strands. it is certainly suggestive that alb ts had a mutation in nsp , which is the c-like proteinase of the virus, but it has yet to be determined if this mutation affects the activity of the proteinase, or if it affects the folding of pp a or pp ab, or if the nsp c-terminal domain itself could have a function in positive-strand rna synthesis. nevertheless, because negative-strand rna synthesis was inhibited in alb ts -infected cells treated with ch at the time of shift to non-permissive temperature, we propose that the alb ts replicase-transcriptase complex does not retain its activity for minus-strand synthesis. rather it fails to gain positive-strand synthesis activity at the non-permissive temperature. we favor a model where the activity that makes positive strands is gained at the expense or loss of the activity to make negative strands. finally, although we are able to rationalize the genotype of alb ts , i.e., a mutation in nsp (the rna dependent rna polymerase) with its phenotype (i.e., an immediate effect on rna synthesis at the non-permissive temperature) we were surprised to find that alb ts , wü ts , wü ts , and wü ts also showed the same phenotype but had mutations in other replicase proteins. generally, it is unusual to find so many ts mutants that show an effect on rna synthesis if the replicasetranscriptase complex is first allowed to assemble at the permissive temperature. most rna-negative ts mutants of alphaviruses, for example, fail to make viral rna when the infection is initiated at the non-permissive temperature but continue to make viral rna if shifted to the non-permissive temperature late in infection (unpublished data). one possibility is that nsp and nsp dissociate or become less tightly associated with the replicase-transcriptase complex after shifting to the non-permissive temperature and this causes the complex to lose elongation activity. another possibility is that the enzymatic activities associated with nsp and nsp are altered in the group iv and group vi mutants. further studies will be required to explain this phenotype. in summary, our detailed phenotypic analysis of mhv-a ts mutants allows us to propose a working model that describes a pathway for viral rna synthesis in mhv-a infected cells. in this model, the replicase-transcriptase complex forms initially and creates a negative-strand template. it is then converted to utilize the negative strand as a template for positive-strand synthesis and, finally, the complex is inactivated by the degradation of negative-strand templates (figure ). our analysis also allows us to place some of our ts mutants at specific points on this pathway. we hope that a more detailed biochemical analysis of these mutants will allow us to identify intermediates in the pathway of rna synthesis and will provide valuable information of the precise function(s) of the viral replicase proteins involved. furthermore, we believe that the characterization of these mutants provides an excellent starting point for the generation of second site reversion mutants. this could be done, for example, by using the recently developed mhv reverse genetic system [ ] to generate ts mutants with codon, rather than nucleotide substitutions. second site reversion mutants may then provide valuable information on protein-protein interactions within the replicase-transcriptase complex. cells and viruses. seventeen clone one ( cl- ) mouse fibroblast cells [ ] were cultured at c in dulbecco's modified eagle's medium (dmem) supplemented with % fetal bovine serum (fbs), % tryptose phosphate broth (tpb), penicillin ( units/ml), and streptomycin ( lg/ml). sac(À) cells [ ] were cultured at c in minimal essential medium (mem) supplemented with % fbs, penicillin ( units/ml), and streptomycin ( lg/ml). the a strain of mhv and a set of ts mutants derived from mhv-a (alb prefix) were originally obtained from the laboratory of l. sturman, wadsworth center for laboratories and research, albany, new york, united states [ ] . mutants prefixed with la (los angeles) and nc (north carolina) were obtained from m. schaad and r. baric, university of north carolina, chapel hill, north carolina, united states and have been initially characterized [ ] . mutants prefixed with ut (utrecht) were obtained from w. spaan, leiden university medical center, leiden, the netherlands and have been initially characterized [ ] . the la, nc, and ut ts mutants were derived from different but related lineages of the albany isolate of mhv-a . mutants prefixed with wü (wü rzburg, germany) were isolated as described below. for our studies, virus stocks were derived from the original mutant isolates after plaque purification and propagation in cl- cells cultured at c or c in low ph dmem (ph . ) containing % fbs, % tpb, penicillin ( units/ml), and streptomycin ( lg/ml) [ ] . revertants were picked from plaques of mutants titered at . c, followed by another plaque purification at . c. the virus stocks used were first passage and were obtained by using virus from a single plaque (; pfu) to infect a dish of ; cl- cells to yield ml of stock virus with a titer of - pfu/ml. isolation of wü ts mutants. sac(À) cells were infected with pfu/ cell of mhv-a (originally obtained from p. carthew, medical research council laboratories, carshalton, united kingdom), and incubated for h at c in medium containing lg/ml of fluorouracil. this concentration of pyrimidine analogue was determined to inhibit virus replication by %. the mutagenized virus stock was diluted to pfu/ml in medium and ll aliquots were incubated with sac(À) cells at c for h. the supernatant was taken from cultures that displayed syncytium formation and used to infect duplicate cultures of sac(À) cells that were incubated at c or . c for h. the supernatant was taken from replica cultures that developed cytopathic effect at c but not at . c, and potential ts mutants were isolated by two plaque purifications. sequence analysis of the wü rzburg strain of mhv-a suggests that approximately , nucleotides at the end of the genome have been exchanged by recombination with a related but different mhv strain (unpublished data). characterization of mutant stocks for titer and eop. cl- cells in mm petri dishes were infected with . ml of -fold dilutions of figure . a model to describe the pathway for viral rna synthesis in mhv-a -infected cells shows a working model that describes a pathway for viral rna synthesis in mhv-a -infected cells. the model proposes that the replicasetranscriptase complex forms initially and creates a negative-strand template. it is then converted to utilize the negative strand as a template for positive-strand synthesis and, finally, the complex is inactivated by the degradation of negative-strand templates. it is also proposed that proteolytic processing of the replicase polyproteins plays a role in regulation of this pathway. also depicted are the putative defects of specific mhv-a ts mutants. it remains to be shown whether or not the group iv and vi mutants (wü ts , alb ts , wü ts , and wü ts ) are defective in negative-strand rna synthesis at the non-permissive temperature. doi: . ] ). the inoculum was removed after min and the cells were overlaid with dmem containing % fbs, penicillin ( units/ml), streptomycin ( lg/ml), and . % gelritee gellan gum (sigma, st. louis, missouri, united states) and incubated at the appropriate temperature in . % co . after incubation for or d at c or d at c or . c, cells were rinsed with . m nacl, fixed with methanol, and stained with a solution of . % toluidine blue, . % azure blue, and % boric acid. the eop was calculated by dividing titers at . c by the titer at c. we found that all of the ts mutants produced the same titer at c as at c and in all cases c was non-permissive for plaque formation of the ts mutants. complementation analysis. classic or genetic complementation was done by infecting cl- cells in mm petri dishes either singly with each mutant or doubly with two mutants at a multiplicity of infection (moi) of - pfu/cell. after incubation at room temperature or c for min, the virus inoculum was removed and the infected cells were rinsed with hbss and re-fed with low ph dmem or dmem supplemented with % fbs, penicillin ( units/ml), and streptomycin ( g/ml). the infected cells were incubated at . c or c in % co . one hour later the cells were rinsed again and re-fed with warmed medium and the dishes returned to the incubator until hpi. the medium was harvested, clarified at , rpm for min, and virus titers were determined by plaque assays at c. cis were calculated using the following formula: a ci greater than two between mutant pairs was consistent with complementation, i.e., -fold difference above background, while a ci less than two was negative for complementation [ ] . biochemical complementation was done by mock-infecting or infecting cl- cells at pfu/cell with mhv-a , or one of the ts mutants, or with a mix of pfu/cell each of two ts mutants. after the adsorption period at room temperature for min, the virus inoculum was removed, ml of prewarmed medium containing dactinomycin and h-uridine ( . mbq/ml) was added and the cells were incubated at c. the wt and the ts mutant-infected cells were harvested at hpi and the h-uridine incorporation into trichloroacetic acid-precipitated rna was determined. viral rna synthesis. viral rna synthesis was measured by determining the amount of h-uridine incorporated in the presence of dactinomycin ( lg/ml) into acid-precipitable material. [ - h] uridine (! . tbq/mmol) was added to the medium at either . or . mbq/ml. after incubation, the radioactive medium was removed and the cells dissolved with % lithium dodecyl sulfate and lg/ml proteinase k in leh buffer ( . m licl, . m edta, . m hepes, [ph . ]) at - cells per ml. the dna was sheared by repeated passage through a -gauge needle attached to a -ml tuberculin syringe. triplicate samples of cells were precipitated with trichloroacetic acid and the precipitates collected on glass fiber filters (whatman incorporated, clifton, new jersey, united states), dried under a heat lamp, and the radioactivity determined by liquid scintillation spectroscopy. to measure negative-strand synthesis, the dissolved cells were extracted with low ph phenol (ph . ), which removed dna from the aqueous phase, and then with cholorofom:isoamyl alcohol ( : ), and the rna was precipitated with ethanol. rf rna was generated by treatment of the rna with rnase t ( u/ug rna, c for min in . m nacl) and collected by chromatography on cf- cellulose and ethanol precipitation. the incorporation of h-uridine into negative strands was measured by denaturing the rf rna with heat and annealing in the presence of . -fold excess of unlabeled rna obtained from purified virions of mhv [ ] . isolation of viral rna. two different procedures were used to obtain viral rna for rt-pcr and sequencing. virions were purified from ; cl- cells that had been infected at a moi of ; pfu/cell and incubated in low ph dmem at - c. the medium from the infected cells (; ml) was collected at hpi and clarified at , rpm for min. the virions were pelleted by centrifugation at , rpm for h at c. the virus pellet was allowed to suspend overnight on ice in . ml/tube of . m nacl and mm hepes (ph . ). the suspended virus from six tubes was pooled and layered over one sw tube containing a linear gradient of % potassium tartrate (bottom) and % glycerol (top), in . . m hepes (ph . ). after centrifugation at , rpm for - h at c, the visible band containing the virions was collected, diluted, and pelleted by centrifugation at , rpm for h at c. the pelleted virions were suspended in . m nacl and mm hepes (ph . ), and lids and proteinase k were added to % and lg/ml, respectively, after incubation at c for min, the viral rna was extracted with phenol followed by chloroform:isoamylalcohol ( : ). viral rna was ethanol-precipitated and the pellet was washed with % ethanol, dried under vacuum, and resuspended in water. alternatively, cl- cells were infected with virus, incubated for h at c to c in . % co . the poly(a)-containing rna was then isolated from the infected cells using oligo-dt dynabeads as described previously [ ] . rt-pcr and sequencing. the entire replicase gene-coding region (orf a and orf b) was sequenced for eight ts mutant and revertant pairs. to do this, we used a set of synthetic oligonucleotides that are complementary to sequences spaced at approximately nucleotide intervals along the positive-and negative-strand copies of the viral rna (sequences available on request). five oligonucleotides, p , p , p , p , and p , were used to prime the rt of viral rna with superscript ii rt (invitrogen, carlsbad, california, united states). the reaction mix ( ll), which contained, in addition to presupplied buffer, ng of primer, - ng of viral rna, mm dntps, mm dtt, u of rnaguard (amersham, little chalfont, united kingdom), and u of reverse transcriptase, was incubated at c for min and then at c for min. the five cdna templates were then amplified using eight primer pairs, p /p , p / p , p /p , p /p , p /p , p /p , p /p , and p /p , and thermostable, recombinant taq dna polymerase. the reaction mix ( ll), which contained, in addition to pre-supplied buffer, ng of primer pair, ll of rt reaction product, lm dntps, mm mgcl , and . u of dna polymerase, was incubated at c for min, then c for s, c for s, c for min, for a total of cycles and a final -min extension at c. the pcr reaction products were purified by ethanol precipitation using ammonium acetate. finally, sequence analysis was done using primers p -p and standard cycle sequencing methods. sequencing reaction mixes ( ll), which contained ng of primer, ng of pcr product, and ll of cycle sequencing mix (bigdye terminator v. . , applied biosystems, foster city, california, united states), were incubated at c for s, c for s, and c for min, for a total of cycles. the reaction products were purified by retention on a size exclusion membrane (montagee seq , millipore, billerica, massachusetts, united states) as described by the manufacturer; eluted and analyzed with an abi prism genetic analyzer. computer-assisted analysis of sequence data was done using the lasergene biocomputing software (dnastar). figure s . plaque morphology of alb ts revertants the plaque morphologies of alb ts l and alb ts s are illustrated. alb ts had a reversion (back mutation) frequency of À and there was a mixture of large and small plaques at c. the virus from the small and large plaques produced progeny that formed uniformly small or large plaques at c, respectively. at c, both r l and r s produced plaques of equal diameter and alb r l produced the same size plaques at c as the parental or wt mhv-a . found at doi: . /journal.ppat. .sg ( . mb ppt). coronaviruses, toroviruses, and arteriviruses characterization and complete genome sequence of a novel coronavirus, coronavirus hku , from patients with pneumonia identification of a new human coronavirus sars-beginning to understand a new virus coronaviruses use discontinuous extension for synthesis of subgenome-length negative strands regulation of relative abundance of arterivirus subgenomic mrnas reverse genetic analysis of the transcription regulatory sequence of the coronavirus transmissible gastroenteritis virus the primary structure and expression of the second open reading frame of the polymerase gene of the coronavirus mhv-a ; a highly conserved polymerase is expressed by an efficient ribosomal frameshifting mechanism characterization of the expression, intracellular localization, and replication complex association of the putative mouse hepatitis virus rna-dependent rna polymerase virus-encoded proteinases and proteolytic processing in the nidovirales unique and conserved features of genome and proteome of sarscoronavirus, an early split-off from the coronavirus group lineage identification of the catalytic sites of a papain-like cysteine proteinase of murine coronavirus identification and characterization of a serine-like proteinase of the murine coronavirus mhv-a the severe acute respiratory syndrome coronavirus nsp protein is an endoribonuclease that prefers manganese as a cofactor adp-ribose- "-monophosphatase: a conserved coronavirus enzyme that is dispensable for viral replication in tissue culture major genetic marker of nidoviruses encodes a replicative endoribonuclease multiple enzymatic activities associated with severe acute respiratory syndrome coronavirus helicase human coronavirus e nonstructural protein : characterization of duplex-unwinding, nucleoside triphosphatase, and rna -triphosphatase activities expression, purification, and characterization of sars coronavirus rna polymerase a complex zinc finger controls the enzymatic activities of nidovirus helicases the nsp replicase protein of sars-coronavirus, structure, and functional insights the severe acute respiratory syndrome-coronavirus replicative protein nsp is a single-stranded rna-binding subunit unique in the rna virus world the crystal structures of severe acute respiratory syndrome virus main protease and its complex with an inhibitor coronavirus minus-strand rna synthesis and effect of cycloheximide on coronavirus rna synthesis coronavirus transcription: subgenomic mouse hepatitis virus replicative intermediates function in rna synthesis the rna structures engaged in replication and transcription of the a strain of mouse hepatitis virus mouse hepatitis virus minus-strand templates are unstable and turn over during viral replication analysis of cis-acting sequences essential for coronavirus defective interfering rna replication characterization of the rna components of a putative molecular switch in the untranslated region of the murine coronavirus genome the stability of the duplex between sense and antisense transcription-regulating sequences is a crucial factor in arterivirus subgenomic mrna synthesis sequence motifs involved in the regulation of discontinuous coronavirus subgenomic rna synthesis regulation of coronavirus mrna transcription identification of a non-canonical signal for transcription of a novel subgenomic mrna of mouse hepatitis virus: implication for the mechanism of coronavirus rna transcription polypyrimidine tract-binding protein binds to the complementary strand of the mouse hepatitis virus untranslated region, thereby altering rna conformation heterogeneous nuclear ribonucleoprotein a binds to the -untranslated region and mediates potential - -end cross talks of mouse hepatitis virus rna mitochondrial aconitase binds to the untranslated region of the mouse hepatitis virus genome evaluation of the role of heterogeneous nuclear ribonucleoprotein a as a host factor in murine coronavirus discontinuous transcription and genome replication clustered charged-to-alanine mutagenesis of poliovirus rna-dependent rna polymerase yields multiple temperature-sensitive mutants defective in rna synthesis genetics of mouse hepatitis virus transcription: identification of cistrons which may function in positive and negative strand rna synthesis temperature-sensitive mutants of mhv-a temperature-sensitive mutants of mouse hepatitis virus strain a : isolation, characterization, and neuropathogenic properties the rate and character of spontaneous mutation in an rna virus complementation between temperaturesensitive mutants of sindbis virus genetic complementation among three panels of mouse hepatitis virus gene mutants recombinant mouse hepatitis virus strain a from cloned, full-length cdna replicates to high titers in vitro and is fully pathogenic in vivo amino acid substitution matrices from protein blocks structure of coronavirus main proteinase reveals combination of a chymotrypsin fold with an extra alpha-helical domain coronavirus main proteinase ( clpro) structure: basis for design of anti-sars drugs phd: predicting one-dimensional protein structure by profile-based neural networks sindbis virus rna-negative mutants that fail to convert from minus-strand to plus-strand synthesis: role of the nsp protein a zinc fingercontaining papain-like protease couples subgenomic mrna synthesis to genome translation in a positive-stranded rna virus the nsp replicase proteins of murine hepatitis virus and severe acute respiratory syndrome coronavirus are dispensible for virus replication major genetic marker of nidoviruses encodes a replicative endoribonuclease the effect of loss of regulation of minusstrand rna synthesis on sindbis virus replication the effect of overproduction of nonstructural proteins on alphavirus plus-strand and minus-strand rna synthesis processing of the coronavirus mhv-jhm polymerase polyprotein: identification of precursors and proteolytic products spanning kilodaltons of orf a mouse hepatitis virus c-like protease cleaves a -kilodalton protein from the open reading frame a polyprotein in virus-infected cells and in vitro the putative helicase of the coronavirus mouse hepatitis virus is processed from the replicase gene polyprotein and localizes in complexes that are active in viral rna synthesis characterization of a human coronavirus (strain e) c-like proteinase activity enhanced growth of a murine coronavirus in transformed mouse cells nonproducer malignant tumor cells with rescuable sarcoma virus genome isolated from a recurrent moloney sarcoma effective amplification of -kb dna by reverse transcription pcr we would like to thank barbara schelle and tamara jones for technical help. key: cord- -mk mzc z authors: morris, cindy e.; bardin, marc; kinkel, linda l.; moury, benoit; nicot, philippe c.; sands, david c. title: expanding the paradigms of plant pathogen life history and evolution of parasitic fitness beyond agricultural boundaries date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: mk mzc z nan how do pathogens, whether they parasitize plants or animals, acquire virulence to new hosts and resistance to the arms we deploy to control disease? the significance of these questions for microbiology and for society at large can be illustrated by the recent worldwide efforts to track and limit the emergence of human transmissible strains of swine and avian influenza virus and of multidrug-resistant lines of human pathogenic bacteria, and to restrain the spread of ug , a strain of stem rust of wheat. recent research in medical epidemiology has elucidated the impact of pathogen ecology in environmental reservoirs on the evolution of novel or enhanced pathogen virulence. in contrast, the evolution of virulence in plant pathogens has been investigated from a predominantly agro-centric perspective, and has focused overwhelmingly on evolutionary forces related to interactions with the primary plant host. here, we argue that current concepts from the field of medical epidemiology regarding mechanisms that lead to acquisition of novel virulence, biocide resistance, and enhanced pathogenic fitness can serve as an important foundation for novel hypotheses about the evolution of plant pathogens. we present numerous examples of virulence traits in plant pathogenic microorganisms that also have a function in their survival and growth in nonagricultural and nonplant habitats. based on this evidence, we make an appeal to expand concepts of the life history of plant pathogens and the drivers of pathogen evolution beyond the current agro-centric perspective. the classification of diseases in terms of their epidemiology is a useful starting point for a comparison of plant and human pathogens [ ] . in medical epidemiology, anthroponoses are diseases trans-mitted among humans that have no other known reservoirs for multiplication. typhoid fever, smallpox, and certain venereal diseases are examples. zoonoses, such as rabies, lyme disease, severe acute respiratory syndrome (sars), and avian and swine influenzas, are transmitted to humans from living animals. sapronoses are diseases transmitted to humans from environmental reservoirs where the pathogen thrives saprophytically. these habitats include soil, water, and decaying plant and animal matter. examples include legionnaire's disease, cholera, aspergillosis, and the emerging epidemics of melioidosis (burkholderia pseudomallei). human pathogens with saprophytic phases or residing in environmental reservoirs are also referred to as ''environmental pathogens'' [ ] [ ] [ ] [ ] [ ] . studies of virulence factors of human pathogens in environmental reservoirs have begun to reveal the importance of alternate hosts, of dual-use virulence factors, and in general of how environmental habitats can select for traits that confer enhanced fitness as human pathogens. for example, interactions with microbial eukaryotes seem to have led to the acquisition of traits useful for pathogenicity to mammalian cells. numerous environmental pathogens, including cryptococcus neoformans, legionella spp., chlamydophila pneumoniae, mycobacterium avium, listeria monocytogenes, pseudomonas aeruginosa, and francisella tularensis, might have acquired virulence traits via their resistance to predation by amoebae. this resistance, associated with the ability to grow inside the amoebae-which are essentially alternate hosts-has likely led to the selection of traits conferring survival in macrophages [ ] . resistance to macrophages involves the capacity of the bacteria to resist or debilitate the macrophage's phagosomes and to multiply in the cytoplasm. many of the traits essential for virulence to humans likewise seem to play roles in adaption to the environments where the organisms are saprophytes (table ). these traits have dual roles in environmental and parasitic fitness and are thus referred to as ''dualuse traits''. melanins, siderophores, and the capacity to form biofilms are among the frequently cited examples. c. neoformans provides one of the richest examples of dual-use traits. this fungus, frequently found in soils that contain high levels of bird guano and in association with certain plants, causes meningoencephalitis. a nonexhaustive list of its dual-use traits includes capsule formation and production of melanin, laccase, phospholipase, proteases, and ureases [ ] . in the environment these traits contribute to survival and in human hosts they contribute to the capacity of c. neoformans to avoid host resistance mechanisms and to attack host tissue. microbial efflux pumps have also evolved dual uses. these transport systems are used for managing toxic compounds in the environment of the microorganism and can have a broad spectra of activity leading to multidrug resistance among environmental microorganisms [ ] . human activities resulting in the disposal of a wide range of chemical products into the environment, including household cleaners that contain the broad spectrum antimicrobial triclosan, may be inadvertently exacerbating the abundance of multidrug-resistant bacteria [ ] . virulence of environmental pathogens has been described as a set of cards, or a diverse set of attributes acquired as a function of the life history of a pathogen and its adaptation to different environments [ , ] . it is becoming increasingly clear that evolutionary forces outside the context of human-pathogen interactions are responsible for the acquisition and maintenance of some virulence factors [ ] . genomics and phylogenetics are revealing the evolutionary link between, for example, commensal strains of escherichia coli and modern pathogens such as enterohaemorrhagic strains of this species (such as o ). the mechanisms proposed to explain how these commensals have become pathogens are grounded in their ecology and life histories, culminating in the notion of ecological evolution (''eco-evo'') [ ] . the eco-evo approach to understanding the emergence of pathogens gives credence, from the perspective of genomics, to evolutionary and adaptive scenarios that are surmised from a thorough understanding of the ecology and life history of pathogens. at present, epidemiological classifications of plant diseases are based on the interaction of the pathogen and the host (biotrophic or necrotrophic, obligate or facultative), on the number of cycles of propagule production (mono-and polycyclic diseases), on the importance of latency in symptom expression, and on the role of vectors, but there is no formalized equivalent of ''sapronoses''. nevertheless, numerous plant pathogens are present in diverse nonagricultural habitats or survive saprophytically in agricultural contexts. these include a range of bacteria, fungi, and stable viruses (a nonexhaustive list of examples is presented in table ). a striking characteristic of many of the virulence factors of these plant pathogens is that they are linked to-or are in themselves-traits critical to adaptation to the nonplant environment, as will be illustrated below. this provides a compelling reason to adopt a holistic view of the life history and evolution of plant pathogens, to move beyond the traditional borders of agriculture and the presumed ''primary'' plant host. adaptation to biotic and abiotic stresses, within or outside of agricultural habitats, likely plays as important a role in the evolution of parasitic fitness of plant pathogens as it does for human pathogens. as illustrated above, traits that confer fitness in response to biotic and abiotic environmental stress can have dual-use as virulence factors in human pathogens. toxins and toxin transport systems (including efflux pumps, in particular) are among the common adaptations for antagonizing and defending against the co-inhabitants of a habitat. in plant pathogens, the transport systems for toxins and antimicrobials can have broad spectrum activity, leading to resistance to agricultural fungicides and also contributing to virulence [ ] . genes coding for wide spectrum efflux pumps are present in the chromosomes of all living organisms [ ] . the efflux pump bcatrb of botrytis cinerea confers resistance to antimicrobials produced by soil and plant microflora ( , diacetylphloroglucinol and phenazine antibiotics) [ , ] and also to the fungicide fenpiclonil and the plant defensive phytoalexin resveratrol [ ] . the transporter abc from magnaporthe grisea protects the fungus against azole fungicides and the rice phytoalexin sakuranetin [ ] . numerous plant pathogenic bacteria, including erwinia amylovora, dickeya spp. (formerly the multiple biovars of e. chrysanthemi), and agrobacterium tumefaciens, also produce efflux pumps that are involved in their resistance to plant antimicrobials (reviewed by martinez et al. [ ] ). toxins themselves can have a broad spectrum of action. for example, mycotoxins, well known for their human and animal toxicity, have broad spectrum activity and are thought to have evolved as a defense against predators (nematodes) and antagonists (other microorganisms) [ ] . one family of these, the trichothecenes, contributes significantly to the virulence of many gibberella (fusarium) species [ ] . adaptation to biotic stress also implicates systems for the detection or inhibition of arms of aggression used by co-inhabitants. recent work on fungi suggests that systems to detect enzymes that degrade fungal cell walls are also deployed as virulence factors. lysin motifs (lysms) are carbohydratebinding protein modules that have been found in mammalian and plant pathogenic fungi as well as in saprophytes [ ] . bolton et al. [ ] demonstrated that the lysm protein ecp acts as a virulence factor in the plant pathogenic fungus cladosporium fulvum. as virulence factors they may suppress host defenses by sequestrating chitin oligosaccharides that are known to act as elicitors of plant defense responses [ ] and also as activators of host immune responses in mammals [ ] . de jonge and thomma [ ] suggest that these proteins may also have a role in the protection of saprophytic fungi against chitinase-secreting competitor microbes or mycoparasites. protection against abiotic stress can involve molecules that have also become virulence factors. siderophores [ ] [ ] [ ] and various pigments including melanins [ ] are virulence factors in some human pathogens. siderophores contribute to resistance to oxidative stress and sequestering iron when it is rare in the environment. in the plant pathogens alternaria brassicicola, cochliobolus spp., fusarium graminearum [ ] , and m. grisea [ ] , siderophores or their precursors are virulence factors. melanins offer protection from extreme temperatures, uv radiation, and antimicrobials. in the plant pathogens m. grisea and colletotrichum spp., melanins are also virulence factors via their essential role in the formation of tissue-penetration structures such as appressoria [ ] . in many cases, toxins and siderophores are produced by nonribosomal peptide synthase or polyketide synthase pathways. these pathways, widely distributed in the microbial world, are highly adaptable and have given rise to a wide range of compounds with a plethora of activities, including many of pharmaceutical importance [ ] . hc-toxin of cochliobilus carbonum, victorin in c. victoriae, and t-toxin in c. heterostrophus are products of these pathways [ ] . the key virulence factor of streptomyces spp., thaxtomin [ ] , and the multitude of host-specific and nonspecific toxins in pseudomonas syringae pathovars [ ] are also produced by these pathways. the capacity to detect changes in conditions of the abiotic environment has also become part of the virulence factors of some plant pathogens. for example, to detect changes in environmental conditions, organisms exploit two-component histidine kinase complexes. these are key elements of the machinery for signal sensing, allowing bacteria, yeasts, fungi, and plants to adapt to changing environments. in the plant pathogen b. cinerea, one of its multiple histidine kinases, bos , not only mediates osmosensitivity and resistance to fungicides, but is also essential for formation of macroconidia and expression of virulence [ ] . recognition and understanding of the full complexity of the life history of plant pathogens will enhance our capacity to evaluate the diversity and intensity of environmental stresses that microorganisms face and will contribute novel hypotheses concerning the role of environmental stresses in the evolution of pathogenicity. stress is considered to play an important role in adaptive evolution in general, in particular via its effect on mutation rates [ ] . for certain fungi and bacteria, including plant pathogens, stress increases the activity of transposable elements [ ] [ ] [ ] and induces the sos response and other systems involved in the modification or repair of dna [ ] . mutations can target the ensemble of the microbial genome. however, it has been suggested that adaptation of bacteria to multiple stresses can lead, in particular, to the acquisition of virulence factors and to the emergence of pathogenic variants [ ] . adaptation to specific habitats-which involves adapting to a particular ensemble of biotic and abiotic parameters-could also influence the evolution of parasitic fitness. available examples focus on soilborne and rhizosphere microorganisms. the rhizosphere is a dynamic soup whose chemistry changes as plants grow, die, and degrade. chemicals in the rhizosphere are food substrates and means of communication, antagonism, and collaboration among microorganisms, among plants, and between plants and microorganisms. to decompose dead plant material and recycle carbon, microorganisms have developed a range of cell wall-degrading enzymes, without which our planet would be quite encumbered by the accumulation of tissue from dead plants. pectolytic, cellulolytic, and lignolytic enzymes are also well-known pathogenicity factors [ ] [ ] [ ] . to hone the efficiency of these enzymes in planta, pectinolytic fungi are adept at modulating the surrounding ph. alternaria, penicillium, fusarium spp., and sclerotinia sclerotiorum also exploit these ph changes to enhance the action of these enzymes as virulence factors [ ] . streptomyces spp. are considered quintessential soil inhabitants. their ability to degrade biopolymers, including cellulose and chitin, contributes greatly to nutrient cycling, and their vast array of antimicrobials contributes to survival and microbial communication in soil [ ] . some streptomyces species are pathogenic to root crops and to potatoes in particular. a recently discovered virulence factor in streptomyces, a saponinase homologue [ ] , may be the result of adaptation to the rhizosphere. saponins are plant glycosides that contribute to resistance against fungi and insect herbivores. bacteria, and especially gram-positive bacteria, can also be sensitive. saponins are also exuded from the roots of some plant species where they have allelopathic as well as antimicrobial activity [ , ] . key vital functions, housekeeping functions, and basic life cycle processes should also be considered for their potential to give rise to pathogenicity factors. traits fundamental to fitness and survival in general can confer or enhance pathogenic fitness. in plant pathogenic bacteria these include flagella, motility, lipo-and exopolysaccharides, o-antigens, fimbriae, mechanisms for iron acquisition and for quorum sensing, toxin production, cell wall-degrading enzymes, and resistance to oxidative stress [ ] . motility, for example, is essential to dispersal and for attaining new resources. in ralstonia solanacearum it is also essential for early stages of plant invasion and colonization during pathogenesis [ ] . in the fungus aschochyta rabiei, kinesins that are essential for polarized growth and transport of organelles are suspected to be a virulence factor [ ] . an f-box protein of giberrella zeae has been reported to be involved in sexual reproduction and in pathogenicity [ ] . the enzymes that allow fungi to detoxify compounds resulting from plant defense mechanisms are probably also simply means of acquiring nutrients [ ] . for example, detoxification of tomatine in tomatoes by septoria lycopercici and by fusarium oxysporum f. sp. lycopersici is achieved by the deployment of glycosyl hydrolases by these fungi; gaeumannomyces graminis detoxifies avenacins in oats via a beta-glucosidase [ ] . another example of adaptation of basic cellular functions into pathogenicity factors concerns elicitins. elicitins are part of one of the most highly conserved protein families in the phytophthora genus and are widespread throughout phytophthora species. elicitins of p. infestans induce hypersensitivity in plants. recent work from jiang and colleagues [ ] suggests that a primary function of elicitins is the acquisition of sterols from the environment. how can we make sense of the processes that have led to the wide variety of pathogenicity factors in plant pathogens and that continue to drive the evolution of pathogens? bacterial plant pathogens are particularly illustrative of the differences in suites of secretion systems [ , , , ] and of effectors [ , , , , , ] among members of different genera, species, or strains of the same species that attack plants. effectors are proteins secreted by plant pathogens that modulate plant defense reactions, thereby enabling the pathogen to colonize the plant tissues. it is tempting to wonder if the effectors and secretion systems have critical roles in fitness elsewhere other than in association with the host plant. the examples listed above that describe traits that play roles in both environmental fitness and virulence to plants provide a compelling incentive to expand our paradigms concerning the forces that drive evolution of plant pathogenicity. the evolutionary forces that have been described to date for plant pathogens [ ] need to be extended beyond the current agro-centric paradigm. to expand this paradigm we propose that the life cycles and life histories of plant pathogens be reconsidered. studies of pathogen ecology, evolution, and life history should include the full range of habitats and reservoirs these organisms can inhabit. this in turn will permit testing a range of novel hypotheses about the role of ecological contexts-other than direct interaction with host plants-as forces of evolution. in table we propose some such hypotheses. for example, rates of mutation and of transposition of insertion sequences or of transposable elements including phages might be different when a microorganism inhabits nonagricultural habitats (biofilms, lake water, or inert surfaces exposed to uv, for example) than when it colonizes plants. the consequences of these mutations for pathogenicity might in turn be markedly different than for fitness in nonagricultural habitats. likewise, the formation of spores or aggregates that can be released into the air and their survival over long distances might be highly influenced by the nature of the reservoir that the pathogen colonizes, resulting in direct effects of habitat on gene flow. furthermore, the biotic and abiotic stresses endured in nonagricultural habitats might exert positive selection for adaptive survival traits that have dual-use as virulence factors as illustrated in the examples above. these questions are clearly pertinent for pathogens that are not obligate biotrophs. however, the complexity of the biotic and abiotic environment perceived by obligate biotrophs during colonization of plants (powdery mildews on leaf surfaces inhabited by other microorganisms, for example) or during their dissemination (survival in air or in association with vectors) are also likely to exert selection independent of that due to the host plant genotype per se. these are only some of the ways in which environmental parameters other than the host plant are expected to have a marked influence on the diversification of plant pathogens. if nonagricultural environments can foster the evolution of traits that contribute to pathogen virulence, other scenarios are also probable where i) crop plants foster the emergence of traits antagonistic to survival outside of agricultural contexts ii) or nonagricultural environments foster the emergence of traits that are detrimental to pathogen virulence in crops. understanding the prevalence and significance of alternative habitats to pathogen life history is crucial to determining the broad costs of virulence for pathogen fitness. the cost of virulence in terms of fitness in association with plants has been explored extensively for several obligate parasites such as rusts and powdery mildews. work by thrall and burdon [ ] has shown clear fitness tradeoffs between pathogen aggressiveness (capacity to induce intense disease symptoms) and dissemination (via intense spore production). for nonobligate pathogens we do not know the cost of fitness outside of agricultural habitats. the interplay between evolutionary forces and habitat has not been explored for plant pathogens and might be a key feature in the emergence of certain diseases. by expanding our paradigms concerning pathogen life history and the selective forces that drive plant pathogen evolution, we will enhance our understanding of how table . novel hypotheses to be tested concerning the impact of substrates other than host plants on the evolutionary potential of plant pathogens. evolutionary force a novel hypothesis arising from expanded paradigms about the evolution of plant pathogenicity concerning: modifications of the genome. relative to its association with cultivated plant hosts, association of the pathogen with a given nonagricultural substrate leads to: n a significantly greater overall mutation rate. n a greater rate of transposition of insertion sequences or of transposable elements. n more frequent mutations or transpositions that target genes involved in pathogenicity. n a higher probability of acquisition of alien nucleic acids. n genetic exchange with more phylogenetically diverse microbes. effective population size. the effective sub-population size of a pathogen associated with a given nonagricultural (or nonplant) substrate is significantly different from that for sub-populations from cultivated host plants. this could lead to genetic and/or phenotypic differentiation of sub-populations based on substrate of origin. dissemination. the habitats occupied by the plant pathogen influence the mode(s) of dissemination, thereby influencing the distance of dissemination and the spatial and temporal scales of gene flow. mode of reproduction (recombination) genetic recombination. the frequency of recombination (via sexual cycle or other means) varies among strains of plant pathogens as a function of the habitat or substrate. selective pressures and impact on fitness. strains of pathogens adapted to a broad range of habitats have the greatest parasitic fitness. a the evolutionary forces listed here are those that have been considered for plant pathogens in agricultural contexts [ ] . these hypotheses concern pathogens with a marked saprophytic phase or for which nonagricultural or nonplant substrates can be a notable reservoir for survival. reservoirs can include irrigation water, natural waterways and bodies of water, biological vectors (animals, fungi, etc.), abiotic vectors (aerosols, clouds, precipitation), wild plants and weeds, soil, and physical structures in agricultural systems (greenhouse materials, tubing, plastics). doi: . /journal.ppat. .t pathogens survive in the absence of hosts, how and where new pathotypes are likely to emerge, and the significance of natural habitats to agricultural epidemics. insights will come from fundamental research to identify the mechanisms that drive the evolution of pathogenic traits and to explore the ecological significance of pathogenic traits to microbial fitness apart from the plant host. distinguishing the role of adaptation sensu stricto in the emergence of plant pathogenicity relative to that of exaptation [ ] , the useful cooptation of phenotypes that have arisen under natural selection due to forces unrelated to interaction with the primary host plant, will yield critical insight into how plant pathogens evolve independently of agricultural practices. a more complete understanding of the forces that drive plant pathogen evolution will be critical to enhancing and diversifying sustainable disease control strategies, and will improve prediction of the conditions that support the emergence of novel pathogens. emerging human infectious diseases: anthroponoses, zoonoses and sapronoses from outside to inside: environmental microorganisms as human pathogens. a report from the american academy of microbiology accidental virulence, cryptic pathogenesis, martians, lost hosts, and the pathogenicity of environmental microbes biofilm formation and dispersal and the transmission of human pathogens the virulence of human pathogenic fungi: notes from the south of france processes controlling the transmission of bacterial pathogens in the environment microorganisms resistant to free-living amoebae ready made' virulence and 'dual use' virulence factors in pathogenic environmental fungi-the cryptococcus neoformans paradigm functional role of bacterial multidrug efflux pumps in microbial natural ecosystems the biocide triclosan selects stenotrophomonas maltophilia mutants that overproduce the smedef multidrug efflux pump bacterial pathogenomics fungal transporters involved in efflux of natural toxic compounds and fungicides fungal abc transporters and microbial interactions in natural environments involvement of the abc transporter bcatrb and the laccase bclcc in defence of botrytic cinerea against the broad-spectrum antibiotic , -diacetylphloroglucinol the abc transporter bcatrb affects the sensitivity of botrytis cinerea to the phytoalexin resveratol and the fungicide fenpiclonil pathogenicity genes of phytopathogenic fungi fungal lysm effectors: extinguishers of host immunity? the novel cladosporium fulvum lysin motif effector ecp is a virulence factor with orthologues in other fungal species tlr- and il- a in chitin-induced macrophage activation and acute inflammation histoplasma requires sid , a member of an iron-regulated siderophore gene cluster, for host colonization iron metabolism in pathogenic bacteria distinct roles for intra-and extracellular siderophores during aspergillus fumigatus infection color me bad: microbial pigments as virulence factors functional analysis of all nonribosomal peptide synthetases in cochliobolus heterostrophus reveals a factor, nps , involved in virulence and resistance to oxidative stress ferricrocin synthesis in magnaporthe grisea and its role in pathogenicity in rice phylogenetic analysis of condensation domains in nrps sheds light on their functional evolution the phylogeny of plant and animal pathogens in the ascomycota evolution of plant pathogenicity in streptomyces pseudomonas syringae phytotoxins: mode of action, regulation and biosynthesis by peptide and polyketide synthetases a class iii histidine kinase acts as a novel virulence factor in botrytis cinerea stress-induced mutagenisis in bacteria heat shock, copper sulfate and oxidative stress activate the retrotransposon maggy resident in the plant pathogenic fungus magnaporthe grisea ihf is the limiting host factor in transposition of pseudomonas putida transposon tn in stationary phase foxy: an active family of short interspersed nuclear elements from fusarium oxysporum evolution of microbial virulence: the benefits of stress mini review: recent advances in the molecular genetics of plant cell wall-degrading enzymes produced by plant pathogenic fungi advances in molecular genetics of plant-microbe interactions the importance of fungal pectinolytic enzymes in plant invasion, host adaptability and symptom type pathogenic fungi: leading or led by ambient ph? the role of root exudates in rhizosphere interactions with plants and other organisms principles and practices in plant ecology: allelochemical interactions comparative genomics reveals what makes an enterobacterial plant pathogen ralstonia solanacearum needs motility for invasive virulence on tomato towards identifying pathogenic determinants of the chickpea pathogen ascochyta rabiei a novel f-box protein involved in sexual development and pathogenesis in gibberella zeae nutrition acquisition strategies during fungal infection of plants ancient origin of elicitin gene clusters in phytophthora genomes genome sequence of the enterobacterial phytopathogen erwinia carotovora subsp. atroseptica and characterization of virulence factors comparison of the genomes of two xanthomonas pathogens with differing host specificities insights into genome plasticity and pathogenicity of the plant pathogenic bacterium xanthomonas campestris pv. vesicatoria revealed by the complete genome sequence roadmap to new virulence determinants in pseudomonas syringae: insights from comparative genomics and genome organization diverse evolutionary mechanisms shape the type iii effector virulence factor repertoire in the plant pathogen pseudomonas syringae groovy times: filamentous pathogen effectors revealed bacterial phytopathogens and genome science the population genetics of plant pathogens and breeding strategies for durable resistance evolution of virulence in a plant host-pathogen metapopulation exaptation -a missing term in the science of form global impact of vibrio cholerea interactions with chitin virulence and the environment: a novel role for vibrio cholera toxincoregulated pili in biofilm formation on chitin legionella pneimophila -a human pathogen that co-evolved with fresh water protozoa identification of a novel virulence factor in burkholderia cenocepacia h required for efficient slow killing of caenorhabditis elegans transmission of yersinia pestis from an infectious biofilm in the flea vector cloning and characterization of smedef, a novel multidrug efflux pump from stenotrophomonas maltophilia multidrug efflux pumps of gram-negative bacteria acinetobacter baumannii: emergence of a successful pathogen diversity of the burkholderia cepacia complex and implications for risk assessment of biological control strains presence of erwinia chrysanthemi in two major river systems and their alpine sources in australia long distance transport of erwinia carotovora in the atmosphere and surface water presence of erwinia carotovora in surface water in north america soft rot erwinia bacteria in surface and underground waters in southen scotland and in colorado pantoea agglomerans, a plant pathogen causing human disease pantoea agglomerans pvs. gypsophilae and betae, recently evolved pathogens? a surprising niche for the plant pathogen pseudomonas syringae the life history of the plant pathogen pseudomonas syringae is linked to the water cycle applied aspects of rhodococcus genetics rhodococcus fascians in herbaceous perennials versatile persistence pathways for pathogens of animals and plants high diversity of fungi in air particulate matter alternaria spp.: from general saprophyte to specific parasite the broad-scale distribution of microfungi in the windmill islands region, continental antarctica a comparative study of siderophore production by fungi from marine and terrestrial habitats habitat and temporal differences among soil microfungal assemblages in ontario lack of host specialization in aspergillus flavus ecology of the fungus, fusarium: competition general ecology of the fusaria biodiversity of the genus fusarium in saline soil habitats effects of water potential on spore germination and viability of fusarium species metabolites of fusarium the stem canker (blackleg) fungus, leptosphaeria maculans, enters the genomic era insectassociated fungi in soils of field crops and orchards diversity, host, and habitat specificity of oomycete communities in declining reed stands (phragmites australis) of a large freshwater lake penicillium mycobiota in arctic subglacial ice detection of infectious tomato mosaic tobamovirus in fog and clouds detection of tomato mosaic tobamovirus rna in ancient glacial ice detection of infectious tobamovirus in forest soils we thank the three anonymous reviewers for their constructive comments and for the suggestion of additional materials to incorporate into the text. we also thank dr. melodie putnam (oregon state university, united states of america) for useful discussions about the ecology of bacterial plant pathogens. key: cord- -liaihahj authors: huang, jiachen; diaz, darren; mousa, jarrod j. title: antibody recognition of the pneumovirus fusion protein trimer interface date: - - journal: plos pathog doi: . /journal.ppat. sha: doc_id: cord_uid: liaihahj human metapneumovirus (hmpv) is a leading cause of viral respiratory infection in children, and can cause severe lower respiratory tract infection in infants, the elderly, and immunocompromised patients. however, there remain no licensed vaccines or specific treatments for hmpv infection. although the hmpv fusion (f) protein is the sole target of neutralizing antibodies, the immunological properties of hmpv f remain poorly understood. to further define the humoral immune response to the hmpv f protein, we isolated two new human monoclonal antibodies (mabs), mpv and mpv . both mabs are neutralizing in vitro and were determined to target a unique antigenic site using competitive biolayer interferometry. we determined both mpv and mpv have higher affinity for monomeric hmpv f than trimeric hmpv f. mpv was co-crystallized with hmpv f, and the mab primarily interacts with an alpha helix on the f region of the hmpv f protein. surprisingly, the major epitope for mpv lies within the trimeric interface of the hmpv f protein, suggesting significant breathing of the hmpv f protein must occur for host immune recognition of the novel epitope. in addition, significant glycan interactions were observed with a somatically mutated light chain framework residue. the data presented identifies a novel epitope on the hmpv f protein for epitope-based vaccine design, and illustrates a new mechanism for human antibody neutralization of viral glycoproteins. human metapneumovirus (hmpv) is a common cause of lower respiratory tract infection in children, the elderly, and the immunocompromised. there is currently no approved vaccine or therapeutic to prevent or treat hmpv infection. the hmpv fusion (f) protein is the sole target of antibodies that neutralize hmpv. here we describe new human mabs that bind a unique epitope on the hmpv f protein. the mabs were mapped to a new antigenic site on the hmpv f protein located within the trimeric interface of the hmpv f protein based on competitive biolayer interferometry and x-ray crystallographic experiments. this is the first example of a human mab that binds inside the trimeric interface of a viral glycoprotein and neutralizes the virus. the results suggest such antibodies may human metapneumovirus (hmpv) is a leading cause of viral respiratory infections in children, the majority of whom are seropositive for hmpv by five years of age [ ] . although hmpv was discovered in [ ] , there are no vaccines or therapeutics approved to prevent or treat viral infection. similar to other respiratory pathogens, children, the elderly, and the immunocompromised are the major groups for which hmpv infection may require hospitalization [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . several reports have demonstrated hmpv infection can be lethal in both adults and children. in particular, haemopoietic stem cell transplant patients are at high risk of severe hmpv infection [ ] [ ] [ ] [ ] , and several outbreaks of hmpv in nursing homes have been reported [ ] [ ] [ ] . in addition, fatal hmpv has been observed in one child during an outbreak of hmpv in a daycare center [ ] . hmpv is also a significant cause of febrile respiratory illness in hiv-infected patients [ ] , and has been linked to exacerbations of chronic obstructive pulmonary disease [ ] . co-circulation of hmpv was observed during the sars outbreak of [ ] [ ] [ ] , and similar observations have been made during the current sars-cov- pandemic [ , ] , suggesting hmpv interacts with other circulating respiratory viruses. hmpv circulates as two genotypes, a and b, and based on the sequence variability of the surface proteins, hmpv is further grouped into four subgroups, a , a , b , and b [ , ] , and two additional subgroups, a a and a b, have been proposed [ ] . hmpv has three surface glycoproteins, the small hydrophobic (sh), the attachment (g), and the fusion (f) proteins. the hmpv sh protein has been demonstrated to have viroporin activity [ ] , while the hmpv g protein has been hypothesized to be involved in cellular attachment [ ] . the hmpv f protein is indispensable for hmpv infection, and is highly conserved among hmpv subgroups [ ] . furthermore, the hmpv f protein is the sole target of neutralizing antibodies [ ] . although the hmpv g protein is thought to interact with proteoglycans, the hmpv f protein can interact with glycans in the absence of hmpv g [ ] . the hmpv f protein contains a highly conserved rgd motif that has been proposed as a key region in receptor binding to cellular integrins [ , ] . the entry of hmpv into the host cell can occur by cell membrane or endosomal membrane fusion [ ] . both hmpv and the related respiratory syncytial virus (rsv) are members of the pneumoviridae family, and share a structurally similar f protein that has approximately % homology between the two viruses. for both viruses, the f protein has two long-lived conformations, the pre-fusion and post-fusion states [ ] . both rsv and hmpv lacking the g protein can infect cells in vitro, although these viruses are attenuated in vivo [ ] . both rsv g and hmpv g proteins are immunogenic, yet only antibodies to rsv g are neutralizing [ , ] . the pre-fusion conformation of the pneumovirus f protein is meta-stable, and stabilized versions of both hmpv f [ ] and rsv f [ , ] have been generated. the rsv f protein was initially stabilized in the prefusion conformation using cysteine substitutions to lock the protein in the pre-fusion state by disulfide bonds, and through cavity-filling mutations to prevent transition to the post-fusion state. this ds-cav construct has been further developed for clinical trials, and has shown promise in a phase i clinical trial [ ] . additional constructs for rsv f have focused on stabilizing the α -α loop through proline mutations [ ] . a similar approach was undertaken for the hmpv f protein, whereby a a p mutation was introduced to stabilize the pre-fusion conformation [ ] . the hmpv f protein contains a single site that is cleaved to convert the polypeptide f protein into the meta-stable disulfide-linked f -f pre-fusion homotrimer. this is in contrast to rsv f, which contains two furin cleavage sites flanking the p peptide fragment. the cleavage enzyme for hmpv f in vivo is currently unknown, although cleavage can be accomplished by trypsin in vitro [ ] . post-fusion hmpv f has been generated by removing the fusion peptide and incorporating one furin cleavage site from rsv f [ ] . x-ray crystal structures of the hmpv f protein from the a subgroup have been determined in the pre-fusion and postfusion conformations [ , ] . both proteins were expressed in cv- cells using a vaccinia virus expression system, although stabilized versions for routine hek f or cho cell line expression have not yet been generated. for rsv f, the pre-fusion conformation contains antigenic sites Ø [ ] and v [ ] located on the head of the rsv f protein, which elicit a more potent neutralizing antibody response as compared to the post-fusion conformation [ , ] . furthermore, the human antibody response to rsv infection is primarily focused on these pre-fusion-specific epitopes [ ] . for hmpv f, data using human serum has shown that the preponderance of hmpv f-specific human antibodies bind both pre-fusion and post-fusion f conformations, which has been proposed is due to differential glycan positioning on the head of the hmpv f protein as compared to the rsv f protein [ ] . although several monoclonal antibodies (mabs) have previously been isolated that recognize the hmpv f protein [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , the predominant antigenic sites targeted by the human antibody response are unclear. a panel of rodent-derived mabs was initially used to map the neutralizing epitopes on the hmpv f protein using viral escape mutants [ , ] . the known antigenic sites on the hmpv f protein include antigenic sites iii, iv, and an unnamed site targeted by mab ds [ ] . ds was isolated from a human phage display library [ ] , and was co-crystallized with a fragment of the pre-fusion hmpv f protein [ ] . several mabs isolated have been found to cross-neutralize rsv and hmpv, including mpe [ ] and p [ ] (site iii), and f [ ] , g [ ] , and e [ ] (site iv). in addition, we have recently isolated a panel of human mabs targeting site iii and the ds epitope [ ] . one of these mabs, mpv , competes for binding at antigenic site iii, but does not cross-react with rsv f, suggesting closer examination of hmpv f epitopes is required to improve knowledge on the optimal epitopes for elicitation of neutralizing antibodies. in this study, we isolated new human mabs to further identify the epitopes on the hmpv f protein recognized by the human immune system. in our efforts to identify the major antigenic epitopes on the hmpv f protein, we isolated mabs from human subjects using hybridoma technology [ ] . as hmpv infection and exposure is not routinely tested in patients, and the majority of individuals are seropositive for hmpv infection [ ] , we isolated mabs from two healthy human subjects. two new mabs were isolated against the recombinantly expressed hmpv b f protein (s table) expressed in hek f cells [ ] . mpv and mpv were isolated from two different donors, and have isotypes of igg and kappa, and igg and lambda, respectively. mpv utilizes v h - , j h , d h , v k - , and j k , while mpv utilizes v h - , j h , d h - , v l - , and j l . the heavy chain complementarity determining region (hcdr) loop length differs dramatically between the two mabs as the hcdr loop for mpv is just amino acids, while mpv has a amino acid long cdr loop (s table) . to identify the antigenic epitopes targeted by the isolated mabs, we performed epitope binning using competitive biolayer interferometry [ ] . previously discovered mabs with known antigenic epitopes were utilized as mapping controls, including mabs f [ ] (site iv), mpv [ ] and ds [ ] (ds epitope), and mpe [ ] and mpv [ ] (site iii) ( fig a; s fig) . these previously discovered mabs bind both pre-fusion and post-fusion conformations of the hmpv f protein with similar affinity, except for mpe , which has higher affinity for the pre-fusion conformation of hmpv f. anti-penta-his biosensors were loaded with the hmpv -bv f [ ] protein (a subgroup, s table) and then loaded with one hmpv fspecific mab, followed by exposure to a second mab. mabs mpv and mpv did not compete with any of the mapping control mabs, yet competed for binding with each other, suggesting these two mabs bind to a unique antigenic site on the hmpv f protein. plaque neutralization assays were performed to determine the neutralization properties of mpv and mpv against hmpv subgroup b (strain tn/ - ) and hmpv subgroup a (strain can/ - ) in vitro (fig b) . mpv neutralized hmpv with % inhibitory concentration (ic ) values of ng/ml for mpv can/ - and ng/ml for mpv tn/ - , while mpv had ic values of and ng/ml, respectively. the neutralization potency of mpv was comparable to mabs mpe and f. we next determined the binding properties of mpv and mpv by elisa and biolayer interferometry. for elisa, the half-maximal effective concentration (ec ) values were used to quantify binding between mabs across multiple hmpv f protein constructs (fig a; s fig; s fig; s table) . generating trimeric hmpv f can be achieved by treating purified protein with trypsin as previously described [ , ] , although this process generates batch to batch variation of both pre-fusion and post-fusion conformations [ ] . mpv binds to hmpv f proteins from all four hmpv f subgroups, while mpv showed binding to hmpv f proteins from subgroups a , b , and b (fig a) . we next assessed binding to exclusively monomeric (presumed pre-fusion) and trimeric (post-fusion) hmpv b f proteins that were treated with trypsin to induce cleavage (fig a; s fig) , and mpv and mpv had higher binding to monomeric hmpv f than to trimeric hmpv f. mpv had a nearly five-fold lower ec to monomeric hmpv b f than to trimeric hmpv b f. mpv bound well to the hmpv b f monomer, while binding was completely abrogated binding to the post-fusion hmpv b f trimer. these data indicate the epitope for mpv and mpv is predominantly exposed on pre-fusion and/or monomeric hmpv f. binding affinity of the mabs was assessed by biolayer interferometry using monomeric hmpv b f protein (fig b) . affinity measurements were completed by cleaving mabs to fab fragments and coupling biotinylated hmpv b f monomer to streptavidin biosensors. association (k on ), dissociation (k off ), and k d values were comparable for mpv and mpv fabs, and the fabs had a - log higher k d than the control mab f. binding k d values were . nm, . nm, and nm for fabs of mpv , mpv , and f to monomeric hmpv b f protein, respectively. to fully define the epitope targeted by the newly isolated mabs, we co-crystallized the fab of mpv in complex with hmpv b f. trypsinization of hmpv b f generated trimeric and monomeric versions of hmpv f as assessed by size exclusion chromatography (s fig). cleavage of mpv and mpv mabs to fab fragments and subsequent addition of these fabs to trypsinized trimeric hmpv b f resulted in monomeric hmpv f-fab complexes (s fig; s fig) . although the hmpv b f trimer appeared to fall apart upon fab binding, we cannot attribute this to binding of mpv and mpv as other fabs also caused trimer dissociation of this construct. the mpv -hmpv b f complex was subjected to crystallization screening antibody recognition of the pneumovirus fusion protein trimer interface and crystals were obtained in . m ammonium sulfate, . m sodium citrate tribasic dihydrate ph . , and . m lithium sulfate monohydrate. crystals were harvested and x-ray diffraction data was collected, and the structure of the complex was determined to . Å (fig ; s table) . the asymmetric unit contained one hmpv f protomer with one mpv -fab molecule. hmpv f was observed in the pre-fusion conformation, although no trimeric structure was observed when viewing symmetry related partners (s fig). mpv targets a unique epitope compared to previously discovered pneumovirus antigenic sites. the primary epitope consists of a single alpha helix of amino acids - of the f region (fig a) . compared to the hmpv f protein, mpv binds nearly perpendicular to the long axis of the f protein. upon overlay with the previously determined x-ray crystal structure of pre-fusion hmpv f, it is clear the major epitope lies completely within the interface between two protomers of trimeric hmpv f (fig b) . this unusual epitope suggests the hmpv f protein is partially monomeric on the surface of the virion envelope or on hmpv infected cells, and/or substantial breathing of the hmpv f protein takes place to allow the antibody to bind and neutralize the virus. as mentioned earlier, mpv has an unusually short hcdr loop of just amino acids. the hcdr and light chain cdr (lcdr) are centered on the - helix region. numerous hydrogen bonding events were clear in the electron density (fig c; fig d; s fig) . the hcdr interacts via asp with arg of hmpv f, while hcdr asn and ser interact with hmpv f glu and arg , respectively (fig c) . the hcdr utilizes arg to interact with hmpv f glu . the light chain lcdr has more hydrogen bonding events than the hcdr , utilizing the backbone amino group of leu to interact with hmpv f thr , arg to hmpv f asp , and asp to hmpv f lys (fig d) . lcdr arg interacts with hmpv f asn , which has an extended n-linked glycan motif. in addition, the lcdr asp interacts with hmpv f thr . the framework loop of the light chain interacts with the glycan motif consisting of nag-nag-bma with branched man residues off the bma glycan, in which tyr interacts with the extended man glycan, while the long-face of tyr sits parallel to the extended glycan, suggesting a favorable interaction with the glycan motif. the - helix of hmpv f is structurally conserved in the pre-fusion and post-fusion conformations, although the helix is exposed on the outer surface in the trimeric post-fusion conformation (fig a; fig b) . upon overlay of the - region of the pre-fusion and post-fusion hmpv f proteins, residues - align well, while the helix breaks on post-fusion hmpv f at residues - (fig c) . this sequence identity of the helix is highly conserved, as residues are identical between the a and b subgroups, except for a lys /arg mutation. as mpv and mpv exhibited binding to post-fusion hmpv f constructs, we further examined binding by attempting to generate a complex between the fab of mpv and trypsinized hmpv b f that was in the post-fusion conformation (s fig; s fig) . no complex was observed as assessed by size exclusion chromatography, while the fab of f readily formed a complex with the post-fusion hmpv f protein. this suggests that although binding is observed by elisa, part of the epitope lies outside the - helix and is incomplete in the post-fusion conformation. since the major epitope is focused on the single helix, we assessed binding by western blot to determine if mpv displayed binding to a linear conformation in the control. data points are the average of three replicates and error bars are % confidence intervals. data are shown from one experiment and are representative of two independent experiments. a mab was considered neutralizing if > % plaque reduction was observed at the highest concentration. https://doi.org/ . /journal.ppat. .g antibody recognition of the pneumovirus fusion protein trimer interface mpv and mpv have lower ec values (higher affinity) for monomeric hmpv b f than trimeric hmpv b f. mpv and mpv bind to hmpv f proteins from all four subgroups. data points are the average of four replicates and error bars are % confidence intervals. > indicates the calculated ec was either not in the range of the curve (due to an overall low signal) or a signal above . absorbance units was not detected at the highest concentration of μg/ml. each data point is the average of four replicates and error bars represent % confidence intervals. data are representative of one experiment from two independent experiments. (b) binding curves from biolayer interferometry. biotinylated monomeric hmpv b f protein coated streptavidin biosensors were exposed to fabs (mpv and mpv : / / / . μg/ml, f: / / / μg/ml) for s before dissociating in buffer for s. binding constants are displayed underneath each graph and were averaged from the four replicates. https://doi.org/ . /journal.ppat. .g antibody recognition of the pneumovirus fusion protein trimer interface denatured hmpv f protein (s fig). binding to hmpv b f was analyzed using reduced and heated protein, and a nonreduced protein. mpv showed binding to all states of hmpv b f, while control mabs f and mpe showed binding to only the nonreduced state. these data suggest the mpv epitope is at least partially linear. as the epitope for mpv lies within the trimer interface, the mechanism by which b cells recognize this epitope is unclear. to determine if the mpv epitope is exposed on the surface of hmpv infected cells, we performed flow cytometry using mpv , mpe , and a negative control pneumococcal-specific antibody (s fig) . both mpv and mpe induced a fluorescent shift in hmpv infected cells, while the negative control mab did not. this indicates the hmpv f protein is either in antibody recognition of the pneumovirus fusion protein trimer interface monomeric form on the surface of infected cells, or that hmpv f trimer exhibits breathing motion that allows for binding of mpv . by comparing the binding sites with previously the corresponding - epitope is colored on the x-ray crystal structure of post-fusion hmpv f ( l x). the - epitope is surface exposed on trimeric post-fusion hmpv f. (c) structural overlay of the - region between pre-fusion (cyan) hmpv f from the hmpv b f + mpv fab complex and post-fusion (red) hmpv f (pdb id: l x). conserved amino acid residues between the b and a subgroups are listed in black, while residues that have mutations or shift positions are colored according to the corresponding structure. (d) structural overlay of mpv on the hmpv f protein with previously structurally characterized hmpv f-specific mabs. mpe (site iii, orange) and f (site iv, red) were aligned onto hmpv f by aligning the corresponding rsv f residues onto hmpv f from the co-complex structures with rsv f (pdb id: u and pbd id: o ). ds was aligned from pdb dag. (e) the structure overlay in (d) is rotated degrees to view the hmpv f protein from the top down. antibody recognition of the pneumovirus fusion protein trimer interface described hmpv f-specific mabs that have been structurally characterized (mpe , f, and ds ), the mpv epitope is distant from all three known antigenic sites (iv, ds -site, and iii), and lies on the opposite face of the monomeric hmpv f protein (fig d; fig e) . this unique epitope was unexpected on the hmpv f protein, although intratrimeric epitopes have been observed on the influenza hemagglutinin protein [ ] [ ] [ ] [ ] [ ] . however, these previously discovered influenza-specific mabs have been nonneutralizing, and a subset were determined to function by disrupting the ha trimer and inhibiting cell-to-cell spread [ , ] . evidence for pneumovirus f protein breathing was previously demonstrated for the rsv f protein, whereby the mab cr that binds at antigenic site v enhances opening of the pre-fusion rsv f protein [ ] , suggesting a similar process may occur for hmpv f. here we demonstrate a new class of neutralizing hmpv f-specific human mabs. the mabs are broadly reactive across all hmpv subgroups, and neutralize viruses from both hmpv genotypes. the mabs were discovered to bind to a novel epitope by competition experiments with previously discovered rodent and human derived hmpv f-specific mabs. the rsv f protein has at least two antigenic sites that are surface exposed on the head of the trimeric surface (antigenic sites Ø [ ] and v [ , ] ), however, such antigenic sites have not yet been identified for hmpv f, likely due to glycan shielding [ ] . furthermore, the x-ray crystal structure of one mab, mpv , was determined in complex with the hmpv f protein and solved to . Å. the structure revealed mpv binds at a newly defined epitope on the hmpv f protein defined by the alpha helical - amino acid region contained within the f fragment on pre-fusion hmpv f. this new epitope is the first defined on the head of the hmpv f protein as previous mabs identified have targeted the lower half of the protein [ , , [ ] [ ] [ ] [ ] . the new epitope is nearly completely contained within the pre-fusion trimeric interface of the hmpv f protein, which is a unique feature among previously discovered human mabs to viral glycoproteins. although the mabs were shown to bind both predominantly pre-fusion and postfusion conformations of the hmpv f protein, preferential binding to pre-fusion hmpv f was observed as evidenced by our attempts to complex mpv and mpv with post-fusion hmpv f. these data indicate that while the - epitope is present in both pre-fusion and post-fusion conformations, the complete structural epitope is present only on pre-fusion hmpv f, as several contacts outside of the - region were observed in our x-ray crystal structure. these additional epitope residues are rearranged in the post-fusion conformation. human mabs targeting the trimeric interface of the influenza ha protein have previously been discovered [ ] [ ] [ ] [ ] [ ] , yet these mabs were nonneutralizing, unlike the mabs described here, which are the first human mabs discovered that bind within the trimeric interface of a viral glycoprotein and neutralize the virus. as epitopes at the trimeric interface have now been determined for influenza virus and hmpv, it is likely such epitopes are important mediators of viral immunity for other class i fusion viral glycoproteins. the mechanism by which mabs mpv and mpv neutralize hmpv remains to be determined. the mabs could inhibit the transition of the hmpv f protein from the pre-fusion to the post-fusion conformation, which is likely the mechanism for the majority of antibodies targeting pneumovirus fusion proteins. alternatively, the mabs could prevent infection by disrupting the trimeric structure of the hmpv f protein. currently, we do not have reliable prefusion constructs that could be used to examine this hypothesis. it is clear that mpv binds to the surface of infected cells as demonstrated by our analysis by flow cytometry, although it is unclear if the mab is binding to trimeric or monomeric hmpv f on the cell surface. since the - epitope is hidden within the trimeric interface of the previously determined x-ray antibody recognition of the pneumovirus fusion protein trimer interface crystal structure of pre-fusion hmpv f [ ] , a mechanism must occur whereby the hmpv f protein motion facilitates exposure of the epitope for mpv binding, and indeed for initial naïve b cell recognition of this epitope since these mabs were derived from seropositive human subjects. this motion, termed "breathing" has previously been demonstrated for the rsv f protein by identification of an alternative conformation of the rsv f protein, whereby the mab cr causes opening of pre-fusion rsv f trimers, and rsv f was also found to be both monomeric and trimeric on the surface of transfected hek f cells [ ] . furthermore, breathing of influenza and hiv glycoproteins has also been described [ , ] , and mabs to the hiv glycoprotein have been shown to destabilize the trimeric structure [ ] . the mab cr targets antigenic site v of the rsv f protein, which was previously defined by the mab hrsv [ ] . mabs to a similar antigenic site v epitope on the hmpv f protein have not been identified, and mpv targets an epitope on the opposite face of monomeric hmpv f. although we have identified a new antigenic site by isolating two mabs from different donors, it remains unclear if such antibodies are a major part of the hmpv f humoral immune response. it also remains to be determined if mabs such as mpv will protect against viral replication in vivo. since the mpv epitope is partially linear, as evidenced by our binding studies to reduced hmpv f, a peptide-based vaccine based solely around this epitope may elicit neutralizing antibodies. additionally, although mpv and mpv target a similar epitope based on epitope binning analysis, the binding properties to trimeric hmpv f are quite distinct. mpv shows binding to both monomeric and trimeric hmpv f constructs, while binding to trimeric hmpv f is completely eliminated for mpv . further structural analysis of the mpv epitope will delineate the differential binding properties. our findings provide novel insights on the human antibody response to the hmpv f protein, and responses to viral glycoproteins. the x-ray crystal structure of the immune complex may guide the development of vaccines against hmpv. in addition, mpv can be potentially applied to the treatment and prevention of hmpv infection if prophylactic efficacy is demonstrated in animal challenge models. this study was approved by the university of georgia institutional review board as study . healthy human donors were recruited to the university of georgia clinical and translational research unit and written informed consent was obtained. after obtaining informed consent, ml of blood was drawn by venipuncture into heparincoated tubes, and ml of blood was collected into a serum separator tube. peripheral blood mononuclear cells (pbmcs) were isolated from human donor blood samples using ficoll-histopaque density gradient centrifugation, and pbmcs were frozen in the liquid nitrogen vapor phase until further use. plasmids encoding cdnas for hmpv f proteins listed in s table were synthesized (gen-script) and cloned into the pcdna . + vector. the plasmids were expanded by transformation in escherichia coli dh α cells with μg/ml of ampicillin (thermo fisher scientific) for selection. plasmids were purified using the ezna plasmid maxi kit (omega biotek), antibody recognition of the pneumovirus fusion protein trimer interface according to the manufacturer's protocol. to generate stable cell lines that express hmpv b f, hmpv b f-gcn , and hmpv f -bv, expi f (thermo fisher scientific) cells were plated into a well plate ( x per well) with ml of growth medium (dulbecco's modified eagle medium (corning), % fetal bovine serum (corning)) day before transfection. for each milliliter of transfection, μg of plasmid dna was mixed with μg of , -molecular-weight polyethylenimine (pei; polysciences inc.) in . μl opti-mem cell culture medium (gibco). after min, the dna-pei mixture was added to hek f cells in opti-mem. after to days, μl of cell culture supernatant was used for western blot to determine protein expression. then, the culture medium was replaced with ml growth medium supplemented with g (geneticin; vwr) antibiotic to a final concentration of μg/ml. after - days, hek f cells were resuspended with the growth medium supplemented with g , and expanded to a -cm cell culture flask. cells were trypsinized once they reach - % confluency and further expanded to a -cm cell culture flask. again, at - % confluency, trypsinized the cells were transferred to a ml flask in ml freestyle medium (gibco) supplemented with g and cultured in shaking incubator at ˚c with % co . for protein expression and purification, the stable cell lines were expanded in ml of freestyle medium supplemented with g . the remaining constructs are expressed by transient transfection of expi f cells. after to days, the cultures were centrifuged to pellet the cells, and the supernatants were filtered through a . -μm sterile filter. recombinant proteins were purified directly from the filtered culture supernatants using histrap excel columns (ge healthcare life sciences). each column was stored in % ethanol and washed with column volumes (cv) of wash buffer ( mm tris ph . , mm nacl, and mm imidazole) before loading samples onto the column. after sample application, columns were washed with cv of wash buffer. proteins were eluted from the column with cv of elution buffer ( mm tris ph . , mm nacl, and mm imidazole). proteins were concentrated and buffer exchanged into phosphate buffered saline (pbs) using amicon ultra- centrifugal filter units with a -kda cutoff (millipore sigma). in order to generate homogeneous cleaved trimeric hmpv f, tpck (l- -tosylamido- -phenylethyl chloromethyl ketone)-trypsin (thermo scientific) was dissolved in double-distilled water (ddh o) at mg/ml. purified hmpv b f was incubated with tame (p-toluene-sulfonyl-l-arginine methyl ester) units/mg of tpck-trypsin for hr at ˚c. trimeric and monomeric hmpv b f proteins were purified from the digestion reaction mixture by size exclusion chromatography on a superdex s , / column (ge healthcare life sciences) in column buffer ( mm tris ph . , and mm nacl). trimeric hmpv b f protein was identified by a shift in the elution profile from monomeric hmpv b f protein. the fractions containing the trimers and monomers were concentrated using -kda spin-x uf concentrators (corning). to obtain fully post-fusion hmpv f, samples were heated at ˚c for minutes to induce conversion of remaining pre-fusion hmpv f proteins to the post-fusion conformation. all samples were purified by size exclusion chromatography on a superdex s , / column (ge healthcare life sciences) in column buffer before they were applied on grids. carbon-coated copper grids (electron microscopy sciences) were overlaid with μl of protein solutions ( μg/ml) for min. the grid was washed in water twice and then stained with . % uranyl formate for min. negative-stain electron micrographs were acquired using a antibody recognition of the pneumovirus fusion protein trimer interface jeol jem transmission electron microscope equipped with a high-contrast k-by- k amt midmount digital camera. for hybridoma generation, million peripheral blood mononuclear cells purified from the blood of human donors were mixed with million previously frozen and gamma irradiated nih t cells modified to express human cd l, human interleukin- (il- ), and human baff [ ] in ml stemcell medium a (stemcell technologies) containing . μg/ml of cpg (phosphorothioate-modified oligodeoxynucleotide zoezoezzzzzoeezoezzzt; invitrogen) and μg/ml of cyclosporine a (millipore-sigma). the mixture of cells was plated in four -well plates at μl per well in stemcell medium a. after days, culture supernatants were screened by elisa for binding to recombinant hmpv b f protein, and cells from positive wells were electrofused as previously described [ ] . cells from each cuvette were resuspended in ml stemcell medium a containing × hat (hypoxanthine-aminopterinthymidine; sigma-aldrich), . × ht (hypoxanthine-thymidine; corning), and . μg/ml ouabain (thermo fisher scientific) and plated at μl per well in a -well plate. after days, cells were fed with μl of stemcell medium a. the supernatant of hybridomas were screened after weeks for antibody production by elisa, and cells from wells with reactive supernatants were expanded to -well plates for week in . ml of stemcell medium e (stemcell technologies), before being screened again by elisa. positive hybridomas were then subjected to single-cell fluorescence-activated sorting into -well plates containing % of stemcell medium a plus % of stemcell medium e. two weeks after cell sorting, hybridomas were screened by elisa before further expansion of wells containing hmpv f-specific hybridomas. for recombinant mabs, plasmids encoding cdnas for the heavy and light chain sequences of f [ ] , mpe [ ] , and ds [ ] were synthesized (genscript), and cloned into vectors encoding human igg and lambda or kappa light chain constant regions, respectively. mabs were obtained by transfection of plasmids into expi f cells as described above. for hybridoma-derived mabs, hybridoma cell lines were expanded in stemcell medium a until % confluent in -cm flasks. cells from one -cm cell culture flask were collected with a cell scraper and expanded to four -cm cell culture flasks in serum-free medium (hybridoma-sfm; thermo fisher scientific). recombinant cultures from transfection were stopped after to days, and hybridoma cultures were stopped after days. culture supernatants from both approaches were filtered using . μm filters to remove cell debris. mabs were purified directly from culture supernatants using hitrap protein g columns (ge healthcare life sciences) according to the manufacturer's protocol. to obtain fab fragments, papain digestion was performed using the pierce fab preparation kit (thermo fisher scientific) according to the manufacturer's protocol. fab fragments were purified by removing igg and fc contaminants using a hitrap mabselectsure (ge healthcare life sciences) column according to the manufacturer's protocol. for determination of mab isotypes, -well immulon hb × elisa plates (thermo fisher scientific) were coated with μg/ml of each mab in pbs (duplicate wells for each sample). the plates were incubated at ˚c overnight and then washed once with water. plates were blocked with blocking buffer ( % nonfat milk, % goat serum in pbs with . % tween (pbs-t)) and then incubated for hr at room temperature. after incubation, the plates were washed three times with water. isotype-specific antibodies obtained from southern biotech (goat anti-human kappa-alkaline phosphatase [ [fc]-ap [catalog number - ]) were diluted : , in blocking buffer, and μl of each solution was added to the respective wells. plates were incubated for hr at room temperature and then washed five times with pbs-t. the pnpp substrate was prepared at mg/ml in substrate buffer ( m tris base, . mm mgcl , ph . ), and μl of this solution was added to each well. plates were incubated for hr at room temperature and read at nm on a biotek plate reader. rna was isolated from expanded hybridoma cells using the enza total rna kit (omega bio-tek) according to the manufacturer's protocol. a qiagen onestep rt-pcr kit was used for cdna synthesis and pcr amplification. for rt-pcr, μl reaction mixtures were designed with the following final concentrations: × qiagen onestep rt-pcr buffer, μm deoxynucleoside triphosphate (dntp) mix, . μm primer mix, μl of qiagen onestep rt-pcr enzyme mix, μg total of the template rna, and rnase-free water. three separate sets of primer mixes were used: gamma, kappa and lambda forward and reverse primers as previously described [ ] . the rt-pcr was performed in a thermocycler with the following program: min at ˚c, min at ˚c, and then a -step cycle with repeats of denaturation for s at ˚c, annealing for s at ˚c, and extension for min at ˚c, followed by min of final extension at ˚c. samples were analyzed by agarose gel electrophoresis and purified pcr products (enza cycle pure kit; omega biotek) were cloned into the pcr . vector using the original ta cloning kit (thermo fisher scientific) according to the manufacturer's protocol. plasmids were purified from positive dh α colonies with enza plasmid dna mini kit (omega biotek) and submitted to genewiz for sequencing. sequences were analyzed using imgt/v-quest [ ] . for mpv , × of hybridoma cells were sent to genscript for antibody variable domain sequencing. for recombinant protein capture elisas, -well plates (greiner bio-one) were treated with μg/ml of antigen in pbs for hr at ˚c or overnight at ˚c. following this, plates were washed once with water before blocking for hr with % blocking buffer. primary mabs or culture supernatants were applied to wells for hr following three washes with water. plates were washed with water three times before applying μl of secondary antibody (goat anti-human igg fc; meridian life science) at a dilution of : , in blocking solution. after incubation for hr, the plates were washed five times with pbs-t, and μl of a pnpp (p-nitrophenyl phosphate) solution ( mg/ml pnpp in m tris base) was added to each well. the plates were incubated at room temperature for hr before reading the optical density at nm on a biotek plate reader. binding assay data were analyzed in graphpad prism using a nonlinear regression curve fit and the log(agonist)-versus-response function to calculate the binding ec values. for all biosensors, an initial baseline in running buffer (pbs, . % bovine serum albumin [bsa], . % tween , . % thimerosal) was obtained. for epitope mapping, μg/ml of antibody recognition of the pneumovirus fusion protein trimer interface his-tagged hmpv -bv f [ ] protein was immobilized on anti-penta-his biosensor tips (fortébio) for s. the baseline signal was measured again for s before biosensor tips were immersed into wells containing μg/ml of primary antibody for s. following this, biosensors were immersed into wells containing μg/ml of a second mab for s. percent binding of the second mab in the presence of the first mab was determined by comparing the maximal signal of the second mab after the first mab was added to the maximum signal of the second mab alone. mabs were considered noncompeting if maximum binding of the second mab was � % of its uncompeted binding. a level of between % and % of its uncompeted binding was considered intermediate competition, and � % was considered competition. for affinity studies, monomeric hmpv b f protein was purified by size exclusion chromatography, biotinylated, and loaded onto streptavidin biosensors at μg/ml for s, and decreasing concentrations of fabs were analyzed for binding by association for s and dissociation for s. octet data analysis software was used to analyze the data. values for reference wells containing no antibody were subtracted from the data and were fit to a : binding model. binding curves were independently graphed in graphpad prism for data visualization. hmpv plaque neutralization assay llc-mk cells (atcc ccl- ) were maintained in opti-mem (thermo fisher scientific) supplemented with % heat inactivated fetal bovine serum and grown in -cm flask at ˚c in a % co incubator. two days prior to neutralization assays, cells were trypsinized and diluted in opti-mem at , cells/ml, . ml of cells were seeded into -well plates. on the day of the experiment, serially diluted mabs isolated from hybridoma supernatants were incubated : with a suspension of infectious hmpv b strain tn/ - or hmpv a strain can/ - for hr. following this, cells were washed twice with pbs to remove serum, and inoculated with μl of the antibody-virus mixture for hr with rocking at room temperature. cells were then overlaid with ml of . % methylcellulose dissolved in opti-mem supplemented with μg/ml trypsin-edta and μg/ml cacl . cells were incubated for days, after which the cells were fixed with % neutral buffered formalin. the cell monolayers were then blocked with blocking buffer ( % nonfat milk supplemented with % goat serum in pbs-t) for hr. the plates were washed with water, and μl of mouse anti-hmpv n primary antibody (catalog number c m; meridian biosciences) diluted : , in blocking buffer was added to each well, and the plates were incubated for hr. the plates were then washed three times with water, after which μl of goat anti-mouse igg-horseradish peroxidase (hrp) secondary antibody (catalog number - ; seracare) diluted : , in blocking solution was added to each well for hr. plates were then washed five times with water, and μl of trueblue peroxidase substrate (seracare) was added to each well. plates were incubated until plaques were clearly visible. plaques were counted by hand under a stereomicroscope and compared to a virus-only control, and the data were analyzed in graphpad prism using a nonlinear regression curve fit and the log(inhibitor)-versus-response function to calculate the ic values. protein samples in reducing condition were mixed with loading buffer containing β-mercaptoethanol and heated at ˚c for min before loading on - % bis-tris plus gels (invitrogen). samples in non-reducing conditions were diluted in loading buffer without any other treatment. samples were transferred to pvdf membranes via iblot system (invitrogen) and blocked with % blocking buffer ( % nonfat milk, % goat serum in pbs-t) at ˚c overnight. antibody recognition of the pneumovirus fusion protein trimer interface primary antibodies were diluted at . μg/ml in pbs-t and hrp-conjugated goat anti-human secondary antibody was diluted at : , in pbs-t. both incubations were hr at room temperature with a x pbs-t wash in between. substrate (pierce ecl western blotting substrate, thermo scientific) was added immediately before the image was taken with chemidoc imaging system (biorad). to generate the complex of hmpv b f + mpv fab complex, purified trypsinized b f trimer was added to mpv fab at a : molar ratio and incubate at ˚c overnight. to crystallize the complex, the sample was subjected to size exclusion chromatography (s , / , ge healthcare life sciences) in mm tris ph . , mm nacl. the fractions containing the complex were concentrated to mg/ml and crystallization trials were prepared on a ttp labtech mosquito robot in sitting-drop mrc- plates (hampton research) using several commercially available crystallization screens. crystals were obtained in the crystal screen ht (hampton research) in condition f ( . m ammonium sulfate, . m sodium citrate tribasic dihydrate ph . , . m lithium sulfate monohydrate). crystals were harvested and cryo-protected with % glycerol in the mother liquor before being flash frozen in liquid nitrogen. xray diffraction data were collected at the advanced photon source ser-cat beamline -id-d. data were indexed and scaled using xds [ ] . a molecular replacement solution was obtained in phaser [ ] using the hmpv pre-fusion f structure (pdb wb ) and the fab structure (pdb q q). the structure of the complex was completed by manually building in coot [ ] followed by subsequent rounds of manual rebuilding and refinement in phenix [ ] . the data collection and refinement statistics are shown in s table. flow cytometry of hmpv infected llc-mk cells llc-mk cells were cultured in -cm flask at - % confluency, and then infected with hmpv (can/ - ) at . moi in opti-mem containing μg/ml cacl and μg/ml trypsin-edta. after hrs, cells were washed twice with pbs and digested with versene (gibco) at ˚c for - minutes. cells were washed once with pbs then transferred to . ml tubes, pelleted and resuspended in ml facs buffer (pbs containing % fbs, inactivated % human serum, inactivated % goat serum, mm edta ph . , % sodium azide) and incubated for min to block fc receptors. cells were washed three times with pbs, then aliquoted in a well u bottom plate for antibody staining. mouse anti-human igg fc apc (biolegend, ) was used for secondary antibody staining. stained cells were fixed in % paraformaldehyde and data was collected with beckman coulter cytoflex flow cytometer. data were analyzed in flowjo. in (a), hmpv f protein loaded biosensors are exposed to each mab displayed in the legend. in (b) and (c), the biosensors loaded with the first mab are exposed to mpv (b) or mpv (c). a decrease in signal was observed when attempting to load or in the presence of biosensors already loaded with mpv or mpv . no competition between mpv or mpv and other control mabs was observed, and competition was observed between mpv and mpv . a newly discovered human pneumovirus isolated from young children with respiratory tract disease human metapneumovirus: review of an important respiratory pathogen human metapneumovirus infections in young and elderly adults prevalence and clinical symptoms of human metapneumovirus infection in hospitalized patients human metapneumovirus-associated lower respiratory tract infections among hospitalized human immunodeficiency virus type (hiv- )-infected and hiv- -uninfected african infants human metapneumovirus in adults human metapneumovirus infection in lung transplant recipients: clinical presentation and epidemiology human metapneumovirus in a haematopoietic stem cell transplant recipient with fatal lower respiratory tract disease fatal human metapneumovirus infection following allogeneic hematopoietic stem cell transplantation human metapneumovirus infections in hematopoietic cell transplant recipients and hematologic malignancy patients: a systematic review human metapneumovirus infection in a hematopoietic stem cell transplant recipient with relapsed multiple myeloma and rapidly progressing lung cancer fatal human metapneumovirus infection following allogeneic hematopoietic stem cell transplantation severe respiratory illness associated with human metapneumovirus in nursing home outbreak of human metapneumovirus in a nursing home: a clinical perspective outbreaks of human metapneumovirus in two skilled nursing facilities -west virginia and idaho epidemiological survey in a day care center following toddler sudden death due to human metapneumovirus infection viral pathogens including human metapneumovirus are the primary cause of febrile respiratory illness in hiv-infected adults receiving antiretroviral therapy human metapneumovirus infection in chronic obstructive pulmonary disease: impact of glucocorticosteroids and interferon human metapneumovirus-associated atypical pneumonia and sars co-circulation of human metapneumovirus and sars-associated coronavirus during a major nosocomial sars outbreak in hong kong human metapneumovirus detection in patients with severe acute respiratory syndrome report of death in children with sars-cov- and human metapneumovirus (hmpv) co-infection: is hmpv the trigger integrin alphavbeta promotes infection by human metapneumovirus the human metapneumovirus fusion protein mediates entry via an interaction with rgd-binding integrins human metapneumovirus is capable of entering cells by fusion with endosomal membranes antibody epitopes of pneumovirus fusion proteins recombinant human metapneumovirus lacking the small hydrophobic sh and/or attachment g glycoprotein: deletion of g yields a promising vaccine candidate humoral response to the central unglycosylated region of the respiratory syncytial virus attachment protein structure and immunogenicity of pre-fusion-stabilized human metapneumovirus f glycoprotein structure-based design of a fusion glycoprotein vaccine for respiratory syncytial virus a highly stable prefusion rsv f vaccine derived from structural analysis of the fusion mechanism a proof of concept for structure-based vaccine design targeting rsv in humans characterization of human metapneumovirus f protein-promoted membrane fusion: critical roles for proteolytic processing and low ph engineering, structure and immunogenicity of the human metapneumovirus f protein in the postfusion conformation rapid profiling of rsv antibody repertoires from the memory b cells of naturally infected adult donors a novel pre-fusion conformation-specific neutralizing epitope on the respiratory syncytial virus fusion protein prefusion f-specific antibodies determine the magnitude of rsv neutralizing activity in human sera isolation and characterization of monoclonal antibodies which neutralize human metapneumovirus in vitro and in vivo identification of antibody neutralization epitopes on the fusion protein of human metapneumovirus a recombinant human monoclonal antibody to human metapneumovirus fusion protein that neutralizes virus in vitro and is effective therapeutically in vivo a broadly neutralizing human monoclonal antibody exhibits in vivo efficacy against both human metapneumovirus and respiratory syncytial virus cross-neutralization of four paramyxoviruses by a human monoclonal antibody structural basis for antibody crossneutralization of respiratory syncytial virus and human metapneumovirus human antibody recognition of antigenic site iv on pneumovirus fusion proteins a potent neutralizing site iiispecific human antibody neutralizes human metapneumovirus in vivo structure of the human metapneumovirus fusion protein with neutralizing antibody identifies a pneumovirus antigenic site use of human hybridoma technology to isolate human monoclonal antibodies high seroprevalence of neutralizing capacity against human metapneumovirus in all age groups studied in bonn structural basis for nonneutralizing antibody competition at antigenic site ii of the respiratory syncytial virus fusion protein characterization of the epitope for anti-human respiratory syncytial virus f protein monoclonal antibody f using synthetic peptides and genetic approaches anti-influenza h human antibody targets antigenic site in hemagglutinin head domain interface influenza antigen engineering focuses immune responses to a subdominant but broadly protective viral epitope antibodies to a conserved influenza head interface epitope protect by an igg subtype-dependent mechanism molecular-level analysis of the serum antibody repertoire in young adults before and after seasonal influenza vaccination a site of vulnerability on the influenza virus hemagglutinin head domain trimer interface transient opening of trimeric prefusion rsv f proteins structure of rsv fusion glycoprotein trimer bound to a prefusion-specific neutralizing antibody conformational dynamics of single hiv- envelope trimers on the surface of native virions intermediate conformations during viral fusion glycoprotein structural transition antibodies to a conformational epitope on gp neutralize hiv- by destabilizing the env spike structure of a major antigenic site on the respiratory syncytial virus fusion glycoprotein in complex with neutralizing antibody f efficient generation of monoclonal antibodies from single human b cells by single cell rt-pcr and expression vector cloning imgt/v-quest: the highly customized and integrated system for ig and tr standardized v-j and v-d-j sequence analysis phenix: a comprehensive python-based system for macromolecular structure solution coot: model-building tools for molecular graphics x-ray data were collected at the southeast regional collaborative access team (ser-cat) -id beamline at the advanced photon source, argonne national laboratory. ser-cat is supported by its member institutions (see www.ser-cat.org/members.html), and equipment grants (s _rr and s _rr ) from the national institutes of health. use of the advanced photon source was supported by the u.s. department of energy, office of science, office of basic energy sciences, under contract no. w- - -eng- .we thank georgia electron microscopy at the university of georgia for assistance with negative-stain electron microscopy, the university of georgia clinical and translational research unit for assistance with donor identification and blood draws, and the university of georgia center for tropical and emerging global diseases flow cytometry core for assistance with cell sorting. the content is solely the responsibility of the authors and does not necessarily represent the official views of the national institutes of health.the structure factors and structure coordinates were deposited to the protein data bank under accession code w . conceptualization: jarrod j. mousa.